Comparison of the pathogenicity of Francisella orientalis in Nile tilapia (Oreochromis niloticus), Asian seabass (Lates calcarifer) and largemouth bass (Micropterus salmoides) through experimental intraperitoneal infection

Francisella orientalis is a highly virulent, emerging bacterium that causes mass mortalities in tilapia. This pathogen also affects numerous other warm-water fish species, including three-line grunt, hybrid striped bass and various ornamental fish. This study sheds light on two new species of fish that are susceptible to F. orientalis. Asian seabass and largemouth bass showed variable levels of susceptibility in a bacterial challenge experiment. After intraperitoneally injected with a dose of 10⁶ CFU/fish, a total of 64.28% and 21.42% mortalities were obtained in Asian seabass and largemouth bass, respectively. Meanwhile, Nile tilapia showed acute mortality of 100%. All fish showed typical lesions of francisellosis, including multifocal granulomas in the spleen and head kidney. Immunohistochemical analysis revealed strong positive signals inside the granulomas of all fish. The bacterial recovery in solid media from infected fish was highest in Nile tilapia (85.71%), followed by Asian seabass (35.71%) and largemouth bass (21.42%). PCR results tested 100% positive for Nile tilapia, and 78.57% and 21.42% for Asian seabass and largemouth bass, respectively. In conclusion, Asian seabass and largemouth bass are susceptible to this pathogen, which warrants new management strategies when employing predation polyculture systems of these species with tilapia.     

Growth response and acquired resistance of Nile tilapia, Oreochromis niloticus (L.) that survived Streptococcus iniae infection

This study determined the growth performance and acquired resistance of Nile tilapia, Oreochromis niloticus (L.) that survived Streptococcus iniae infection. Tilapia were challenged with three doses of S. iniae (8.8 × 10³, 8.8 × 10⁴ and 8.8 × 10⁵ CFU fish^?1 for low, medium and high challenges respectively). Groups of non‐injected and tryptic soy broth‐injected fish were maintained as controls. Significantly (P<0.05) higher mortality (45.0%) occurred in the high challenge treatment than in the low challenge treatment group (29.6%). The medium challenge group had mortality (36.3%) that did not differ significantly from the high or low treatment. Few fish died in the non‐injected and broth‐injected treatments (3.4% and 0.8% respectively). The tilapia that survived S. iniae infection used to assess growth performance were selected from survivors without gross clinical signs of disease. These fish were randomly stocked at a rate of 30 fish into each 57 L aquarium in triplicate and fed to apparent satiation for 8 weeks. No significant differences were detected in weight gain, feed intake, feed efficiency ratio or survival between S. iniae‐survived tilapia and the control treatments following the 8‐week growth performance trial. Following the 8‐week feeding study, tilapia were challenged with 1 × 10⁶ CFU fish^?1 of S. iniae to assess acquired immunity. Mean cumulative mortality was significantly higher (P<0.05) in the control treatments (41.7% for the non‐injected and 43.3% for the broth‐injected fish) than in the low, medium and high challenge treatments (7.4%, 3.3% and 8.3% respectively). Serum protein was significantly (P<0.05) elevated in the S. iniae‐survived tilapia that were subsequently challenged when compared with controls challenged for the first time. Agglutinating antibody titre was significantly higher in the fish in the medium and high challenge treatments, compared with the control fish challenged for the first time. The results suggest tilapia that survive S. iniae challenge without showing overt disease signs performed as well as non‐infected tilapia. Further, the S. iniae‐survived tilapia challenged following the 8‐week growth performance trial gained acquired resistance to homologous S. iniae challenge.     

