Part 3: Artemia Cyst Production in Solar Earthen Ponds in Vinh Chau, Vietnam, and Its Associated Microbiome

4. Discussion

This study provides an update for Artemia cyst production in Vinh Chau, Vietnam — one of the most successful Artemia aquaculture practices globally [3]. The average cyst productivity of all the experimental ponds in our study was 112.0±22.3kg/ha/crop. This is significantly higher than 83.8±30 kg/ha/crop as reported by Vinh et al. [23] for 94 Artemia farms in the same production area in 2019. However, large variation still existed among these production ponds as reflected by the large CV of cyst productivity (52.8%). The highest and lowest cyst productivity was 190.0 and 15.0kg/ha/crop, respectively. This great variation prompts the need for improving husbandry and pond management for more reliable Artemia cyst production. Also, it may indicate possible increasing impacts of extreme weather or climate changes on Artemia, either directly or via other influencing factors, such as food availability and water quality. The success of Artemia cyst production depends on the operator’s capabilities of creating/ maintaining a good microalgae bloom and keeping salinity within the desirable range of 80–120ppt throughout the crop. Chicken manure in combination with inorganic fertilizers, such as urea, diammonium phosphate (DAP), and nitrogen phosphate potassium (NPK) has been used to provide nitrogen (N) and phosphorus (P) for algae growth in Artemia ponds [23, 48]. Dunaliella spp. and diatoms are considered the most popular and important foods for Artemia in solar salterns [49, 50] or specialized production ponds [25]. Failures to bloom these microalgae and maintain the bloom throughout the crop will result in the lack of food for Artemia. The results of our study showed that both fertilization rate and feeding rate strongly correlated to cyst productivity (Figure 6), as mentioned in previous studies [23, 51]. The amount of chicken manure and shrimp feed crumbles used explained 89.0% and 79.3% of the observed variations of cyst productivity in our study. Generally, higher fertilization rate and feeding rate resulted in higher cyst productivity up to a certain level. As shown in Figure 6, no more than 85kg of shrimp feed crumbles or 3600 kg of chicken manure should be used per ha per crop for possible highest cyst productivity within the ranges reported in our study. These figures are equivalent to approximately 0.9kg of feed/ha/day and 38.7kg of chicken manure/ha/day. Excessive fertilization and/or feeding rates can result in eutrophication, which com promises water quality and Artemia survival in shallow solar ponds where oxygen is often limited [49].

The heatwave that occurred on DOC85 (17 days after the first sampling) and lasted for 4 weeks in the research area had detrimental effects on Artemia in our study. Artemia mortality was first observed on DOC89 only 4 days under extreme heat, followed by algal crashes. As the survived Artemia stopped producing cysts, cyst collection ceased from DOC94. Artemia are extremophiles. However, they are more tolerant of extremely high salinities than high water temperatures. For Artemia, optimal water temperature for growth and survival is 20–28°C [52] and 20–26°C [53], respectively. Reproduction is possible within 15–30°C but preferably below 25°C [54]. Saygı and Demirkalp [55] reported that total mortality of Artemia sp. originated from Tuz Lake in Turkey occurred after 15 days at 32°C and 25 days at 30°C. In Tuz Lake, Artemia biomass sharply decreases in mid-June when water temperature approaches 30°C. According to [56] Artemia consumed more oxygen at higher temperatures and higher salinities. Thus, high densities of Artemia in the production ponds of our study could easily result in the depletion of oxygen, which is detrimental to Artemia. Previous studies have shown that A. franciscana can survive and reproduce well at 180ppt [52, 54]. In our study, salinity at the end of the crop was 154–158 ppt. Therefore, high water temperatures were likely causing Artemia mortality.

