A Practical and Effective Artemia Hatching Method to Eliminate Covert Mortality Nodavirus (CMNV)

Abstract

The covert mortality nodavirus (CMNV), an emerging pathogen that infects several species severely in aquaculture, including marine shrimps, freshwater prawns, and crabs, has been detected in both wild Artemia and commercial Artemia cysts. Utilization of Artemia from unknown sources can impose considerable biosecurity risks in hatchery operations; therefore, effective hatching methods to eliminate the pathogenic potential from CMNV-positive (CMNV+) Artemia cysts are urgently needed. In this work, we assessed the efficacy of three treatment methods of disinfecting CMNV+ Artemia cysts during hatching: (1) decapsulation of Artemia cysts with Na₂CO₃ (sodium carbonate) and NaClO (sodium hypochlorite) at various durations before hatching; (2) application of different concentrations of formalin in hatching water; (3) combinations of decapsulating Artemia cysts and formalin-treated hatching water. Hatching CMNV+ Artemia with disinfected seawater only served as the control. The virus located on the cyst shells attached/infected Artemia larvae during hatching and remained active for a prolonged time of 7 days. The viral load of empty shells decreased with the decapsulation treatment time. After a 45 min treatment of decapsulation, CMNV on shells, as well as larvae, were eliminated successfully. Furthermore, decapsulation shortened the hatching time of the cysts from 19 h to 12 h. Ten ppm formalin in the hatching water could block the transmission of CMNV from the shells to the newly hatched larvae, although at this level it was insufficient to eliminate the virus from the shells completely. While use of 30 ppm formalin or higher dosage could eliminate CMNV, however, it also reduced the hatching rates of the Artemia cysts. A combination of decapsulation (treated with Na₂CO₃-NaClO for 15 min) and 10 ppm formalin in hatching water effectively eliminated the CMNV. This study developed a practical, effective, and reliable treatment method for hatching Artemia to ensure biosecurity in aquaculture hatcheries.

1. Introduction

In 2008–2009, a disease called covert mortality disease (CMD) broke out in shrimp farming areas in several southern provinces of China. Shrimps infected with CMD often exhibit symptoms such as paler body color, empty intestines, atrophy of the hepatopancreas, slow growth, and eventually die covertly in deep water areas at the bottom of the aquaculture pond [1]. The disease can result in a 60–80% mortality rate and has caused serious economic loss to China’s shrimp farming industry. A new nodavirus named covert mortality nodavirus (CMNV), which mainly infects shrimp hepatopancreas and muscle tissues, was later identified as the viral pathogen of CMD [2]. CMNV belongs to the genus Alphanodavirus (Nodaviridae) RNA virus [2]. Besides CMNV, several nodaviruses, e.g., Macrobrachium rosenbergii nodavirus (MrNV), movement disorder nodavirus (MDNV), and Korea shellfish nervous necrosis virus (KSNNV), also cause diseases in aquatic animals [3]. Epidemiological investigations showed that CMNV was not only prevalent in China but also in other shrimp farming countries, e.g., Vietnam, Ecuador, India, Thailand, Indonesia, and Mexico [4,5]. In addition to shrimp, CMNV can also infect other cultured crustaceans, such as the freshwater prawn Macrobrachium rosenbergii [5,6]. In shrimp ponds, several coexisting species, including brine shrimp Artemia sinica, barnacle Balanus sp., the rotifer Brachionus urceus, the polychaete Perinereis aibuhitensis, and the amphipod Corophium sinense, could be the vectors and reservoirs of CMNV [7,8]. We can also detect CMNV in commercial Artemia cysts, the important aquatic feed; therefore, the utilization of Artemia from unknown sources can impose considerable biosecurity risks on aquaculture.

