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Quantitative relationship of two viruses (MrNV and XSV) in white-tail disease of Macrobrachium rosenbergii.

Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) were purified from diseased freshwater prawns M. rosenbergii and used to infect healthy post-larvae (PL) by an immersion method. Three groups of prawns were challenged with various combined doses of MrNV and XSV. Signs of white-tail disease (WTD) were observed in Groups 1 and 2, which had been challenged with combinations containing relatively high proportions of MrNV and low proportions of XSV. By contrast there was little sign of WTD in Group 3, which had been challenged with a higher proportion of XSV than MrNV. A 2-step Taqman real-time RT-PCR was developed and applied to quantify viral copy numbers in each challenged PL. Results showed that genomic copies of both viruses were much higher in Groups 1 and 2 than they were in Group 3, indicating that MrNV plays a key role in WTD of M. rosenbergii. The linear correlation between MrNV and XSV genome copies in infected prawns demonstrated that XSV is a satellite virus, dependent on MrNV, but its role in pathogenicity of WTD remains unclear.

Animals↗

In situ hybridization and RT-PCR detection of Macrobrachium rosenbergii nodavirus in giant freshwater prawn, Macrobrachium rosenbergii (de Man), in Taiwan.

Between January and March 2006, suspected outbreaks of white tail disease were observed in post-larvae and juveniles of Macrobrachium rosenbergii in hatcheries and nursery ponds at Kaohsiung and Pingtung Counties in southern Taiwan. Pathognomonic lesions showed the presence of large oval and/or irregular basophilic, cytoplasmic inclusion bodies in the infected muscles and hepatopancreas. Reverse transcriptase polymerase chain reaction (RT-PCR) assay and in situ hybridization (ISH) revealed evidence of M. rosenbergii nodavirus (MrNV) infection but did not detect extra small virus. Phylogenetic analysis indicated that there were very high identities between nucleotide sequences among six strains obtained in this study (99.5-100%), moderate identities with Caribbean and Indian strains (98.2-98.6%), but slightly lower identity with a Chinese strain (95.2%). This is the first confirmation of MrNV in giant freshwater prawns, using an RT-PCR and ISH, in Taiwan.

Abdominal Muscles↗

Genome-based detection methods of Macrobrachium rosenbergii nodavirus, a pathogen of the giant freshwater prawn, Macrobrachium rosenbergii dot-blot, in situ hybridization and RT-PCR.

The availability of specific and rapid detection methods is essential for monitoring the health status of farmed species, particularly in viral diseases as in this case early diagnosis is a critical factor in containing disease outbreaks. Three complementary genome-based methods were developed for the detection of Macrobrachium rosenbergii nodavirus (MrNV), i.e. dot-blot hybridization, in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR). Detection limits were established for dot-blot hybridization and RT-PCR and are c. 7 fg and 8 pg of viral RNA, respectively. In situ hybridization indicated that infection was confined to the striated muscle tissue. As a result of its sensitivity, RT-PCR can be used for in-depth investigations to examine the extent of the viral infection and establish the onset of infection in hatcheries. The application of RT-PCR on samples collected from prawn farms in China showed the possible use of this method in routine health monitoring.

Animals↗

Artemia as a possible vector for Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus transmission (XSV) to Macrobrachium rosenbergii post-larvae.

Five developmental stages of Artemia were exposed to Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) by immersion and oral routes in order to investigate the possibility of Artemia acting as a reservoir or carrier of these viruses. The second objective was to determine if virus-exposed Artemia were capable of transmitting the disease to post-larvae (PL) of M. rosenbergii. There was no significant difference in percent mortality between Artemia control groups and groups challenged with these viruses. On the other hand, all the developmental stages of Artemia were positive for both viruses by nested RT-PCR, regardless of the challenge route. In horizontal transmission experiments, 100% mortality was observed in M. rosenbergii PL fed with Artemia nauplii exposed to MrNV and XSV by either challenge route. However, no mortality was observed in PL fed with virus-free Artemia. RT-PCR analysis of the M. rosenbergii PL confirmed the presence of MrNV and XSV in the challenge group and absence in the control group.

Animals↗

Rapid detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), the pathogenic agents of white tail disease of Macrobrachium rosenbergii (De Man), by loop-mediated isothermal amplification.