In coastal areas, large variation among aquaculture production ponds has been commonly observed even with ponds built with a standard design and operated by the same operator using the same inputs for farming. Differences in their microbial assemblages, which are usually invisible to farm operators and quite stochastic, are likely the most logical explanation for these observed large variations. Molecular studies on rearing water microbiomes have shown that the community composition of these systems is dynamic over time and exhibits large variability across replicate cultivations [57]. Prior to the implementation of our study, the owner of the farm already classified the experimental ponds into two groups by cyst productivity, that is, HIGHER and LOWER using his experience. The results of our study confirmed this classification. The HIGHER group outperformed the LOWER one regarding the number of harvest days, amount of cysts per harvest day, and total cyst yield (Table 2). Cyst productivity was 160.6±10.3kg/ha/crop for the HIGHER group, 2.5 folds significantly higher than that for the LOWER one (63.2±18.7kg/ha/crop) (p< 0:05). Since pond preparation, water source, Artemia origin and stocking density were the same for both pond groups, the differences of their microbial fauna and how they reacted to extreme weather may explain the observed difference of cyst productivity.

The results of our study showed no connection among the bacterial communities between pond sediment, pond water, and Artemia gut either mid-crop or at the end of the crop. At the same time, a lot of similarities were found between the two pond groups regarding bacterial communities of pond sediment, pond water, and Artemia gut. These observations altogether question the roles of bacteria as foods for Artemia. It is believed that bacteria can supply supplementary micronutrients for A. franciscana [58], but feeding experiments con ducted by Makridis and Vadstein [32] demonstrated that bacteria are too small for Artemia to graze on. Artemia is a filter feeder of small food particles ranging 1–50μm in size [59]. However, their preferred particle size is 3–8µm, far larger than most single-celled bacteria. Thus, only halophilic heterotrophic bacteria which at high densities form bioflocs may be grazed more efficiently by Artemia [25, 60, 61]. Even though, research has shown that bioflocs can only replace up to 50% of micro algae when feeding Artemia in laboratory conditions [62]. Moreover, the inclusion of bioflocs or reduction of microalgae in feeding compromises Artemia’s reproductive performance at 120ppt.

Tkavc et al. [63] reported that Halomonas, Salinivibrio, and Vibrio dominated the Artemia microbiota in solar salterns in Israel. Furthermore, their relative abundance changed substantially with increasing salinity. Halomonas abundance in Artemia gut increased from 11% at 110ppt to 94% at 300ppt, but Vibrio disappeared at 140ppt. In our study, Halomonas dominance in Artemia gut was confirmed regardless of sampling time or cyst productivity. This taxon accounted for 23.3%–33.9% mid-crop at 115–120ppt and 62.3%–72.7% at the end of the crop when salinity was 154–158ppt. This could be the effect of increasing salinity and likely extreme temperature as the result of the 4-week heatwave. Lopes-dos-Santos et al. [60] reported that Artemia nauplii grow well when being fed with Halomonas ventosae alone. Furthermore, apart from being a good food source, Halomonas are able to accumulate the biodegradable polymer polyhydroxybutyrate (PHB) [64]. PHB protects Artemia by enhancing the expression of the heat shock protein 70 (Hsp70) [65]. The absolute dominance of Halomonas that we found in Artemia gut at the end of the crop might be highly beneficial for the surviving Artemia after that 4-week heatwave. It also explains why Vibrio abundance in Artemia gut was significantly reduced under increasing dominance of Halomonas. Sui et al. [66] found that feeding Artemia with Halomonas species reduced microbial diversity in the gut, particularly decreasing the abundance of Vibrio.

