The samples of the cephalothoraxes and muscle segment were fixed with 4 % PFA for 24 h at 4 ◦C and then transferred to a graded ethanol series for dehydration, followed by treatment with 100 % xylene and infiltrating in paraffin.The sections were obtained and stained with conventional H&E staining according to the previous procedures.Subsequently, the sections were scanned through the PANNORAMIC Pathology Diagnostic Scanners to obtain good quality images.In situ hybridization was performed on serial tissue sections.In briefly, the sections were dewaxed in xylene, followed by rehydration with successively dilute solutions of ethanol.Then ISH using CMNV as a probe was performed on three sections according to the protocols described previously.After color reaction, counter staining of the sections was carried out by using the Nuclear Fast Red solution , followed by dehydration in alcohol and mounting with water-soluble sealant.Finally, the sections were scanned to obtain extra-quality images by PANNORAMIC Pathology Diagnostic Scanners.The tissue of hepatopancreas was sampled as rapidly as possible and immediately transferred to a 1.5 mL EP tube containing fixative, 2.5 % glutaraldehyde in 0.1 M PBS , and held overnight at 4 ◦C in fixative.Subsequently, the sample was secondarily fixed with osmium tetroxide,grow table hydroponic dehydrated with graded ethanol, and then embedded in Spurr’s resins.
Ultrathin sections were cut with a diamond knife and collected on collodioncoated grids by staff in the Equipment Center of the Medical College of Qingdao University.The sections were stained with uranyl acetate and lead citrate and then observed with a JEOL JEM-1200 electron microscope operating at 80–100 kV.After conventional PCR amplification, all secondary PCR products were detected by running agarose gel, and single bands of the 413 bp targeted gene amplicons were detected in the all the samples, as well as in positive control.The sequences of the PCR products were subjected to BLAST analysis.BLAST analyses indicated that all sequences of CMNV RdRp gene from the collected samples showed as high as 98–100 % nucleotide identity with the original CMNV isolate from P.vannamei.The phylogenetic analysis showed that all the CMNV target fragments from three different farm’s isolates were clustered tightly into a branch of known CMNV isolate, which demonstrated higher similarity with genus Alphanodavirus rather than Betanodavirus.Histological examination confirmed that histopathological changes occurred in multiple tissues and organs of diseased shrimp infected with CMNV.Hepatopancreatic tubules underwent necrosis with atrophy and sloughing of tubular epithelium cells.Meanwhile, haemocytic infiltration, karyopyknosis and eosinophilic inclusion bodies were observed between the atrophic hepatopancreas tubules.In addition, extensive karyopyknosis and severe muscular lysis and myonecrosis of muscle fibers that the cell boundaries disappeared were observed in the whitish muscle lesions.What’s more, inspection of Fig.3j indicated massive vacuolation in the cytoplasm of the abdominal nerve.Furthermore, necrosis and exfoliation of intestine epithelial cells were also observed in the diseased shrimp.For further confirmation of CMNV infection in diseased shrimp, ISH was performed by using CMNV-specific RNA probes.
The results showed that blue-violet hybridization signals of CMNV probes were evident in the hepatopancreas, striated muscle, abdominal nerve and intestinal epithelium of diseased shrimp.Massive purple signals of CMNV probes were observed in the tubular epithelium of hepatopancreas, especially the inclusion.Notably, the probes reacted intensely with the karyopyknosis in the necrotic abdominal muscle and vacuolated nerve cells.Meanwhile, purplehybridization signals were also detected in the intestinal epithelial cells.Outbreaks of disease that cause significant morbidity and/or mortalities due to high-density farming and environmental changes in an aquaculture operation are always a major concern.This case report specifically confirmed via PCR, histopathology, ISH, and TEM outbreaks of disease causing by CMNV in local semi-intensive farms in Dongying City and Weifang City, China.Detection results of suspicious pathogens showed that all 28 samples from 4 farms were detected to be CMNV-positive and the viral load of 82 % of samples exceeded 103 copies/μg total tissue RNA.Among them, the frozen bait samples from all of three farms were detected high viral loads, especially the sample of C5-Artemia sp.as high as 2.1 × 108 copies/μg total tissue RNA.Meanwhile, it is worth noting that CMNV-positive was detected in all frozen baits from more other local shrimp farms by investigators.Further, the sequences of PCR amplicons both from the disease shrimp and the Artemia in the phylogenetic tree were highly identical to that of the original CMNV isolate.What’s more, the challenge test results showed that CMNV purified from Artemia can infect healthy P.vannamei and cause a 31.5 % mortality of the infected shrimp within 7 days.Considering that the shrimp post-larva used in the farms were Specific Pathogen Free, and aquaculture water used in diseased farms was underground brine which was free of known pathogens, the CMNV from the frozen bait, Artemia sp.and Acetes sp., was highly suspect to be the origin causative agent of disease on the investigated farms.