A loop-mediated isothermal amplification (LAMP) procedure is described for rapid diagnosis of white tail disease, a viral disease caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), in the giant freshwater prawn, Macrobrachium rosenbergii. This method was more sensitive than conventional RT-PCR for detecting the two viruses. A set of four primers, two outer and two inner, were designed for MrNV detection. An additional pair of loop primers was also used in an accelerated LAMP reaction for detection of XSV. Time and temperature conditions were optimized for detection of the two viruses. The LAMP reaction is highly suited for disease diagnosis in developing countries as amplification of DNA can be detected without the use of agarose gel electrophoresis, by the production of whitish precipitate of magnesium pyrophosphate as a by-product.

Animals↗

Seawater type shapes larval development, intestinal microbiota, and water quality in Macrobrachium rosenbergii.

Giant freshwater prawn (Macrobrachium rosenbergii) has high economic value and extensive aquaculture prospects, and larval quality critically restricts the sustainable development of the industry. To investigate the effects of different seawater sources on larval survival and growth, artificial seawater, natural seawater from Beibu Gulf (China) and East China Sea (China) were used as culture media to compare their influences on larval development and intestinal microbiota of M. rosenbergii. Significant intergroup differences were observed in cultivation outcomes. The larval emergence rate reached the maximum value of 52.1% in Group G (Beibu Gulf natural seawater), followed by 43.57% in Group R (artificial seawater), and only 36.4% in Group Z (East China Sea natural seawater), indicating distinct larval emergence gaps and a gradual decline in larval development efficiency. Intestinal microbiota analysis indicated that Pseudomonadota, Actinomycetota and Bacillota were the dominant phyla in all three groups. The relative abundance of Bacillota increased significantly in Group G on day 10. LEfSe analysis proved that this group owned the most stable microbial structure and highest community diversity, and pronounced structural fluctuations occurred in Group R, suggesting that intestinal microbial profiles can reflect larval developmental conditions. Correlation analysis showed that gut microbial composition was closely correlated with water environmental parameters, and microbial metabolic pathways were strongly associated with larval growth and development. This study indicates that natural seawater from the Beibu Gulf of China is more suitable for healthy larval cultivation of M. rosenbergii. Structural stability and diversity of intestinal microbiota are key factors affecting larval growth. The results offer theoretical references for optimizing artificial seawater formula and improving larval rearing efficiency.

16S rRNA↗

Effects of nitrite exposure on acid-base balance, respiratory protein, and ion concentrations of giant freshwater prawn Macrobrachium rosenbergii at low pH.

Macrobrachium rosenbergii that had been exposed individually for 24 h to 0 (control), 2, 5, 10 mg/L nitrite-N (nitrite as nitrogen) at 4. 3 and 7.7 pH levels were examined for hemolymph nitrite-N, oxyhemocyanin, protein, acid-base balance, ion concentrations, and ammonia-N (ammonia as nitrogen) excretion. Hemolymph oxyhemocyanin, protein, pH, HCO3- , TCO2, osmolality, and ion concentrations were inversely related to ambient nitrite-N concentration and were lower at pH 4.3. However, hemolymph nitrite-N, PO2 and PCO2 levels, and ammonia-N excretion were directly related to ambient nitrite-N, and were higher at pH 4.3. Ambient nitrite-N and pH level interacted to cause changes in hemolymph nitrite-N, oxyhemocyanin, protein, PO2, and pH levels. It is concluded that for M. rosenbergii following nitrite exposure, the incorporated nitrite causes a decrease of pH and an increase of PO2 in the hemolymph where it reduces oxyhemocyanin level; disturbs nitrogen excretion, ion regulation, and respiratory gas exchange; and may lead to a decrease of oxygen-carrying capacity, which are affected more at low pH.

Acid-Base Equilibrium↗

Extra small virus-like particles (XSV) and nodavirus associated with whitish muscle disease in the giant freshwater prawn, Macrobrachium rosenbergii.