In aquaculture breeding, Artemia (brine shrimp) cysts are widely used due to their easy storage, convenient hatching, and high nutritional value [9]. Before hatching into nauplii, Artemia cysts were usually decapsulated (i.e., removal of the outer layer of the cyst shell), which can bring many advantages, such as disinfection of the cysts and possible direct ingestion and digestion of decapsulated cysts by the larvae [10]. The decapsulation solution usually contains hypochlorite. Although hypochlorite can disinfect the cysts [10], whether CMNV can be eliminated during decapsulation is still unknown. Here we developed an effective and reliable treatment method for decapsulating and hatching Artemia cysts to ensure biosecurity in aquaculture hatcheries.

2. Materials and Methods

2.1. Artemia Cysts and the Detection of CMNV

2.1.1. Artemia Cysts

A can of Artemia cyst (500 g, trademark Hongda, manufactured by Haixing County Hongda Artemia Cysts Factory, Cangzhou, China) was provided by Guangdong Haid Group Ltd. (Guangzhou, China).

2.1.2. RNA Extraction

Extraction of RNAs containing the putative nodavirus genome was conducted using the TaKaRa MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 (TaKaRa, Beijing, China) according to kit instructions. Briefly, 10 mg of Artemia cysts was ground in liquid nitrogen. After grinding, 200 μL of PBS solution, 200 μL of Buffer VGB, 20 μL of Proteinase K (20 mg/mL), and 1.0 μL of Carrier RNA were added to the ground tissue. And then the solution was incubated at 56 °C for 10 min. After centrifuging at 12,000 rpm for 2 min, the precipitate was washed with Buffer RWA and Buffer RWB. Finally, the RNA was eluted in a spin column by centrifuging at 12,000 rpm for 2 min and stored with RNase-free dH₂O at −20 °C.

2.1.3. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

The CMNV detection was performed using the CMNV Fluorescent RT-PCR Detection Kit (Qingdao Lijian Bio-Tech Co., Ltd., Qingdao, China). Firstly, to prepare the RT-PCR reaction liquid, the RT-PCR enzyme reaction liquid was mixed with the TaqMan fluorescence primer probes, which target on the CMNV RNA1 and RNA2 genome segments [11]. Each PCR reaction tube was added with 20 μL RT-PCR reaction liquid, and then 5 μL RNA sample. The RT-PCR was performed on a StepOne Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) and the procedure was as follows: reverse transcription at 50 °C for 15 min, initial denaturation at 95 °C for 1 min, and a total of 40 reaction cycles, including denaturation at 95 °C for 10 s and extension at 60 °C for 30 s. FAM fluorescence signals were collected at 60 °C.

2.1.4. Result Determination

When the sample had a typical amplification curve and the cycle threshold (Ct) value ≤ 37, it could be judged as CMNV positive (CMNV+). When there was no Ct value or typical amplification curve of the sample, it was determined as CMNV negative (CMNV−). The Artemia cysts in this study showed a typical amplification curve and had a Ct value of 33.0; we therefore determined that they are CMNV+. These CMNV+ Artemia cysts were then used for the following treatments and assessing the efficacy of disinfecting CMNV.

2.2. Treatments of CMNV+ Artemia Cysts

The following treatments were applied to assess their efficacy of disinfecting CMNV+ Artemia cysts during hatching: (l) Before hatching with disinfected seawater, the Artemia cysts were decapsulated with 1% Na₂CO₃ (sodium carbonate) and 3.2% NaClO (sodium hypochlorite) for 5, 15, 30, and 40 min, respectively; (2) application of different concentrations (10, 30, and 60 ppm) of formalin in the hatching water; (3) combinations of decapsulating Artemia cysts and formalin-treated hatching water, in which two combinations were applied, ① 5 min decapsulation followed by adding 5 ppm formalin in the hatching water; ② 15 min decapsulation followed by adding 10 ppm formalin in the hatching water. Hatching CMNV+ Artemia with disinfected seawater only served as a control.

For each treatment, 0.8 g of cysts was used, and the cysts were incubated at 25–26 °C. After hatching, 100 larvae/unhatched cysts were randomly selected and observed under a stereomicroscope to count the hatching rate. The Artemia larvae were fed with green algae (Chlorella sp.) after hatching.

