Features of Artemia aquaculture technology in Russia, prospects for its use in other temperate and tropical climates

. The results of long-term monitoring of the abundance and biomass of Artemia on the example of several model lakes in the south of Western Siberia are presented. Based on this, conclusions are drawn about the dynamics of the density of Artemia crustaceans characteristic of shallow lakes of temperate climate, associated with low live birth: a high number Artemia shrimps of the first generation and a low following. It is proposed to inoculate naupliuses in lakes during the period of catastrophic decline in the number of crustaceans, which will create a new powerful generation of Artemia. The results of such experiments are shown on the example of two lakes for two years. An assumption is made about the possibility of using this technology of aquaculture of artemia in other temperate countries. Laboratory and field studies on reducing the incubation time of cysts and early release into the brine of lakes are presented. The influence of brine salinity of lakes on the results of early release of nauplius and non-hatched cysts is shown. The technology of reducing the incubation period of cysts can be used in subtropical and tropical climate.


Introduction
Aquaculture, as one of the main sources of protein food supply for humanity, needs a stable supply of live starter feed.Artemia cysts have earned recognition as the most easy-to-handle food among all currently available live foods for fish larvae and crustaceans.Thanks to these cysts, according to the latest data [1], more than 10 million tons of valuable aquaculture species (shrimp, crabs, various marine fish) are grown annually.At present, almost no sturgeon farm in Russia can do without the use of brine shrimp for feeding fish larvae [2,3].The main world reserves of artemia cysts are concentrated on the territory of several countries (USA, China, Russia, Kazakhstan, Uzbekistan), where from 3.0 to 4.5 thousand tons of cysts in dry weight are harvested annually.A characteristic feature of commercial Artemia reservoirs is that they are drainless relatively shallow lakes located in a temperate continental arid climatic zone.In a changing climate, the natural reserves of artemia are very vulnerable.
In many countries with a tropical and subtropical climate (Thailand, Vietnam, Kenya, Mozambique, Sri Lanka, Peru, Philippines, Iran), attempts are being made to grow Artemia in coastal seasonal salt ponds.For temperate water bodies, such technologies are not suitable.The need to conduct research to improve the technology of growing artemia at the local level is mentioned in many publications [1,4,5,6,7].Our tasks were to study the possibility of growing artemia in natural salty reservoirs with a local artemia population to obtain additional cysts.Laboratory and field studies were carried out, the results were evaluated and prospects for work on inoculation of nauplius in salt lakes were outlined.

Monitoring of Artemia biomass and abundance in model lakes
In the period from 2001 to 2004, year-round studies of Аrtemia population density were conducted in 5 lakes (Vishnyakovskoye, Nevidim, Bolshoe Medvezhye, Maloe Medvezhye and Ebeyty), in 3 lakes (Novo-Georgievskoye, Cherdynskoye) -during the development of artemia crustaceans (from April to October) (Fig. 1).A total of 227 comprehensive studies were carried out, which also included abiotic factors: temperature, oxygen, and salinity (Table 1).The results of these field studies are presented in more detail in the literature (Van Stappen, Litvinenko et al., 2009).To summarize the data on monitoring studies, indicators of the ratio of the actual values of the average monthly biomass and the number of artemia crustaceans to the seasonal average values were used.In 2019-2020 in lakes Okunevo and Karasye, studies of the abundance and biomass of crustaceans and Artemia cysts were carried out from June to August, that is, before and after the experiments on inoculation of nauplii.

Laboratory experiments on incubation of cysts and survival of nauplii in brine with different salinity
The material for the experiments was cysts from Filatovo Lake, collected in September 2011.All experiments were carried out in triplicate.The temperature during the experiments was 23-24 0 C. The percentage of nauplii hatching was determined by standard methods (Sorgeloos et al., 1986) in the water of natural saline reservoirs with salinity of 150‰ (Filatovo Lake), 198‰ (Ebeyty Lake) and 240‰ (Medvezhye Lake), which was diluted with fresh water in concentrations from 1 to 100‰.
In experiments to study the survival rate, cysts were incubated in solutions of brine from Lake Ebeity diluted with fresh water.Concentrations of 0.5-195‰ were tested.
In experiments to reduce the incubation period, every 2 hours, 25 ml of a sample was taken from the incubation medium, which was passed through a gas sieve.Filtered cysts (cysts and nauplii) were placed in 50 ml vessels with lake brine.These samples were observed on day 1 from the start of incubation and on day 2 after being placed in the brine.The number of cysts and nauplii was counted using a stereoscopic microscope.