A disease of Macrobrachium rosenbergii, the giant freshwater prawn, farmed in China was recently recorded in Zhejiang, Jiangsu, Shanghai, Guangxi and Guangdong provinces. The clinical sign of the disease, which develops in post-larvae (PL), is a whitish appearance of the muscles, particularly noticeable in the abdomen. Mortalities may reach 100% in some hatcheries. Investigations by transmission electron microscopy after negative staining of diseased PL homogenates showed the presence of two types of viral particles: one, unenveloped, icosahedral in shape, 26-27 nm in diameter, the second, much smaller, about 14-16 nm in diameter, designated extra small virus particle (XSV). The large virus has a genome with two pieces of ssRNA (RNA-1 and RNA-2), of 3 and 1.2 kb, respectively. Hybridization tests confirmed that this large virus is closely related to M. rosenbergii nodavirus (MrNV) which was isolated from diseased prawns in a hatchery in the French West Indies. Its very small size and hypothesized biochemical and biological characteristics suggest XSV is a new type of crustacean virus. As XSV has always been found associated with the larger virus (nodavirus) and is located in muscle and connective cells of diseased animals, it could be an autonomous virus, a helper-type virus or a satellite-like virus.

Animals↗

Androgenic gland cell structure and spermatogenesis during the molt cycle and correlation to morphotypic differentiation in the giant freshwater prawn, Macrobrachium rosenbergii.

The freshwater prawn Macrobrachium rosenbergii shows three male morphotypes: blue-claw males (final stage having high mating activity), orange-claw males (transitional stage showing rapid somatic growth), and small males (primary stage showing sneak copulation). This morphotypic differentiation is considered to be controlled by androgenic gland hormone, which is probably a peptide hormone. However, its physiological roles are not fully understood. In the present study, we examined the correlation of androgenic gland cell structure to spermatogenic activity and morphotypic differentiation histologically in M. rosenbergii. spermatogenic activity showed close correlation to the molt cycle in orange-claw males and small males. spermatogonia increased in number in the late premolt stage, becoming spermatocytes in the postmolt stage, and spermatocytes differentiated into spermatozoa in the intermolt and early premolt stages. Ultrastructure of the androgenic gland was additionally compared among the molt stages, but, distinct histological changes were not observed in relation to spermatogenesis during the molt cycle. On the other hand, among the three morphotypes, the androgenic gland was largest in the blue-claw males, containing developed rough endoplasmic reticulum in the cytoplasm. These results suggest that, during spermatogenesis which is related to the molt cycle, the androgenic gland hormone is at rather constant levels and plays a role in maintaining spermatogenesis rather than directly regulating the onset of a specific spermatogenesis stage and that, during the morphotypic differentiation, the androgenic gland is most active in the blue-claw males and plays a role in regulating the observed high mating activity in M. rosenbergii.

Analysis of Variance↗

Interaction of host gene-gut microbiota in male grading of Macrobrachium rosenbergii.

UNLABELLED: The giant freshwater prawn (GFP; Macrobrachium rosenbergii), a crustacean of high nutritional and economic value, is crucial for aquaculture. During the same growth cycle, male GFPs develop into three distinct forms: small males, orange claw males, and blue claw males. These morphotypes display varying social behaviors, which severely constrain their industrial development. To address this, this study collected male GFP samples at critical developmental time points (100, 110, and 120 days post-hatching) for phenotypic trait measurement and analysis to obtain external morphological data. Through gut microbiota diversity analysis, we identified key gut bacteria (Lactococcus garvieae and Lactobacillus taiwanensis) influencing male morphotype differentiation. Transcriptomic analysis revealed host Kyoto Encyclopedia of Gene and Genome pathways and key genes (Wnt-6, CTSB, CTSL, PPAE, and TP53) associated with morphotype differentiation. The interactions among phenotypic traits, gut microbiota, and key genes were systematically studied through association analysis. Weighted gene co-expression network analysis was employed to construct co-expression modules, from which critical gene modules influencing phenotypic variation were identified. Through association network analysis, we established an "Achromobacter-CD-TRINITY_DN93139_c0_g2 (calpain clp-1)" interaction model. Our findings provide novel insights into the genetic enhancement of GFPs and offer guidelines for future research regarding gut symbiotic bacteria and breeding initiatives. IMPORTANCE: Male Macrobrachium rosenbergii (giant freshwater prawn [GFP]) in the same growth cycle will develop into small males, orange claw males, and blue claw males. This individual heterogeneity in growth significantly impacts the benefits of aquaculture. However, the factors influencing the differentiation of male GFP morphotype remain unclear. This study analyzed the phenotypic data of various GFP levels, the structure of the intestinal microbiota, and the differential genes within the gonadal transcriptome at critical time points of male GFP-level type differentiation. The aim was to explore the potential role of intestinal microbiota and differential genes in this phenomenon. This study offers new insights into the research on the phenomenon of male GFP-level type differentiation.