2.3. CMNV Detection and Viral Load Quantification

CMNV detection and viral load quantification were performed for (1) the newly hatched larvae (L); (2) the empty shells picked out after hatching (S); and (3) the larvae cultured for 3 days (the shells stayed, and the larvae were cultured with the shells; we name this group as 3M). For the control group, we also examined the larvae cultured for 5 days (5M) and 7 days (7M).

CMNV detection was the same as described in Section 2.1. To determine the viral load, we diluted the purified CMNV plasmids (the positive control in the kit, 3560 copy number/μL) into 10⁰, 10¹, 10², and 10³ copy numbers/μL, and ran the RT-PCR, respectively. A standard curve was generated by plotting a graph between copy numbers and Ct value [12]. Based on the standard curve, the viral copy number of samples was then calculated according to their Ct value, and the RNA concentration of samples was analyzed by a NanoDrop One spectrophotometer (Thermo Scientific, Waltham, MA, USA).

3. Results

3.1. No Treatment (The Control Group)

After hatching, CMNV was detected positive in both the shells (S) and the newly hatched larvae (L) samples, but the viral load of the larvae is only 0.03 copy number/ng RNA, which is 13.7 times lower than that of the shells (Figure 1 and Supplementary Table S1). This result indicates that the viruses originally attached to the shells only and then were transmitted to the larvae during hatching. The viral load of 3-day larvae (3M) is much higher than that of newly hatched larvae, suggesting that the viruses were reproducing. CMNV was also detected on 5-day larvae (5M) and 7-day larvae (7M), although the viral load decreased (Supplementary Table S1).

3.2. Decapsulation with Na₂CO₃-NaClO

The result of decapsulating with Na₂CO₃-NaClO for 5 min was similar to that of the control group. The CMNV was still detected positive on the shells (S), in newly hatched larvae (L), and in 3-day larvae (3M) samples, but with a reduction in viral load (Figure 1 and Supplementary Table S1). When treated for 15 min, the shells were CMNV+, but CMNV was not detected on the newly hatched larvae, indicating that the treatment might block the virus’ transmission from shells to larvae during hatching. But 3-day larvae (3M) were CMNV+, indicating that the CMNV+ shells are still capable of contaminating the larvae. When treated for 30 min, newly hatched larvae and 3-day larvae (3M) were both CMNV−, although shells were still CMNV+. When treated for 45 min, the shells, newly hatched larvae and 3-day larvae, were all CMNV− (Figure 1 and Supplementary Table S1).

The hatching rates of Na₂CO₃-NaClO treating groups and the control group were all above 94% (Supplementary Table S1), suggesting that the decapsulating procedure does not affect the hatching rate. Moreover, the decapsulating may shorten the hatching time; when decapsulating for 5 min, the hatching time decreased from 19 h to 17 h; when the treatments were ≥15 min, the hatching time reduced to 12 h (Supplementary Table S1).

3.3. Adding Formalin in the Hatching Water

When 10 ppm formalin was applied, the newly hatched larvae (L) were CMNV−, but the shells (S) and 3-day larvae (3M) were CMNV+. When formalin concentrations were ≥30 ppm, the newly hatched larvae and 3-day larvae (3M) were both CMNV−, although the shells were still CMNV+ (Figure 2 and Supplementary Table S1), demonstrating that the treatments can disinfect the viruses on the shells and block their transmission to the larvae. However, these treatments decreased the hatching rate (Figure 3 and Supplementary Table S1). When 60 ppm formalin was applied, the hatching rate decreased markedly from 94% to 40%.

3.4. Combination of Decapsulation and Adding Formalin in the Hatching Water

For the treatment of 5 min decapsulation followed by adding 5 ppm formalin in the hatching water, the newly hatched larvae and 3-day larvae were both CMNV− although the shells were CMNV+ (0.29 copy number/ng RNA, Supplementary Table S1). For the treatment of 15 min + 10 ppm formalin, the shells, the newly hatched larvae, and 3-day larvae were all CMNV−. Furthermore, this treatment shortened the hatching time to 12 h and did not affect the hatching rate, and the larvae developed faster than the control group (Figure 4).

