A Method for In vivo Propagation of the Necrotizing Hepatopancreatitis Bacterium (NHPB) in Litopenaeus vannamei

The necrotizing hepatopancreatitis bacterium (NHPB) is difficult to preserve and has not been propagated in vitro. NHPB can be maintained continuously by simply adding specific pathogen free (SPF) shrimp to tanks containing NHPB‐infected shrimp. However, obtaining large amounts of highly concentrated infectious material free of contaminating bacterial flora for repeating challenge experiments was found to be exceedingly difficult using the current published methods. Therefore, a system was implemented using visible implant elastomer (VIE) tags to identify animals within a propagation tank by introduction group. Utilizing this method allowed for continuous reproduction of NHP within propagation tanks and provided researchers with access to infectious material allowing for consistent replication of challenge experiments and concentrated material for preservation.     

Productive performance of juvenile freshwater prawns Macrobrachium rosenbergii in biofloc system

The aim of this study was to compare two rearing systems for freshwater prawn Macrobrachium rosenbergii: one with use of a recirculating aquaculture system with biofilters (RAS) and another with use of microbial flocs (F). Thirty postlarvae of freshwater prawn with an initial average weight of 0.13 ± 0.05 g were randomly stocked in six experimental units with 0.20 m² and volume of 50 L. The experiment lasted thirty days. Dissolved oxygen, temperature and pH were monitored daily; ammonia concentration was determined three times per week; nitrite concentration, alkalinity and hardness were measured weekly. For the formation of microbial floc, molasses was used to keep the ammonia concentrations within safe levels for prawn farming. The variables of water quality remained within the suitable range for the production of the species, except for ammonia concentrations at the F treatment, which exceeded the safe levels. At the end of the experiment, the following parameters were evaluated: survival, specific growth rate, weight gain and feed conversion rate. Differences were found only in feed conversion rate with better values on RAS treatment. The microorganisms present in the RAS and F treatment were also evaluated. The densities of rotifers, amoebas and total bacteria were higher at the F treatment although the same organisms were found at the RAS treatment. The results of this study showed the possibility of rearing M. rosenbergii in biofloc system technology.     

Mode of resistance to respiration inhibitors at the cytochrome bc1 enzyme complex of Mycosphaerella fijiensis field isolates

Field isolates of Mycosphaerella fijiensis, causing black Sigatoka of banana, were characterised for their sensitivity to different inhibitors of the cytochrome bc₁ enzyme complex (Qo respiration inhibitors, strobilurin fungicides), using physiological, biochemical and molecular genetic methods. Strobilurin‐resistant isolates exhibited very high resistance factors both in mycelial growth inhibition and NADH consumption assays. Cross‐resistance was observed among all Qo inhibitors, including trifloxystrobin, azoxystrobin, famoxadone, strobilurin B and myxothiazol. However, the Qi and the cytochrome aa₃ inhibitors, antimycin A and potassium cyanide, respectively, were not cross‐resistant to Qo inhibitors. In sensitive but not in resistant isolates, mixtures of Qo inhibitors and SHAM, an inhibitor of the alternative oxidase (AOX), were more active than the components alone, indicating that the alternative pathway is essential in metabolism, but not causal for resistance. In the cell‐free NADH‐consumption assay, the Qo inhibitors affected the sensitive but not the resistant isolates, suggesting that AOX was not active in sub‐mitochondrial particles. In whole cells, however, the AOX has a basic expression level and is probably not inducible by trifloxystrobin. Sequencing of the cytochrome b gene of sensitive and resistant M fijiensis isolates revealed a difference in the nucleotide sequence leading to a single amino acid change from glycine to alanine at position 143 in the resistant isolate. This change is known to occur also in the naturally tolerant basidiomycete Mycena galopoda. It is suggested that the field isolates of M fijiensis can acquire resistance to Qo inhibitors due to a target site alteration with a single base pair change. Resistant isolates do not seem to contain a mixture of mutated and non‐mutated DNA, indicating a complete selection of resistant mitochondria and a maternally donated mode of resistance. © 2000 Society of Chemical Industry     

