The reproductive patterns of Oryzias woworae Parenty & Hadiaty, 2010, the endemic fish of Muna Island, Southeast Sulawesi, Indonesia, in rearing with varied sex ratios

. The endemic freshwater ornamental fish Oryzias woworae Parenty & Hadiaty, 2010 is an endangered species, and the biological information is very rare. This research aims to study the effectiveness of sex ratio on reproduction in the limited number of broodstock. Research was conducted at RC for Limnology and Water Resources, BRIN Laboratory, in April 2022 for 3 weeks with two replicates. The sex ratio used (♂:♀) included (1:1)/A, (2:3)/B, and ( 2:5)/C. All treatments were 100% on the percentage of hatching to spawning (H/S). Treatments A & B had a fertilization rate of 100%, respectively. Meanwhile, treatment A had the highest hatching rate (91.88%). Treatment C had the highest score on the number of spawning and hatching, 5 ± 1.41 times, respectively; the average number of eggs per spawning (5.6±2.27); and the average number of larvae per hatching (4.8±1.23). However, this value is almost the same as the other treatments (A & B). For the effectiveness of using a limited number of broodstock, it is recommended to use the sex ratio 1:! for O woworae reproduction. Sex determination has been seen in the fish since the 14th of the week old. Male's size tends to be larger than female.


Introduction
Oryzias woworae Parenty & Hadiaty, 2010 is a relatively recently discovered genus of Oryzias.The discovery of this type through the activities of the Indonesian Institute of Sciences (LIPI) in 2010 on Sulawesi island and was described by Parenty and Hadiaty in 2010, the name O. woworae is a tribute to Dr. Daisy Wowor, the Senior researchers from Muzeum Zoology Bogoriensis, Research Center for Biology, Indonesia Institute of Sciences, the first person who collected these new species in their habitat [1].Therefore the O. woworae species has the common name Daisy's Blue Ricefish or Daisy's Ricefish [2].The fish is the smallest type of Oryzias in Sulawesi (3−4 cm in total length) and endemic of Muna Island, Southeast Sulawesi, Indonesia, at Fotune Oe and Baleno Oe (Oe=spring water).Besides that, it is also found in the Lawe River, Motokano Ke, Waleale River as a new locality [1, 3,4,5].The discovery of this endemic fish further proves that Sulawesi Island has a high diversity of ichthyofauna and a high level of endemicity [6,7] and supports Indonesia's statement as a megadiversity country with 565 endemic freshwater [8].
Oryzias woworae is a benthopelagic and schooling fish.The fish has a very attractive color appearance.Male or female fish have a metallic blue color on the sides of the body, that tends to fade in the ventral area and bright red color on various body part (Fig. 1), so when a group of fish swims in clear water, it looks very beautiful.According to [2] not long since it was described, this fish is very well known as an ornamental fish which is traded both domestically and abroad, so the need for it is very high.It is known that the individual price in the local market is around Rp. 8,000.00 to Rp. 14,000.00 per individu, while the price in international markets is 18 times that [9] Exploitation activities, anthropogenic factors, the presence of invasive species, and a decrease of their habitat are thought to have decreased the population at their habitat so that it has been recorded as an endangered species by IUCN [3] while the biological information of the fish is still very rare, and [1] have stated that this endemic species really needs to be conserved.Thus, to maintain its existence both ecologically and economically, efforts are needed to increase the population.One effort that can be done is ex-situ conservation or domestication [10].In the domestication process, various biological information could be explored, such as growth patterns, reproduction patterns, sex differentiation, resistance to various conditions, and others.
Research on O. woworae to support ex-situ conservation is still relatively rare.However, several studies have been conducted on the O. woworae of the species, among others, the development of their embryo [11,12], spawning behavior [9] and some reproductive aspects of the fish [13].Research on sex ratios in reproduction in domestication efforts really needs to be done to address the limited number of fish and the relatively little number of male individuals.Therefore, information on the best composition of sex ratio is needed to get the optimal reproduction process.
Several studies have been conducted regarding the influence of sex ratio on the reproductive ability of fish in aquaculture, including on an endemic single fish species of River Maros, South Sulawesi, Indonesia (Marosatherina ladigesi) [14,] also on the Rasbora argyrotaenia that the native spesies of Lake Maninjau, West Sumatra, Indonesia [15].Similar studies have also been reported on zebra fish, Helostoma, Anabas, Arapayma [ 16,17,18,19], and of the Ajamaru Lake endemic fish Melanotaenia ajamaruenis [20].According to [21] in order to maintain the species existence in future breeding, the ratio of sex is one of the most essential parameters in demography.
Based on this information, research on manipulation biology with sex ratios of O. woworae reproduction needs to be conducted, namely to obtain the best composition of broodstock to efficiently the broods so the fish could reproduce optimally.Several reproductive components observed in this study are in accordance to [10].Which includes spawning frequency, hatching frequency, total number of eggs produced, fertilization rate, and hatching rate.In addition, to increase information on captive O. woworae, growth patterns and sizes achieved from larva to adult were observed and the age of fish when the sex differentiation was identified.This study provides an important basis for ex-situ breeding or domestication to support population increase and even to support programs for restocking activities of O. woworae in the future.