Animals↗

Effects of intrinsic and extrinsic factors on the haemocyte profile of the prawn, Macrobrachium rosenbergii.

The giant freshwater prawn Macrobrachium rosenbergii was investigated for its total haemocyte count (THC) based on season, sex, size and feeding rate. The THC, when the prawns were subjected to injections of foreign materials was also investigated. The prawns displayed the highest and lowest THC in autumn and winter respectively, with no significant difference between male and female, or among animals with a body weight range of 7-115 g. The prawns displayed the lowest THC at D3 stage, and the highest in C stage. The prawns displayed the lowest THC when they were fed at 0.1% feeding rate among feeding rates of 0.1%, 0.5%, 1.0% and 1.5% body weight x day(-1). Prawns injected with carbon powder and Enterococcus showed increased THC during the first 6 h. Prawns injected with saline and carbon powder had the lowest THC after 30 h, and recovered to the normal value after 54 h. Prawns injected with Enterococcus showed the lowest THC after 42 h, and showed delayed recovery.

Animal Feed↗

A high-quality chromosome-level genome assembly and annotation of the giant freshwater prawn (Macrobrachium rosenbergii).

The giant freshwater prawn, Macrobrachium rosenbergii, is native to Southeast Asia and is used in aquacultural practices worldwide. It is considered advantageous because of its rapid growth, high nutritional value, and economic benefits. As one of the three major freshwater aquaculture shrimp sources in China, a high-quality genome resource is of great significance for promoting the germplasm improvement of varieties. This study presents a high-quality chromosome-level genome assembly of M. rosenbergii that was generated by combining PacBio, MGI, and Hi-C reads. The assembled genome was 2.96 Gb in size, with a contig N50 of 0.64 Mb and a scaffold N50 of 55.76 Mb, which was positioned on 59 pseudo-chromosomes. The Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis for genome assembly reached 94.37%. In total, 27,111 protein-coding genes were identified, of which 25,470 were functionally annotated. These results provide a foundation for future research into adaptive evolution, genomics, and molecular breeding in M. rosenbergii.

Animals↗

Appendage deformity syndrome--a nutritional disease of Macrobrachium rosenbergii.

Culture of the freshwater prawn Macrobrachium rosenbergii as an alternative to penaeid shrimp has recently increased in coastal areas of southern India in order to avoid numerous problems, particularly with white spot syndrome virus (WSSV). However, M. rosenbergii culture is now threatened by a new disease, appendage deformity syndrome (ADS), that also results in high mortality. Analysis of ADS prawns for viruses such as WSSV, monodon baculovirus (MBV) and infectious hypodermal and hematopoeitic necrosis virus (IHHNV) gave negative results. ADS prawns were also negative for bacterial pathogens and affected animals did not respond to antibiotic therapy. A study of potential nutritional deficiency revealed that carotenoid supplementation in the diet led to a significant decrease in ADS prawns.

Animal Nutritional Physiological Phenomena↗

Effect of androgenic gland ablation on morphotypic differentiation and sexual characteristics of male freshwater prawns, Macrobrachium rosenbergii.

Mature males of the freshwater prawn, Macrobrachium rosenbergii (de Man), may change from one to another morphotype, according to a set sequence. Small males may develop into orange-claw males and orange-claw males into dominant blue-claw males. Each of the three morphotypes demonstrates distinctive reproductive behavior and secondary sexual characteristics. The role of the androgenic gland in this morphotypic transformation was examined experimentally by bilateral androgenic gland ablation (andrectomy) of small males and orange-claw males. For andrectomy initiated in the small male morphotype, transformation to the next morphotype was permitted (orange-claw), but subsequent transformation to the blue-claw morphotype was blocked. Andrectomy of orange-claw males did not prevent transformation into the blue-claw. Andrectomy on both small and orange-claw males caused disappearance of the genital papillae and atrophy of the sperm ducts and testes. The growth rates of the andrectomized small and orange-claw males were significantly lower than those of the unoperated and sham-operated controls. We conclude that androgenic gland factors control not only the differentiation of male secondary sexual characteristics but also morphotypic differentiation. Bioassays based on the results of this study will be instrumental in the characterization of such a factor(s).

Animals↗

Gross signs and histopathology of branchiostegal blister disease (balloon disease): an idiopathic disease of farmed Macrobrachium rosenbergii (De Man).