Field studies during the period of incubation of cysts and the release of nauplii into a natural salt lake
The experiments were carried out: July 09-26, 2019 and July 14-20, 2020 on Okunevo Lake; July 08-13, 2020 and July 14-24, 2021 -on Karasye Lake.The cysts were incubated in 10 m 3 frame pools located on the shore of a salt lake.The incubation medium with a salinity of 20-50‰ was a mixture of fresh water and water from a salt lake.During incubation, lighting installations, aeration with the help of airlifts, and generators with a total capacity of 7.5 kW were used.Figure 2 shows a diagram and a photo of the incubation unit.The installation was located on the lake shore for convenient release of nauplii into the lake and pumping water from the lake into the installation.Fresh water for dilution was supplied either from nearby freshwater lakes using water pumps or water carts.

Monitoring of saline lakes in the south of Western Siberia
Long-term year-round monitoring of abiotic and biotic factors of 5 model hypergaline lakes Vishnyakovskoye, Nevidim, Bolshoye Medvezhye, Maloye Medvezhye and Ebeity, as well as 3 lakes studied only during the growing season: Novo-Georgievskoye, Cherdynskoye, Ulzhay located in the southern part of Western Siberia (Fig. 3) allowed to reveal regularities in the development of Artemia populations [8,9,10]: -wintering as cysts, -the first generation of crustaceans is the most powerful due to hatching of naupliuses from overwintering cysts at water warming up to 5 0 C (mid-April), -subsequent 2nd and 3rd generations are fading due to low live births, -die-off of crustaceans in the middle of October at stable transition through the temperature of 5 0 C.

Fig.3. Seasonal dynamics of the total number of brine shrimp in model lakes
According to the conducted studies, during the period when commercial stocks of cysts should be formed in the reservoir (August-September), as a rule, sexually mature crustaceans occur in a small amount.
Figure 4 shows the total indicators of the dynamics of abundance and biomass of Artemia crustaceans expressed through the ratio of actual values to average values.It turned out that the maximum abundance of Artemia crustaceans, exceeding the seasonal average by 3 times, is observed in late April, followed by a sharp decline until the end of the growing season.During the period of the 2nd and 3rd generation of the crustaceans (June and September), this decline slows down somewhat.In the dynamics of Artemia biomass, the first peak is observed in June, i.e. in the period when the crustaceans of the first generation reach the sexually mature stage.The biomass of Artemia at this time exceeds the average seasonal values by 2 times.The next peak of Artemia biomass (smaller in magnitude than the first and below the seasonal average) is observed in September.Thus, from June to August there is a sharp decrease in the number and biomass of Artemia crustaceans due to dying off of adult stages of the 1st most powerful generation of crustaceans.At the same time, the high-fed water body (due to a large amount of organic matter of dead crustaceans and slow decomposition processes due to high salinity) is mostly empty in the second half of summer.Inoculation of naupliuses during this period allows creating additional powerful generation and thus doubling the productivity of water bodies.
The idea of Artemia nauplius inoculation during the period of natural population decline was first voiced at the International Conference on Salt Lakes in 2017 [11].In the following years, a number of experiments were carried out to put this idea into practice.

Laboratory studies to determine the optimal salinity for cyst incubation
First, laboratory studies on incubation of cysts in water with different salinity were carried out.Natural lake brine diluted with tap water to the required concentrations was used: 1-100‰.The hatched naupliuses were planned to be released into a natural water body with relatively high salinity from 100 ‰ and more.Therefore, it is necessary to select the most acceptable salinity to reduce osmotic shock.Figure 5 shows that maximum hatching is observed at the lowest salinity values (1-15‰), with salinity greater than 45‰ showing a significant decrease in hatching percentage.Thus, salinity of 35-45‰ was recommended for incubation of cysts and inoculation of nauplii into natural water body.The study of nauplius survival rate after their release into the medium with salinity from 0.5 to 195‰ showed (Fig. 6) that on the second day (48 h) after release the survival rate of 100% was registered in experiments with salinity 45-180‰, on 60 h from the beginning of the experiment the maximum survival rate (about 80%) was observed at salinity 45-120‰.Decrease in survival rate to 48-59% was observed at both low and high salinity.In water with salinity 135-165‰ the survival rate of crustaceans was within 67-69%.