Methods
The research conducted is two separate activities.The first research on reproductive ability by sex ratio.Other research is the growth pattern of larvae to adults and identification of the age when sex differentiation takes place.All research was conducted at the RC Laboratory of Limnology and Water Resources, BRIN, Cibinong, Bogor, Indonesia.To rearing the fish during the research using an aquarium equipped with an aeration system.All aquariums are placed on iron shelves, which are located in a roofed room but are open on one side without walls.

Sex ratio experiment
The broodstock were obtained from fish cultivators in Serang, Banten, which were about 4 months old.The fish were then adapted in the laboratory for one month before being used for research.Reproduction research was carried out in April 2022 for 3 weeks with two replications.The number of repetitions used is adjusted to the availability of the limited broodfish.Observations of reproductive abilities were carried out including the number of spawning that took place (S), the number of eggs produced (TNoE), fertilization ability (FR), number of hatches (H) and egg hatchability (HR), length (days) of egg incubation period (LIP).and the number of larvae that can live up to the first 7 days (SR1-7) (Table 1).The research was started by measuring the length and weight of the sows.These measurements were only carried out randomly on each of 5 male and 5 female parents (Table 2).Parent fish were kept with a sex ratio of each kept in an aquarium measuring 45x40x40 cm as many as 6 pieces.Before the observation began, the broodfish were acclimatized for 5 days to new rearing media.During the study, broodfish were fed frozen bloodworms in the morning and evening.
To collect the eggs produced, in the aquarium containing the broodfish, an artificial substrate made from bundles of pieces of raffia rope that have been broken down until they are soft so that they resemble the roots of aquatic plants.The laying of the substrate was carried out starting on the 6th day after raising the fish.The substrate was observed 24 hours after placement.Females spawn their eggs and lay on the substrate, followed by fertilization by the male.To calculate the number of eggs produced, every morning at around 08:00 am, the substrate is taken.The attached eggs were then observed and, counted and separated between live and dead (failed) embryos.Eggs (live embryos) are characterized by their ability to attach firmly to the substrate and appear shiny clear.
Meanwhile, eggs (embryos) that fail to develop have an opaque color and low stickiness.The substrate containing the eggs was transferred to a hatchery aquarium measuring 25x18x20 cm.Then, the development of the eggs is observed to determine when hatching takes place.Larvae were reared continuously, and survival data were collected during the first 7 days after hatching.Data analysis was carried out descriptively.All data on the two replicates for each parameter are summed, and then the average value, standard deviation, and range are taken.

Growth pattern and Sex determination
Observations of the size at certain ages and the growth of O woworae were carried out from the larval to adult stages, namely at the age of 0 days (shortly after hatching), 4, 8, 10, 14, and 18 weeks old.Measurements were carried out by the sampling method on a number of 24 to 60 individuals.In the initial period, measurements were only made on the total length (TL) of the larvae and juveniles, and in the final stage, when the juveniles started to appear large, observations were also made on the weight size of the larvae.Measurement of the length of the larvae was carried out by placing scaled paper (mm blocks) on the bottom of the transparent container containing the larvae.Observation of the numbers that indicate the length seen from the shooting results.Meanwhile, for larger fish, measurements were taken in a small (10 cm) glass container with a scale (mm), and the results of the observations were seen directly.While the measurement of weight (g) was carried out using the digital scale [Ohaus] with an accuracy of two decimal places.During the measurement, the fish sample remained in water.
From the data obtained, the average value and standard deviation were sought, then a graph of the growth pattern was made (Fig. 3 & 4).Whereas the daily growth value (Daily GR) is calculated from the average length at time (t) minus the value at time (t-1) divided by the number of rearing days.(Table 3).
Daily GR (mm/day) = Lt -Lt-1 (1) t Description: GR =growth rate Lt = length (mm) of juvenile at time t; Lt-1 = length (mm) on one period before; t= time (days) Observation of sex determination in juveniles began when the juveniles were 10 weeks, 14, and 18 weeks old.Observations were conducted on the phenotype of the fish.Male fish tend to be brighter in color and relatively larger in body size.The main characteristics of the adult male of O. woworae are the dorsal and anal fins which tend to be wider and the back side is hairy.Observations were conducted by placing fish in a glass container filled with water.Then, point to the light.When seen the characteristic fine hairs on the outer side of the anal and dorsal fins, then the individual is marked as a male fish.Female fish are relatively small, the color is less bright, and the sides of the fins are plain (Fig. 2)