The giant freshwater prawn, Macrobrachium rosenbergii, is facing increased threat due to disease as its culture becomes more widespread. A disease characterized by the swelling of the branchiostegal region and deformities of the appendages, named balloon disease by farmers, has caused considerable economic loss in the Nellore region of Andhra Pradesh, India. Clinical signs of diseased animals include a voluminous hypertrophy of some gill filaments and the inner area of the branchiostegite. By histology, hypertrophied areas at the level of the gill filaments or branchiostegite had an identical structure corresponding to a large cyst filled with a fluid containing a few free haemocytes, limited on one side by the cuticle and on the other by the subcuticular epithelial layer. Analysis of the diseased prawns did not reveal any pathogenic agent leading us to conclude that the disease is idiopathic, probably due to suboptimal water quality conditions.

Animals↗

Engulfed pathogen-induced apoptosis in haemocytes of giant freshwater prawn, Macrobrachium rosenbergii.

Haemocytes of the giant freshwater prawn, Macrobrachium rosenbergii, were investigated for the induction of apoptosis after phagocytosis of pathogenic yeasts, bacteria and non-pathogenic latex beads in vitro. Isolated haemocytes of M. rosenbergii were cultured at a ratio of 1:50 haemocytes to pathogen with the yeast Debaryomyces hansenii, the bacteria Aeromonas hydrophila or Enterococcus faecium, or with latex beads at 25 degrees C for 2 h, followed by washing to remove free particles. At least 200 haemocytes were counted to determine the phagocytosis rate, and the results showed that haemocytes engulfed latex beads at a higher rate than the aquatic pathogens. By transmission electron microscopy, the yeast- or bacterium-engulfing haemocytes displayed morphological changes characteristic of apoptosis, including formation of cytoplasmic vacuoles, chromatin condensation and fragmentation of nuclei. This pathogen-induced apoptosis was further confirmed by DNA laddering and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end-labelling) assays. Neither haemocytes treated with latex beads nor uninfected haemocytes (control group) showed signs of apoptosis after 48 h in culture.

Aeromonas hydrophila↗

[Intraspecific DNA sequence polymorphism and genetic markers in the mitochondrial CO I gene from three populations of Macrobrachium rosenbergii].

The mitochondrial COI gene was PCR amplified and sequenced from 17 samples obtained from three populations of Macrobrachium rosenbergii (F1 of the Burma wildtype population, Jiangsu cultured population and F2 of the Guangxi breeding population). A 498-bp long partial gene segment was acquired and used to study the genetic diversity among the three populations. Results indicated that the COI gene locus was relatively more polymorphic in the F1 of Burma wildtype population, while the polymorphism in Jiangsu cultured population and Guangxi breeding population were very poor. A total of 10 polymorphic sites and 5 haplotypes were found in the sequences of the 17 samples. The average nucleotide divergence in the three populations was 0.88%0.07% and 0 respectively. The UPGMA phylogenetic tree suggested that F2 of the Guangxi breeding population and Jiangsu cultured population were closest genetically, and their haplotypes could be gathered together to a genetic branch while F1 of the Burma wildtype population diverged and could form another relatively independent branch. For these distinct nucleotide differences, COI gene could be suitable as a genetic marker for distinguishing these two branches of the Macrobrachium rosenbergii population.

Animals↗

Characteristics of the monocistronic genome of extra small virus, a virus-like particle associated with Macrobrachium rosenbergii nodavirus: possible candidate for a new species of satellite virus.

White tail disease (WTD) causes a high mortality rate in the freshwater prawn Macrobrachium rosenbergii. The pathogenic agent is a small virus, 25 nm in diameter, Macrobrachium rosenbergii nodavirus (MrNV), associated with extra small virus (XSV), a virus-like particle,15 nm in diameter. Sequencing of the XSV genome showed that it consists of a linear single-stranded RNA of 796 nucleotides, encoding a single structural protein, the capsid CP-17. The genome is in sense orientation, ended by a short poly(A) tail at the 3'-end. Sequence comparison did not allow XSV to be affiliated to known virus families. The hypothesis that XSV is a satellite virus, such as those described in the plant kingdom, is put forward based on its characteristics. It would constitute, therefore, the first satellite virus associated with a nodavirus.

Amino Acid Sequence↗