CD40 induces an antimicrobial response against the intracellular pathogen Streptococcus agalactiae in Nile tilapia,Oreochromis niloticus

Streptococcus agalactiae is a Gram‐positive facultative intracellular bacterium that leads to severe economic loss of tilapia worldwide. Previous studies demonstrated that CD40 contributes to host protection against intracellular injection. In this study, CD40 was characterized from Nile tilapia (Oreochromis niloticus), named OnCD40. Sequence analysis showed that open reading frame of OnCD40 was 933 bp, containing a single peptide, a transmembrane domain and four cysteine‐rich domains. The qRT‐PCR revealed that OnCD40 was expressed in all examined tissues with the most abundant ones in spleen and thymus. After S. agalactiae stimulation, the expression of OnCD40 was significantly induced in most of the detected organs. Moreover, OnCD40‐overexpressing fish elicited significant protection against subsequent S. agalactiae challenge; approximately 10000‐fold fewer bacteria were detected in spleen of OnCD40‐overexpressing fish in comparison with control fish. Thus, CD40 had protecting function in Nile tilapia against intracellular pathogens.     

Experimental assessment of the utility of visible implant elastomer tags in a stock improvement programme for giant freshwater prawn (Macrobrachium rosenbergii) in Vietnam

An important requirement of many breeding programmes for aquaculture species is the ability to identify organisms individually or at least by family. While a variety of external and internal tagging methods have been developed that can provide efficient identifications systems, most have specific drawbacks. The present study assessed the efficiency of an internal tagging method that can be applied to family selection programmes in crustacean species. Experiments were conducted to test the efficacy of applying visible implant elastomer (VIE) tags to juvenile giant freshwater prawn (GFP). The first experiment assessed performance of VIE tags in juvenile GFP of different size classes and stability of tags placed in different positions in the abdomen with different numbers of tags implanted. The second experiment applied VIE tags in a long term, large scale, field‐based farming experiment. The third experiment tested the reliability of the system. Results showed that juvenile GFP at 2 g were of suitable size for VIE tags with no negative effects evidence on growth and survival. Tag retention rates were above 97.8% in all experiments and tag readability rates were 100% with a correct assignment rate of 95% through to mature animal size of up to 170 g.     

Efficacy of an experimentally inactivated Streptococcus agalactiae vaccine in Nile tilapia (Oreochromis niloticus) reared in Brazil

Tilapia aquaculture is one of the fastest‐growing segments of fish production in Brazil. Nile tilapia (Oreochromis niloticus) is largely cultivated in the state of Parana, where Streptococcus agalactiae is the cause of severe disease outbreaks. The objective of this paper was to evaluate an inactivated S. agalactiae vaccine in tilapia for the control of streptococcal disease outbreaks. Tilapia, weighing approximately 20 g each, were intraperitoneally (i.p.) inoculated with 0.1 mL of the vaccine at a dose of 2.0 × 10⁸ colony‐forming unit (CFU) mL^?1. One group of tilapia (treatment 1) received one vaccine dose, and the other group of tilapia (treatment 2) received two doses, with an interval of 21 days. The control group was i.p. inoculated with 0.1 mL tryptic soy broth fish^?1. Immunized and control tilapia were i.p. challenged with 0.1 mL of 3.0 × 10⁷ CFU mL^?1 at 30 days post vaccination. The fish were monitored daily for disease signs and for mortality for 16 days post challenge. A statistically significant difference (P=0.0045) was found between the mortality of treatments 1 and 2. The value of relative per cent of survival of 83.6% and 96.4%, respectively, indicate that this vaccine was efficient in Nile tilapia.     

Effects of Dietary β-Hydroxy-β-Methylbutyrate on Growth and Survival of Nile Tilapia,Oreochromis niloticus,Vaccinated Against Streptococcus iniae

Abstract

β-hydroxy-b-methylbutyrate (HMB), a leucine catabolite, has been shown to cause increased disease resistance and growth in animal production. A vaccine produced from formalin killed bacteria and concentrated extracellular products of the ARS-98-60 Streptococcus iniae isolate has been used for the prevention of streptococcal disease in Nile tilapia, Oreochromis niloticus. In the present study, the effects of feeding HMB were determined in tilapia vaccinated by intraperitoneal (IP) injection of the S. iniaevaccine or unvaccinated (controls). Nile tilapia were fed diets containing either 0, 12.5, 25, or 50 mg HMB/kg diet for 14 days. The mean daily growth rate and feed efficiency showed no significant (P> 0.05) differences between the treatment groups. Dietary HMB supplementation did not enhance antibody production in unvacci-nated Nile tilapia following challenge. Dietary HMB supplementation did not enhance the survival of vaccinated Nile tilapia following challenge injection with 1 X10⁸ CFU of S. iniae.     