These results indicated that most likely CMNV was derived from frozen baits, and then played a significant role in the outbreak of CMNV-infection and high mortality of in indoor farming shrimp that were investigated.In previous reports, at farm ponds level, the cumulative mortality of diseased P.vanname with CMNV-infected could reached up to 80 %.Whereas, the cumulative mortality of CMNV infected shrimp in the challenge test in indoor farming in present study was significantly lower than intensive pond farming.We deduced that the stable and good farming environment might be conducive to reducing the mortality of shrimp infected with CMNV.And this result is also consistent with a recent report in which the result indicated that the lethal capacity of CMNV was related with the farming environment, and the stable farming environment was conducive to reducing the mortality of shrimp caused by CMNV infection.Although EHP was detected in the samples, which may be related to the slow growth of shrimps in local farms , there is no report that EHP can cause obvious shrimp death.Whether the prevalence of EHP might aggravate mortality in shrimp infected with CMNV is unclear.Additionally, in this case, VpAHPND, IHHNV, IMNV, WSSV and SHIV were not the causal agents causing mass mortality of shrimp because none of these five pathogens were detected in all samples.Shrimp sampled from local farms during the outbreak period showed severe clinical and pathological symptoms, typically related to CMNV infections.The moribund shrimp with whitish plaques on the abdominal muscle at the viral infection acute stage was commonly found in the bottom of the pond instead of swimming to the surface or edges.This phenomenon was consistent with that previously observed in P.vannamei infected with CMNV.Although several RNA viruses have been found to cause typical muscle whitening and necrosis of farmed shrimp, it is somewhat different from CMNV infection.For instance, shrimp infected with Infectious Myonecrosis Virus will display evident signs of extensive white necrotic areas in skeletal muscles, especially in the sixth abdominal segment and tail fan, which can become necrotic and reddened in some shrimp.Likewise, Penaeus vannamei nodavirus infected P.vannamei causing muscle necrosis,but the mortality rate was lower than CMNV infection.
What’s more, even though P.vannamei is another species that is susceptible to Macrobrachium rosenbergii nodavirus , this situation usually only occurs under low temperature together with low salinity of aquaculture water leading to significant mortality.The principal target tissues or organs of CMNV are not completely consistent with those of IMNV, MrNV, and PvNV.In this case, the results of histopathology together with ISH showed that atrophic and necrotic tubule epithelium of hepatopancreas with massive purple CMNV positive hybridization signal was observed , which provides reliable evidence that hepatopancreas is the one of the important target organs of CMNV.Meanwhile, CMNV-like particles also were observed in hepatopancreas tissue under the TEM.But, until now, there was no report demonstrated that hepatopancreatic atrophy and necrosis caused by MrNV, PvNV and IMNV infection.The evidences above strongly supported that CMNV was the causative agent of shrimp epidemic in Dongying and Weifang City.CMNV, a novel member of Alphanodavirus, has been found to have a wide susceptible host range, including invertebrates and poikilothermic vertebrates.Initially discovered in P.vannamei , the virus has been reported to naturally infect Mugilogobius abei , Danio rerio , Larimichthys polyactis ,grow table and Apostichopus japonicas , etc.causing damage to multiple tissues and organs.Additionally, previous studies had shown that a variety of cultured crustaceans and invertebrates from shrimp ponds affected by viral covert mortality disease can be infected by CMNV.In this case, through artificial infection experiments, it was confirmed that healthy P.vannamei could be infected with CMNV isolated from Artemia.Although VCMD is a disease that can cause high mortality in farmed shrimp at acute infection stage, and its pathogens also have a wide range of hosts, it is not listed as a notifiable or significant disease of shrimp by OIE at present.VCMD outbreaks have been related to high temperature together with stressful events such as sudden temperature or salinity changes or even high levels of NO2 – – N caused by poor farming environment, and sudden weather changes, even the operation of dividing growing shrimp population to different ponds for reducing density.Meanwhile, CMNV has been shown to be endemic in many countries around the world and caused significant production losses at a national or regional level.This case reported another significant typical VCMD in shrimp culture of China since 2014.