Early stages of Margaritifera margaritifera glochidiosis in Atlantic salmon: Morphopathological characterization

Freshwater mussels of the order Unionida encyst into the fish mucosa to metamorphose and complete their life cycle, causing a parasitic disease known as glochidiosis. This parasitic stage represents a bottleneck for the survival of naiads, particularly for critically endangered species as Margaritifera margaritifera; however, little is known about the events occurring during this critical stage. Therefore, this study aimed to histologically characterize the development of M. margaritifera glochidiosis in Atlantic salmon to get insight into the pathogenesis of this interaction. Fish exposed to glochidia were sampled during the first 44 days post-exposure, and organs were observed by stereomicroscopy and light microscopy. Glochidia attached to the gills by pinching the lamellar epithelium, whereupon an acute proliferative branchitis engulfed most of the larvae. However, during the first 14 days, a severe detachment of unviable glochidia occurred, associated with the presence of pleomorphic inflammatory infiltrate and epithelial degeneration. In the cases where larvae remained attached, a chronification of the lesions with none to scarce inflammation was observed. These results provide key information to better understand the complex host–parasite interaction during the early stages of glochidiosis and provide valuable information to optimize artificial rearing of naiads in conservation of threatened freshwater mussel populations.     

Amino acid changes in the capsid protein of a reassortant betanodavirus strain: Effect on viral replication in vitro and in vivo

Betanodavirus reassortant strains (RGNNV/SJNNV) isolated from Senegalese sole harbour an SJNNV capsid featuring several changes with respect to the SJNNV‐type strain, sharing three hallmark substitutions. Here, we have employed recombinant strains harbouring mutations in these positions (r20 and r20 + 247 + 270) and have demonstrated that the three substitutions affect different steps of the viral replication process. Adsorption ability and efficiency of viral attachment were only affected by substitutions in the C‐terminal side of the capsid. However, the concurrent mutation in the N‐terminal side seems to slightly decrease these properties, suggesting that this region could also be involved in viral binding. Differences in the intracellular and extracellular production of the mutant strains suggest that both the C‐terminal and N‐terminal regions of the capsid protein may be involved in the particle budding. Furthermore, viral replication in sole brain tissue of the mutant strains, and especially double‐ and triple‐mutant strains, is clearly delayed with respect to the wt strain. These data support previous findings indicating that the C‐terminal side plays a role in virulence because of a slower spread in the fish host brain and suggest that the concurrent participation of the N‐terminal side is also important for viral replication in vivo.     

Towards an objective evaluation of persistency of <Emphasis Type="Italic">Lolium perenne</Emphasis> swards using UAV imagery

Perennial ryegrass (Lolium perenne) is a perennial crop used in temperate regions as forage. In L. perenne breeding programs, persistency is an important trait. Poor persistency results in sward degradation and associated yield and nutritive value losses. Breeders assess persistency of accessions using visual scoring in field plots during the 2nd or 3rd growing season. This evaluation system is easy and cheap but is not free from human bias. In this study, the correlation between the scoring done by different breeders was only 0.243. As an alternative we have developed a methodology to assess persistency of L. perenne breeding materials based on vegetation indices (VIs) derived from Unmanned Aerial Vehicle (UAV) imagery. The VIs Excess green (ExG2), Green Leaf Index and Normalized green intensity (GCC) were found to provide consistent results for flights carried out under different light conditions and were validated by ground reference information. The correlation between the VIs and the percentage of ground cover extracted from on-ground imagery was 0.885. To test the implementation of the method we compared the ExG2 value based approach to selection with a visual score based selection methodology as applied by two breeders. The breeding decisions of Breeder A agreed well with decisions based on ExG2 values (74.6%), but those of Breeder B displayed a lower agreement (54.0%). In contrast, agreement between decisions based on different flights was very high (91.6%). The methodology was validated for general applicability. In summary, the results demonstrate that basing persistency selection in L. perenne breeding programs on ExG2 values from UAV imagery is likely to be more objective in comparison to the currently-used visual scoring method.     