Molecular and histopathological characterization of Photobacterium damselae in naturally and experimentally infected Nile tilapia (Oreochromis niloticus)

Mass mortality has occurred among cultured Nile tilapia, Oreochromis niloticus, on fish farms in Manzala, Dakahlia province, Egypt, in the summer season, 2019. Moribund fish were reported with deep ulcers, septicaemic lesions and sampled for bacterial isolation. In this study, most isolates were subjected to bacteriological examination, antibiotic sensitivity test, 16S rRNA gene sequencing and histopathological examination. Following isolate identification, intraperitoneal challenge of Nile tilapia with a bacterial suspension 2 × 10⁶ CFU/ml was performed. Samples from liver, spleen and kidney were collected for histological and biochemical analysis. The results showed a high similarity (99%) to Photobacterium damselae strains using phylogenetic analysis of 16S rRNA. P. damselae exhibited resistance to amoxicillin and erythromycin, as well it was highly sensitive to chloramphenicol and doxycycline. Moreover, haemorrhage, oedema, hemosiderosis and melanomacrophage activation in the liver and head kidney of infected fish were detected by light and electron microscopy. Also, significant higher levels of CAT and SOD in the spleen and head kidney, as well as the serum levels of NO were observed in experimentally challenged O. niloticus, compared to the control fish. Our data identified P. damselae for the first time from infected Nile tilapia, describing its sensitivity to a variety of antibiotics, histopathological alterations and oxidative stress impact, and it could be useful indicators for understanding P. damselae pathogenesis, which might provide a preventive efficacy for P. damselae.     

Interactive effects of dietary protein and live bakery yeast, Saccharomyces cerevisiae on growth performance of Nile tilapia, Oreochromis niloticus (L.) fry and their challenge against Aeromonas hydrophila infection

Yeasts used as a probiotic in fish diets could stimulate fish resistance against bacterial infection and could enhance the activities of digestive enzymes in fish guts. In addition to yeast importance, dietary protein is another important part in fish diets that should be carefully optimized to meet fish requirement. It is proposed that the yeast supplementation may enhance the dietary protein turnover and reduce the protein requirement for fish. Therefore, the interactive effects of dietary protein and yeast levels on the growth performance of Nile tilapia, Oreochromis niloticus (L.) fry and their challenge against Aeromonas hydrophila infection was evaluated. In the present study, ten experimental diets were formulated to contain either 35% or 45% crude protein (CP). For each protein level treatment, bakery yeast (Saccharomyces cerevisiae) was supplemented at 0.0, 0.50, 1.0, 2.0, or 5.0 g/kg diet. Fish (0.25–0.48 g) were distributed at a rate of 25 fish per 140-L aquarium. For each diet, triplicate aquaria were fed twice a day, 5 days a week for 12 weeks. Fish growth and feed utilization were significantly affected by either dietary protein or yeast levels alone, while no significant effect of their interaction was observed. The highest fish growth was obtained at 1.0–5.0 g yeast/kg diet at both protein levels; however, the fish performance at 45% CP was better than that fed on 35% CP diets. The optimum feed conversion ratio (FCR) was obtained when fish fed on 1.0–5.0 and 2.0–5.0 g yeast/kg diet at 35 and 45% CP, respectively. The cumulative fish mortality, after interperitoneal injection with A. hydrophila for 10 days, and bactericidal activity was significantly higher in fish fed 35% CP diets than those fed 45% CP diets. Both variables decreased significantly with the increase in yeast levels. The lowest bacterial count and bactericidal activity were obtained in fish fed 5.0 g yeast/kg diet irrespective to dietary protein levels. It could be concluded that the inclusion of live bakery yeast in practical diets could improve the growth performances, feed utilization, and physiological status of Nile tilapia fry and their challenge against A. hydrophila infection. Moreover, fish performance when fed 45% CP diet was better than those fed 35% CP diet. Based on these results, the most suitable yeast level for maximum Nile tilapia growth was determined to be 2.0 g yeast/kg diet with 45% CP diet; however, this level was recommended to stimulate their productive performance and enhances their resistance against A. hydrophila infection.     