Field studies of nauplius inoculation into saline lakes
The first experiment on nauplius introduction into Ulzhai Lake was conducted in August 2015 (see Table 1).The lake is drainless, located in the steppe zone, salinity varied from 49 to 235 g/l in different years and periods of the season.During the period of experiments the salinity was about 120 ‰.A total of two experiments were conducted.In the first one 50 kg and in the second one 30 kg of dry cysts were incubated.The quality of cysts was relatively high: impurities were 3%, hatching was 78%, and moisture content was 6%.The experimental conditions are summarized in Table 2 Incubation occurred for 24 hours in the first experiment and 36 hours in the second experiment.The longer incubation duration in experiment 2 was due to low air temperature.Hatching exceeded 60% in both cases.The high cyst density of 5 g/L dry cysts resulted in a decrease in oxygen content to 0.7 mg/L by the end of the experiment.Therefore, the cyst density was reduced in the next experiment.Simultaneously, hatching experiments were conducted in lake brine (salinity 120‰).No hatching of naupliuses was observed within 48 h.
At the end of incubation, the hatched naupliuses were released into a pen (a section of the lake fenced with fine mesh fabric) and the ratio of live to dead naupliuses was used to determine the survival rate in the lake brine.On the 4th day the survival rate in experiment 1 was 47%, in experiment 2 -59%.
Thus, the possibility of incubation of cysts in large volumes (10 m3) near a water body and the success of nauplius inoculation into a natural water body were proved.
The following studies were devoted to refining the technology for such cultivation.
In 2019 and 2020 on Lake Solenoye and in 2020 and 2021 on Lake Karasie (see Table 1), experiments were conducted to determine the optimal incubation parameters: incubation start time and its duration, cyst density, amount of hydrogen peroxide for cyst activation, and the method of releasing Artemia nauplii into a water body (passive, active).The most interesting of these indicators: reduction of incubation time is given in this article.
Under standard conditions the duration of incubation of cysts used as starter live food is 24-48 hours.This is due to the need for complete separation of cyst shells from nauplii, because when these shells and uninoculated cysts enter the intestinal tract of fish and crustacean larvae, they become blocked, leading to death.In the case of inoculation of naupliuses into the water body there is no such need.We tested the possibility of hatching of dry cysts in lake brine without incubation and with incubation for 2-23 h both in laboratory conditions (with brine salinity 101, 125, 225 and 333‰) and in incubation facilities on salty lakes Solenoe and Karasie.
Both laboratory and field studies have shown the absence of cysts hatching in the brine of lakes with salinity more than 100‰.It is known that complete hydration of cysts is necessary for nauplius hatching.To clarify the hydration time, we conducted studies during the incubation of dry cysts in incubation medium with salinity 42‰ at 27 0 C (Litvinenko et al., 2021).It turned out that by 6 h of incubation hydrated cysts were more than 80%, by 10 h -almost 100%.The beginning of hatching characterized by shell rupture and embryo emergence occurs en masse at 14-16 h of incubation, starting from 18 h -the first freeswimming nauplii appear.Starting from 22 h of incubation, dead nauplii appear (Fig. 7).
In experiments to reduce the incubation time of cysts (the results are presented in Figure 8), important answers to the question of the possibility of such a reduction were obtained.The experiments were conducted with cysts from two populations (1 -Ebeyty and 2 -Maloe Yarovoe).Some differences in the results indicate the influence of cyst and origin on these processes.Common for the populations is high mortality of naupliuses at 330‰ brine salinity already on the second day of the experiment, which gives a good reason to exclude lakes with high salinity from inoculation experiments.
The comparison of test data 24+ , i.e. under standard conditions (placement in the brine after 24 h of incubation) with earlier releases into the brine on the second day of the experiment is a more illustrative indicator not only of the possibility of reducing the incubation period, but also of the necessity.In Experiment 1, it is shown that at 100‰ brine salinity the best results were obtained in the 16+, 18+, 20+, 22+ tests, while at 125‰ and 225‰ salinity the best results were obtained in tests 20+ and 22+.In Experiment 2, at 100‰ brine salinity the best results were observed in tests 10+, 12+, 16+, at 125‰ salinity -in all tests starting from 12+ (except 22+), at 225‰ salinity -in test 16+.When comparing the data on nauplius hatching at 24 h and 48 h, it can be seen (see Fig. 8) that nauplius hatching occurs in lake brine, i.e. embryos at the umbrella stage develop into full-fledged naupliuses in lake brine.This process is particularly intensive in experiment 1 in brine with salinity 100‰, and in experiment 2 in brine with salinity 100 and 125‰.
Experiments on shortening the incubation period were also conducted in field conditions, conducted in 2019-2021 on lakes Okunevo and Karasie.The results of these experiments can be clearly judged by hydrobiological samples on the eve of the experiments and after (Fig. 9).