Response of saproxylic insect communities to logging history,tree species,stage of decay,and wood posture in the central Nearctic boreal forest

Saproxylic insect assemblages are essential functional components of forest ecosystems that can be affected by forest management.We used a split-plot ANOVA design to analyze differences in selected saproxylic insects(all arthropod orders and dipteran and parasitic hymenopteran families) emerging from dead wood of sites with different logging histories(horse-logged,mechanically-logged and unlogged),tree species(Populus and Picea),stage of decay(early-and late-decay stages) and posture(standing and downed logs) in the boreal forest of central Canada.No clear effects of logging history were seen for the studied taxa; however,interaction between logging history and other dead wood features was apparent.Cecidomyiidae consistently emerged more from Populus than from Picea dead wood.Most of the studied saproxylic families were more abundant in late-decay than in early-decay wood.Dipterans of the Cecidomyiidae,Ceratopogonidae,Empididae,Mycetophilidae and Sciaridae families,and hymenopterans of the Diapriidae and Ichneumonidae families were significantly more abundant in downed than in standing dead wood.In contrast,Mymaridae was most abundant in standing dead wood.Our study provides evidence that some insects at high taxonomic levels respond differently to dead wood quality,and this could inform future management strategies in the boreal forest for the conservation of saproxylic fauna and their ecological functions.     

First description of a natural infection with spleen and kidney necrosis virus in zebrafish

Zebrafish has become a popular research model in the last years, and several diseases affecting zebrafish research facilities have been reported. However, only one case of naturally occurring viral infections was described for this species. In 2015, infectious spleen and kidney necrosis virus (ISKNV) was detected in zebrafish from a research facility in Spain. Affected fish showed lethargy, loss of appetite, abnormal swimming, distention of the coelomic cavity and, in the most severe cases, respiratory distress, pale gills and petechial haemorrhages at the base of fins. Cytomegaly was the most relevant histopathological finding in organs and tissues, sometimes associated to degenerative and necrotic changes. ISKNV belongs to the relatively newly defined genus Megalocytivirus, family Iridoviridae, comprising large, icosahedral cytoplasmic DNA viruses. This is the first case of naturally occurring Megalocytivirus infection in zebrafish research facilities, associated with morbidity. The virus has been identified based on both pathologic and genetic evidence, to better understand the pathogenesis of the infection in zebrafish and the phylogenetic relationship with other iridoviruses. Given the ability of megalocytiviruses to cross‐species boundaries, it seems necessary to implement stringent biosecurity practices as these infections may invalidate experimental data and have major impact on laboratory and cultured fish.     

Changes in Gut Microbiota Linked to a Reduction in Systolic Blood Pressure in Spontaneously Hypertensive Rats Fed an Extra Virgin Olive Oil-Enriched Diet

Fat type in diet is responsible for specific changes in gut microbiota (GM). Extra virgin olive oil (EVOO) has been shown to be beneficial for blood pressure and to produce effects on GM. To analyze the cause-effect relationship between intestinal microbial changes and blood pressure, we studied the effect of EVOO on fecal microbiota and systolic blood pressure (SBP) levels in spontaneously hypertensive rats (SHR). SHR were fed either an enriched EVOO diet or a standard diet for a period of 12 weeks. At the end of the experimental period, the microbial profiles in the feces were studied in both groups by using PCR-denaturing gradient gel electrophoresis. Real-time PCR was used to quantify the selected bacterial groups. The results demonstrated significant differences when using Lactobacillus (p<0.05), clostridia XIV (p<0.01) and universal (p<0.05) primers. A significant (r=?0.475; p=0.04) inverse correlation between the abundance of clostridia XIV and SBP, which depends on the type of diet, was also observed. Finally, the results suggested an increase in the microbial diversity of the feces of the animals fed the EVOO diet. These results strongly connect the pattern of GM in SHR fed a diet enriched with EVOO to the lower levels of SBP observed in these animals at the end of the feeding period.