Comparison between the intestinal microbiome of healthy fish and fish experimentally infected with Streptococcus agalactiae

Nile tilapia (Oreochromis niloticus) farming is an economic activity that is soaring in the whole world. Septicemia due to Streptococcus agalactiae is the main disease impacting fish farming. The aim of this study was to compare the gut microbiome of healthy animals and animals experimentally infected with S. agalactiae strain 21171A. The microbiome was established with 16S ribosomal DNA next‐generation sequencing (NGS). One hundred Nile tilapias, with an average weight of 35 g, were distributed into two groups. Fifty fish from the challenged group were orally inoculated with 100 μl of a bacterial solution containing 1.98 × 10³ CFU/ml of S. agalactiae strain 21171A, while 50 controls were orally inoculated with sterile saline. After the experiment, 24 fish from the challenged group and 27 fish from the control group were analysed. For both groups, bacteria attached to the mucosa (M) and present in faeces (F) were analysed. The mean of the number of taxa identified in the infected group (M + F) (45.87 ± 30.13) was lower than in the control (M + F) (67.70 ± 21.10) (p < .01). Nineteen bacterial taxa were more abundant in faecal samples from the infected group when compared with the control group (p < .01). Thirty‐nine taxa were associated with mucosa samples from the challenged group when compared to the control samples (p < .01). No OTU was associated with healthy samples. The results demonstrate that the infection with S. agalactiae reduces the variability of the gut microbiota. Moreover, some bacteria proliferate during the infection.     

Effect of visible implant elastomers on the growth,survival and tag retention in juvenile Atlantic sturgeon (Acipenser oxyrinchus) in laboratory conditions

This study identifies the possibility of using visible implant elastomers (VIE) to tag juvenile Atlantic sturgeon (Acipenser oxyrinchus). The mean body weight was 2.4 ± 0.4 g, and mean body length 5.5 ± 0.5 cm. Markers in three colours were used: blue, orange and green. Implant elastomers were injected in the rostrum and in the tail shaft. The experiment was conducted over a period of 10 weeks and included also a control group, that is, no tagging. No significant effect of tagging on the specific growth rate, feed conversion ratio and condition factor of juvenile Atlantic sturgeon was observed. The fish in all groups increased their body weight more than 10 times, reaching body weight of 25–28 g. The final survival in all of the groups was approximately 40%. The final tag retention varied depending on the implant colour. The highest retention of 100% was obtained with orange VIE and was significantly different from blue and green. Tag retention was dependent on the place of marking (P > 0.05). A correlation was observed between the applied dye and the place of marking (P = 0.0166). The best results were obtained when marking with orange VIE in the rostrum as all markers were visible after 70 days even without the use of UV light. According to our research, marking with VIE does not negatively affect growth and survival of juvenile Atlantic sturgeon and can be recommended for fish with an initial body weight of approximately 2.5 g in the case of short‐term research.     

Evaluation of VIE (Visible Implant Elastomer) and PIT (Passive Integrated Transponder) physical tagging systems for the identification of red porgy fingerlings (Pagrus pagrus)

The effect of two physical tagging systems, Visible Implant Elastomer (VIE) and Passive Integrated Transponder (PIT), was evaluated in red porgy (Pagrus pagrus) fingerlings, at different sizes. For VIE tagging, the weight classes were <1 and 1?C5?g and no tag effect was detected on fish growth. There was a significant difference in mortality between tagged and untagged <1?g fish, but not for the 1?C5?g fish. The tag loss rate was null for all sizes, however, tags showed fragmentation. This partial tag loss was evaluated and quantified. Seven VIE colours were compared and arranged from best to worst visibility: green, red, pink, orange, yellow, white and blue. For PIT tagging, 2?C6?g, 5?C10?g and 10?C20?g weight classes were analysed. There was no significant tag effect on fish growth, for any size, nor on mortality from 10?g. Tag loss rate ranged from 2.9 to 5.9%. These results demonstrate that, in red porgy, VIE is a successful tagging system from 2?g onwards whereas PIT system is successful from 10?g onwards. The combination of both systems allows traceability of fish from a very small size on, which is necessary for the implementation of genetic breeding programmes.     