Water quality
Water quality parameters that are measured are only the main factors that are very necessary in fish farming.The parameters measured consist of temperature ( °C), pH, and dissolved oxygen content (mg/L) (Table 5).Measurements were carried out weekly using a water quality checker [Horiba-Japan].

Result and Discussion
The size of the broodstock shown in Table 2 is the result of the average value of each of the 5 male and female parents which are measured randomly as a sample.It can be seen that females are smaller than males.Meanwhile, the ability of reproduction of O. woworae on the sex ratio varies as shown in Table 3 Table 3 shows that treatment C ( ♂ : ♀ = 2:5) gave the highest score for spawning frequency (S) and hatching frequency, namely 5.00±1.41(2−6) times, TOE (eggs) values 5.60±2.27(2-9), and SR1-7 values was 88.57±19.52(60-100)%.Treatment A gave the highest score at HR: 91.88±11.31(75-100) %.The three treatments had the same value on H/S (%), namely 100%.,Meanwhile, treatment A and B have the same FR value (100%).The size of the male and female broodstock used in this study is in accordance with the size of the O. woworae fish in its natural habitat [1,2].Research by Said et al., 2022 [13] also found that the size of the broodstock used in their research was in the range of 31 mm.This activity shows that the broodstock size from captive breeding O woworae is almost similar to the size of adult fish in their natural habitat.
When the average value of the spawning distance is taken of treatment C, it can be interpreted that spawning takes place on average every 4.2 days.Whereas in other treatments (A and B), the number of spawning was about 4 times or equivalent to a spacing of 5 days.Likewise, treatment C had an average number of TEO (eggs) of 5.6 (2-9) the number of eggs obtained from 5 female parents.Whereas in treatment A, the 1 female was able to produce 4 eggs for each spawning (Table 3).This shows that the addition of the female parent does not provide an increase in the spawning frequency or number of eggs that are produced.According to [9], the increase in the number of female will eat the eggs produced by another female.The other research that spawning using a greater number of broodstock (14-15 fish) or mass spawning with the same sex ratio as this study resulted in spawning and egg numbers that were not proportional to the increase of female numbers [22].These results are in line with what happened in the study by [23] that an increase in the female population of golden cuttlefish Sepia esculenta actually reduced fecundity in females.According to [5] in spawning of O woworae with a large number of males, it can be seen that male fish tend to chase other male.And the female O. woworae tends to choose her partner compared to the O. sakaizumii.This is very different from the spawning behavior of M. ajamaruensis, in that an increase in the number of female ( ♂ : ♀ ) = 1:3 increases the number of spawning and the number of eggs ovulated [20], likewise for zebra fish, the sex ratio ( ♂ : ♀ )= 1:3 gives the best value [24].This shows that each fish species has unique characteristics in its reproductive behavior.
Figure 3 shows the size of O. woworae at a specific time period from hatching to adulthood (0 days-18 weeks old).Here, we can see an increase in the size achieved over time.This phenomenon illustrates the ongoing growth of O woworae during the observation period.At the age of 18 weeks, the weight of the fish can be seen increasing.Meanwhile, figure 4 shows the size of male and female fish after identifying sex differences.Phenotypic sex differences begin to appear when the juvenile is 14 weeks old (Fig. 4).It can be seen here that the size (total length and weight) of male fish is higher than the average value when the sizes are combined.At the same time, the size of female fish is smaller than the average value (Fig. 3 & 4).All experiments show that the H/S value is the same as 100%.The FR values for A and B were 100 each and the FR values for C were 97.78±6.67%(Table 3).The value is not remarquable diffèrent.This shows that for all treatments, the number of sperm from male fish was able to fertilize the eggs produced by female fish.Treatment A also gave the highest score on the HR score, namely 91.88±11.31%.This HR value is higher than the HR value of O. woworae in previous studies using broodstock 5 months old, even slightly higher than the HR value of 7 month old broodstock [13].The value of HR shows that the embryo can live well during the hatching process.The SR1-7 and LIT values in this study were not remarkably different, even the LIT values were shorter than in previous research [13].Such values are commonly found in other scholling fish in cultivation, such as O marmoratus and M. .ajamruensis[10,20].