Besides Halomonas, we found two more taxa dominant in Artemia gut including Roseovarius (38.7%–40.8%) and Vibrio (10.0%–20.1%) around mid-crop. Interestingly, these dominant taxa only presented at small proportions in pond sediment and pond water, suggesting that either the intestine environment of Artemia is more suitable for them to harbor or they may have some important roles in the gut of Artemia. Previous feeding study also showed that the bacterial assemblages of feed/water are very different from that of Artemia gut [61]. In our study, the dominance of Roseovarius and Vibrio would have been the result of the application of chicken manure to promote microalgal bloom and the use of shrimp feed crumbles for supplementarily feeding of Artemia. Roseovarius belong to the Rhodobacteraceae — an important group of marine heterotrophic bacteria. Rhodobacteraceae are able to metabolize a large variety of organic compounds and often associated with microalgae. Since microalgae are the major food sources of Artemia, Roseovarius might have been coincidentally grazed (with microalgae) by Artemia and ended up in large quantities in their guts. Furthermore, it is believed that Roseovarius help digest microalgae [60]. Roseovarius survive best at 20–50ppt [67]. This explain why they were more abundant in Artemia gut around mid-crop in our study when salinity was 115–120ppt than at the end of the crop when salinity reached 154–158ppt.

Vibrios are pivotal components in the gut microbiome of a wide variety of marine species including Artemia [64]. Their symbiotic interactions contribute to nutrient acquisition and other critical functions of their hosts. Some Vibrios, such as V. parahaemolyticus, V. campbellii, and V. proteolyticus CW8T2 are harmful to Artemia [68], but some are considered beneficial. has isolated Vibrio sp. ArtGut-C1—a PHB producer from Artemia gut that can be used as probiotics in marine crustacean hatcheries. This species yielded 72.6% PHB of cells’ dry weight at 25°C. More importantly, the analysis of its genome revealed a number of genes functionally related to PHB metabolic pathways and to competing and colonizing abilities. Their findings may help explain why Vibrio dominated the Artemia gut microbiota in our study and why the relative abundance of Vibrio was two folds higher in the HIGHER pond group compared with the LOWER one. Pond with higher abundance of Vibrio and lower abundance of Roseovarius in Artemia gut tended to have higher cyst productivity. It would be very helpful to explore what species/strains of Vibrio that colonize the gut of Vinh Chau Artemia via further research. Any beneficial Vibrio strains, if found, can be used as probiotics to enhance the performance of the system.

Both diatoms and Dunaliella were found in Artemia ponds in our study and revealed interesting patterns in relation to sampling time and cyst productivity. According to [49] the most abundant aerobic phototrophs in solar salterns are the green microalgae Chlorophyceae, typically represented by Dunaliella salina, diatoms (Nitzschia, Amphora), dinoflagel lates (Gymnodinium, Gonyaulax), and Euglenophycaea. They are considered the major food source for Artemia [62, 69–71]. Our observations showed that water color and Artemia’s appearance at the first sampling time were visually different between the two pond groups. The HIGHER group had a thicker bloom of microalgae which was bright brownish or greenish in color. In these ponds, Artemia were present along the pond edge at high densities. They looked healthy and brightly pinkish in color while those in the LOWER group looked pale and whitish. Limited bloom of microalgae was observed across all ponds of the LOWER group. Our micro biome analysis showed that Bacillaphyceae was dominant mid crop but replaced by Chlorophyceae. This typical shift has been reported by Pedrós-Alió [72] and Guermazi et al. [73] as salinity approaches 150ppt. Dunaliella in many cases then become is the sole primary producer and food for Artemia. In our study Dunaliella was more abundant in ponds with no Artemia (i.e., across both pond groups at the end of the crop) or less Artemia (e.g., in the LOWER pond group around mid-crop). These imply that Dunaliella is the major food source for Artemia as stated by several previous studies [32, 49, 73]. Although Artemia is a nonselective filter feeder, their grazing efficiency is best with food particles of 3–8µm across different stages of their development [32]. The small-sized Dunaliella sp. (8–12µm) together with Chlamydomonas sp. (4–7μm), coccoid cyanobacteria (1–2 μm) or centric and pennate diatoms (20 40μm) are Artemia’s most common foods [32]. Thinh Luong-Van et al. [71] have used Cryptomonas sp. successfully to culture Artemia. However, Cryptomonas was not found in the Artemia ponds of our study. In solar salterns Dunaliella can tolerate to a wide range of salinity from 3 to 300ppt. Its population exhibits a negative relationship with that of Artemia. Grazing experiments confirm that Artemia exercise a top down control on Dunaliella populations [73]. This explains why Dunaliella was not detected mid-crop in pond water or pond sediment, but at the end of the crop in our study. We did not find Dunaliella in Artemia guts in our study. First, that was probably because, unlike Bacillariophyceae, Dunaliella do not have cell walls. They can be digested quickly, and thus, remained undetected. Second, it is possible that the density of Dunaliella was relatively low during the crop as part of the poorer microalgal bloom reported by the farm’s owner compared to the previous years. According to the farm’s owner, cyst productivity of this crop was only 50% his usual achievement.