Those results also demonstrated that CMNV is a serious threat to the sustainability of penaeid shrimp and other aquatic organisms’ aquaculture.With the continuous increase in the production of farmed fish, the pollution of the nitrogen-containing excrement of fish in culturing water bodies is getting worse.Aquaponics is a sustainable system that integrates aquaculture and hydroponics technology.By building a synergetic system of fish, bacteria and plants, aquaponic farming pattern converts fish nitrogen excrement into less toxic nitrate nitrogen for plant growth.It is capable of yielding two products in one area, and achieves the dual purpose of agricultural production and wastewater treatment through the conversion and utilization of nitrogen.This pattern has been widely demonstrated to control the nitrogenous dissolved waste as well as purifying water quality , and has great potential for development.Tons of works have delineated that fish intestinal microbiota plays a cardinal role in health maintenance, including metabolic promotion, energy utilization and storage, immune function, endocrine and neuromodulation and enterocyte proliferation.Fish gut microbiota can be generally divided into two types: The native microbiota and the foreign microbiota.The native microbiota, including many probiotics, enhances intestinal absorption and digestion by secreting digestive enzymes and synthesizing vitamins ; meanwhile, it inhibits the proliferation of pathogens and resists the invading pathogens to keep host healthy.In addition, native probiotics have positive effects on improving the structure of gut microflora and establishing the intestinal microbiome balance.Fish farming is threatened by numerous diseases, with the intestine acting as a key transmission route and entrance site for foreign pathogens.Moreover, the composition, structure and function of intestinal microbiota are susceptible to various environmental factors.In specific, under normal circumstances, the foreign microbiota can maintain the dynamic balance of gut microbial community; but when the farming environment deteriorates, these foreign bacteria are likely to have malignant proliferation, resulting in destroying the gut balance and leading to bacterial diseases.Based on these, understanding the composition, modulation and ecological function of intestinal microbiota in various culture environments and culture patterns, is of great significance for aquaculture productivity and sustainability.The diversity and stability of microbiomes in aquaponic systems have received considerable attention recently.In terms of microbiota, researches on aquaponics have mainly focused on rhizosphere microorganisms.There have also been several studies on exploring bacteria living in aquaponics water , pointing out the presence of abundant aquatic probiotics in culture water.However, to our knowledge, scant attention has been paid to the fish intestinal microbiota in aquaponics.Therefore, in the present study, the floating-raft aquaponics were established under controlled laboratory conditions to avoid interfering factors in the open environments, and to further investigate the characteristics of fish intestinal microbiota in aquaponics and traditional farming.Goldfish , as an ornamental variant of crucian carp, is commonly farmed in aquaponic systems.Crucian carp , belonging to the family Cyprinidae, is one of the most widely-farmed freshwater fish in China due to its strong adaptability and high disease resistance.As for the plants, leafy greens are the priority choice for aquaponic systems, accounting for short growth periods and low nutritional requirements.In consideration of these, crucian carp and goldfish were chosen as the culturing fishes, lettuce and water spinach were chosen as hydroponic vegetables, and four different aquaponic systems were established through cross combination.Meanwhile, two corresponding systems without plants were also set up as the control.