4. Discussion

CMNV has a wide range of hosts, multiple vectors, and various modes of transmission, leading to long-term widespread of the virus in crustacean culture areas [8]. In shrimp ponds, in addition to cultured shrimp, CMNV was also detected positive in the coexisting annelid worm Perinereis aibuhitensis and a related brine shrimp Artemia sinica, which are the important fresh food for shrimp culture [7]. Yang et al. (2023) recommended disinfection of P. aibuhitensis eggs with peroxymonosulfate (15 ppm for 1 min) to eliminate CMNV [13]. In this work, we also detected the presence of CMNV in the commercial Artemia cysts. Furthermore, we demonstrated that the CMNV probably attached to the cyst shells and then contaminated the larvae during the hatching period, as the viral load of shells are 13.7 times higher than that of newly hatched larvae.

To disinfect the CMNV on the shell of Artemia cysts, the easiest way definitely is decapsulation, which also brings the advantages of direct ingestion and digestion of decapsulated cysts by shrimp larvae [10]. In this study, we also added formalin in the hatching water to assure the elimination of CMNV. We found that the best combination is a 15 min decapsulation process plus a 10 ppm formalin treatment in the hatching water, which not only effectively eliminated the CMNV but also shortened the hatching time, and the treated larvae developed faster than the control (i.e., larvae hatched from untreated CMNV+ cysts).

Artemia are highly nutritious; their dormant eggs (the cysts) are easy to preserve, and the hatching process is simple, making them widely used in aquaculture breeding, especially for shrimp breeding [14]. However, Artemia and shrimp both belong to crustaceans, so naturally there is a risk of Artemia-spreading shrimp pathogens [15]. Artemia can be infected by pathogenic bacteria and a eukaryotic parasite commonly found in aquaculture [16,17]. At present, it has not been confirmed that Artemia are susceptible to common shrimp viruses [15]. In this study, we found that the CMNV+ Artemia cysts of the control group can also develop normally (although slower) into larvae and then grow up to adult with no significant sign of viral infection. However, the risk of Artemia serving as vectors to transmit shrimp pathogens cannot be overlooked. Besides CMNV, Artemia may also be the vectors of multiple shrimp viruses [18,19]. Therefore, utilization of Artemia from unknown sources must be cautious, especially in the culture of specific-pathogen-free (SPF) shrimps [20].

Currently, SPF technology has been widely used in livestock and aquaculture breeding. It is also because of the establishment of the SPF breeding technology that the white leg shrimp (Penaeus vannamei) has become the world’s most widely cultivated and highest-yielding aquaculture species [21]. During the breeding and culture of SPF shrimps, we detected the commercial Artemia cysts for the presence of common specific pathogens, including CMNV, AHPND (acute hepatopancreatic necrosis disease), EHP (Enterocytozoon hepatopenaei), WSSV (white spot syndrome virus), MrNV (Macrobrachium rosenbergii nodavirus), XSV (extra small virus), DIV1 (decapod iridescent virus 1), and IHHNV (infectious hypodermal and hematopoietic necrosis virus), and we found the Artemia cysts are CMNV positive in this work. Therefore, we developed this hatching protocol to eliminate CMNV to ensure the biosecurity of producing SPF shrimps. It is yet to be known whether the disinfection protocol and hatching method for Artemia cysts described in this study can eliminate other common pathogens and therefore would confer much broader application prospects in the aquaculture industry.

5. Conclusions

CMNV was detected in commercial Artemia cysts, an important aquatic feed. The viruses were located on the cyst shells and attached/infected Artemia larvae during hatching, and they remained active for a prolonged time of 7 days. The combination of decapsulation and adding formalin in hatching water effectively eliminated the CMNV. The recommended treatment is decapsulation with Na₂CO₃-NaClO for 15 min, and the concentration of formalin is 10 ppm.