Discussion
The dynamics of Artemia population density revealed in our study of shallow lakes in the south of Western Siberia (maximum during the first generation and low density of subsequent generations) due to low live births is not specific only to shallow water bodies.For example, similar dynamics have been observed in relatively deep water bodies of Russia in some years, as well as in the largest commercial water body Great Salt Lake [13].Therefore, the geography of use of the proposed method of increasing cyst production can be expanded.
Of the vast number of publications on Artemia, the main topic concerns the use of naupliuses as food for fish and crustacean larvae [14].The need for incubation of dry cysts to obtain naupliuses is determined by their dehydrated state.It is known [12] that only fully hydrated cysts can initiate metabolism.The use of wet cysts instead of dry cysts (to accelerate incubation time) causes difficulties in storage of such cysts and, at high temperature, rapid spoilage.Therefore, the procedure of incubation of dry cysts in a medium with lower salinity is a necessary element in the technology of Artemia inoculation in saline water bodies.During incubation cysts pass the stage of hydration, embryo stage 1 and 2, nauplius.Optimizing this process is very important for good results and cost savings.
Inoculation of cysts into salt ponds without Artemia was first performed in 1977 in Macau (Brazil) with 250 g Artemia franciscana cysts.A few months after this inoculation, the first ton of cysts was collected [4,7,15].Since then, introduced Artemia has been found continuously in Brazilian saltwater bodies.Such examples of high productivity indicate the considerable potential of Artemia under favorable conditions for development.The relatively low annual production of Artemia cysts in Brazil of 2-4 tons of cysts and 25-30 tons of Artemia biomass is attributed limitation of effective management practices [7].
The technology of Artemia cyst and biomass production by inoculation of Artemia nauplii in saline evaporation ponds is well developed in India, Iran, Thailand, Vietnam and other countries with tropical and subtropical climates of the Eastern Hemisphere.According to literature data [1], the greatest results in obtaining stable yields of cysts have been achieved in Vietnam, where about 40 tons of cysts are obtained annually.This technology involves the use of organic and inorganic fertilizers and fodder, which allows obtaining 17-25 kg/ha of cysts from a pond area of 1200 ha [16].
Significant Artemia harvests have been observed in coastal, highly eutrophic ponds in China, located near Bohai Bay.As early as the 2000s, these ponds with small areas (less than 100 ha) had cyst yields of 3-60 kg/ha raw weight (~1.5-30 kg/ha dry cysts).In contrast to Vietnam and Thailand, Artemia cultivation in them was conducted in an extensive way, i.e. without fertilizer and feeding [4].This method of Artemia cultivation in ponds is relatively similar to the cultivation in our studies.The differences are that saltwater lakes have a significant supply of cysts in spring, providing a strong first generation of Artemia.Beginning in July, due to low live births, Russian salt lakes are generally sparsely populated with Artemia, which brings them closer to the ponds of Bohai Bay.The differences are related to the fact that Bohai ponds use not only local Artemia nauplii, but also A. franciscana nauplii for inoculation.In Russia, only local Artemia populations are used for this purpose.Artemia nauplius hatcheries have recently been built in China, which, unlike the hatcheries we used, have a smaller volume and are located indoors at a considerable distance from the nauplius release sites.
The introduction of Artemia cysts and naupliuses into natural saline lakes is poorly covered in the literature.As a rule, a water body with salinity suitable for Artemia development is naturally colonized by this crustacean species after some time (waterfowl, wind).Such Artemia-free lakes with conditions suitable for Artemia development have been found in high-mountain Tibet.The introduction of Artemia sinica into the Tibetan lake Dangxiong Co (China), located at an altitude of 4475 m above sea level, with an area of about E3S Web of Conferences 431, 01047 (2023) ITSE-2023 https://doi.org/10.1051/e3sconf/20234310104756 km2 and an average depth of 12 m, salinity 120-180 g/l was studied in 2004.According to literature data [17], the introduction of naupliuses obtained from 850 g of cysts was sufficient to increase the abundance of crustaceans in 2006 to 20 ind./m3 and in 2013 to 1950 ind./m3.Based on the results of such introduction, preliminary calculations were presented, which showed that up to 150 tons of cysts could be collected from the lake per year, as well as 3.2 thousand tons of frozen or 350 tons of dried biomass of Artemia adults.
We found no data on early release of embryos into lake brine in the literature.The intentional shortening of cyst incubation and release of unhatched embryos into lake brine in order to speed up and cheapen the procedure of lake inoculation in our study showed that this technology is promising.
Thus, the use of the presented Artemia aquaculture technology in other temperate countries is possible, provided that the live birth rate of local Artemia populations is low and the abundance and biomass of the crustaceans decrease sharply after the first generation.Population of naupliuses in optimal concentrations can form a generation as powerful as the first one and thus double the productivity of such water bodies.
The results of studies on reduction of cysts incubation time and early release into lake rapa allow reducing the release time and cheapening this procedure of incubation and inoculation.This part of the technology may well be used in countries of subtropical and tropical climates.

Fig. 2 .
Fig. 2. Scheme of the incubation plant (A), photo of the plant (B), two plants at night (C), experience of hatching in lake brine (D), release of naupliuses into the lake (E)

Fig. 4 .
Fig. 4. Seasonal dynamics of the total number of brine shrimp in model lakes

Fig. 6 .
Fig. 6.Survival of nauplii after their inoculation into brine with different salinityThus, the resistance of Artemia nauplius to hypergalyne shock was clearly demonstrated.

- Ulzhai Table 1 .
Characteristics of saline lakes during the study period .