The observed differences of microbiome between the two samplings in our study appear to be a mere effect of increasing salinity, from within the optimal range for cyst production mid-crop to unfavorable levels at the end of the crop. Recent studies have indicated a reduction in abundance, species richness, and diversity of solar salterns’ microfauna as salinity increases (for review see [50]). In our study, diversity of bacteria, fungi, and microalgae all reduced at the end of the crop when salinity of pond water reached 154–158 ppt on average due to the prolonged heatwave. The largest reduction of diversity and shift in composition were observed in pond water. Of these, the most apparent effect of salinity was evident via the shift of phytoplankton communities across both pond groups. It is fair to say that Artemia pond water and its microbial communities are more vulnerable to extreme weather, particularly extreme salinity and heat.

More recently, halotolerant and halophilic fungi have also been recognized as regular inhabitants of solar salterns [49]. The dominant group is black yeasts Hortaea werneckii, Aureobasidium pullulans), but filamentous species (Aspergillus sp., Penicillium sp.) have also been described [74, 75]. There is, however, no information about their roles to Artemia. Our study is the first one that investigates the presence of fungi in Artemia ponds. Previous studies on halophilic fungi were conducted in solar salterns in Slovenia, Botswana, China, India, Israel, Puerto Rico, Spain, South Africa, and Thailand (for review see [74]. We found fungi across all three micro biotas: pond sediment, pond water, and Artemia gut at both samplings in Vinh Chau. The dominant taxa include Malasseziomycetes, Wallemiomycetes, Eurotiomycetes, and Agarimycetes. At the genus level we detected Aspergillus, which has been reported commonly for solar salterns in different countries. Aspergillus were found at low frequencies in pond water and Artemia gut, and more at the end of the crop than around mid-crop. They could originate from the chicken manure used to bloom microalgae [76] or were available locally [74]. Overall, in our study, fungi were found in greater relative abundance in Artemia gut and pond sediment than in pond water. This makes sense, as pond water does not provide enough substrates or organic matter, like Artemia gut or pond sediment to support fungi growth. It is likely that from pond sediment fungi were dispersed into the water column via daily disturbance of the pond bottom by the farmer and associate with suspending particles before being eaten by Artemia. Their relatively large presence in Artemia gut may suggest that they are not easy to digest. According to Chung et al. [74] halotolerant and halophilic fungi are good sources of enzymes and novel bioactive compounds that exhibit antimicrobial, cytotoxic, immunostimulatory, or antioxidant activities. It is not known yet if those compounds help create unique nutritional values of Artemia to marine larvae. More research on the roles of fungi to Artemia nutrition or development is considered helpful for further development of Artemia farming.

By Tung Hoang, Binh Thai Nguyen, Thu Thi Minh Vo, Tham Thi Hong Le, and Long Minh Tran

Read more:

• Part 1: Artemia Cyst Production in Solar Earthen Ponds in Vinh Chau, Vietnam, and Its Associated Microbiome
• Part 2: Artemia Cyst Production in Solar Earthen Ponds in Vinh Chau, Vietnam, and Its Associated Microbiome
• Evaluating the effect of protists in diets for Pacific white shrimp