Based on this phenomenon, for O. woworae spawning it is recommended to use a ratio of ( ♂ : ♀ ) = 1:1 individually.This is to get the number of spawning and the optimal number of eggs as well as for the effectiveness of the use of limited or rare brood fish The total length investigated of larvae aged 0 days was around 3.20 mm (Fig. 3).This size is similar to the size of O. marmoratus larvae in 0 days old, namely 3.3±0.483mm [10] also with the size of O. woworae in previous studies of 3.125±1.158(3−5) mm [13].It is thought that Oryzias tend to have larger egg diameters and a long initial larval size.In the early stages, the measurement of growth patterns was only carried out on the length parameter.Length measurements were more accessible to do than weight measurements.
At the juvenile age of 10 weeks old, weight and length measurements were carried out.At the age of 14 weeks, the male and female sexes could be identified, and at the age of 18 weeks, these characteristics were even more pronounced with the characteristics as shown in Figure 2. From these conditions it can be seen that the female fish are smaller than the male (Fig. 4).However, it is not yet known whether at the age of 18 weeks the fish are capable of reproducing.The [13] study was conducted on fish aged 5 months, 6 and 7 months, and high viability was obtained in fish aged 7 months.According to [25], ent species have their initial sexual maturity, which depends on the age and gonad development of each species.In the same population, the time of early and late sexual maturation for individuals is related to changes in their growth rate and living environment.Gonadal maturation may reflect the influence of external factors on the population such as water movement, water temperature, or hardness.When the rearing water temperature is suitable and the growth is good, it will stimulate the growth of gonadal maturity.Table 4 shows that the male daily growth is more significant than the female.This is in line with Figure 4.There is a tendency that the older the age, the greater the daily growth.This means that up to 18 weeks, O woworae fish are still growing.Somatic growth is still ongoing even after reaching reproductive age.Daily growth in each age period tends to be higher from age to age (Table 4).The size daily growth of O. woworae in previous studies at 12 weeks old was 20.375±3.623(20−24) mm equivalent to daily growth rate of 0.243 mm/day.The growth of male individuals looks faster than the growth of female fish.This is presumably by the maximum size of the male and female parents achieved, where the female is smaller than the male at the same age.This size is also in line with the daily growth of O. marmoratus [10] with a daily growth of 0.271 mm/day at 16 weeks old.In line with the results of [14], the daily growth rate of Marosatherina ladigesi (Celebes Rainbow) in controlled habitat was 0.21−0.29 mm/day at 12 weeks old.The results of this study indicate that the endemic fish O. woworae has been adapting to grow and reproduce in a controlled habitat or cultivation environment.However, to increase reproductive performance, growth, and survival of O woworae fish, development research is needed, such as the use of various types of natural feed or content enrichment in feed, or optimization in the cultivation environment.
Several values of pH, temperature, and dissolved oxygen content in the O. woworae rearing water are shown in Table 5.This shows that O. woworae can tolerate these parameter values, as indicated by their ability to grow and reproduce.This research took place in reared water with conditions of temperature, pH and dissolved oxygen as shown in table 5.The data obtained it shows that O woworae fish are able to tolerate these conditions to reproduce and grow well.Different conditions of rearing water quality are expected to give different results of fish growth and development [11,12].The rearing water temperature and the pH value are within the natural habitat water condition.According to [2] the natural habitat conditions of the O woworae are temperature: 23-27 °C, pH range: 6.0−7.5; and Hardness: 90-268 ppm.

Conclusion
For Oryzias woworae reproduction, it is recommended to use a sex ratio Male:Female (♂:♀= 1:1).The fish is able to reproduce and grow at ex-situ habitat and sex differentiation begins at 14 weeks (3.5 months) old.Male size tends to be bigger than the female.It is hoped that the fish could be developed to support domestication programs in the future, but further research of the fish is needed.

Fig. 2 .
Fig. 2. (A) Observation activity of sex determination and ( ) characteristics of male

Table 2 .
The mean and range size of broodstock

Table 3 .
The reproductive ability of O. woworae on various sex ratio

Table 4 .
Daily (length)growth rate (mm/day) of O woworae from larva to adult size

Table 5 .
The value of several water quality parameters of rearing water