In vivo and in vitro assessment of Lactobacillus acidophilus as probiotic for tilapia (Oreochromis niloticus,Perciformes:Cichlidae) culture improvement

In this study, the use of Lactobacillus acidophilus as a probiotic for Nile tilapia (Oreochromis niloticus) culture was studied. Fish survival and the expression of some genes involved in the immune response were assessed. Diet supplementation with L. acidophilus for 15 days caused a significant increase in fish survival during a challenge with Aeromonas hydrophila and variations in immune response related to IL‐1β and transferrin expression in Nile tilapia spleen and kidney. Moreover, extracellular products (ECPs) of L. acidophilus showed high antibacterial activity against fish pathogens such as A. hydrophila and Streptococcus agalactiae in vitro. It was also observed that viable L. acidophilus was able to disrupt quorum sensing activity in Chromobacterium violaceum.     

Oral and parenteral vaccines against <Emphasis Type="Italic">Flavobacterium columnare</Emphasis>: evaluation of humoral immune response by ELISA and in vivo efficiency in Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

Flavobacterium columnare is a bacterial pathogen for many freshwater fish species. It is responsible for outbreaks in fish farms worldwide, causing high mortality rates. Fish vaccination is a potential approach for prevention and control of disease, with oral vaccines suitable for fish because of their easier application, low cost and minimum stress to fish. Alginate microparticles have been widely used as controlled release systems, including for fish vaccination. The aim of this study was to evaluate the capacity of oral and parenteral vaccines against F. columnare to induce a humoral response, as well as the in vivo efficiency in Nile tilapia fingerlings. The fingerlings were immunized with bacterin by intraperitoneal (i.p.), intramuscular (i.m.), oral and immersion routes, as well as orally with alginate microparticles containing formalin-killed bacteria. A sandwich ELISA was developed to detect specific antibodies against F. columnare. The animals were challenged with pathogenic strain BZ-1 to determine the relative percentage of survival. A significant humoral response was induced by bacterin administered by i.p. and i.m. routes (P < 0.05). However, none of the vaccine preparations were effective in protecting fish against F. columnare infection (P < 0.05). In spite of high antibody levels, there was no relation between immunoglobulin titers and resistance to columnaris for Nile tilapia fingerlings. These data suggest that use of serological analysis as the only method to determine vaccine efficiency against F. columnare infection in Nile tilapia can lead to imprecise results for the usefulness of these products in vivo.     

Dietary supplementation of garlic (Allium sativum) modulates gut microbiota and health status of tilapia (Oreochromis niloticus) against Streptococcus iniae infection

This study was conducted to characterize the causative agent of streptococcosis in tilapia (Oreochromis niloticus) and control of Streptococcus infection by means of garlic (Allium sativum) supplementation. The morphological, biochemical and polymerase chain reaction amplification confirmed 11 isolates belong to Streptococcus iniae from the infected fish eyes and tissue samples. Random screening of 12 well‐known medicinal plant parts against S. iniae revealed the garlic extract as the most effective herbal recovery against Streptococcus infection. In vivo challenge test with dietary supplementation of garlic powder significantly improved survival rates of fish against S. iniae infections, and modulate the microbial community and cytokine gene expression profiling in the intestine of the experimental tilapia. Among the two garlic supplemented treatments, 1.0 g garlic supplemented diet significantly increased (p < 0.05) the survival rates of tilapia and the gut bacterial operational transitional units abundance for Proteobacteria and Tenericutes, the phyla associated with healthy intestinal flora. The bacterial diversity index also found high with garlic supplemented diets. Significant upregulations of IL‐10 and IL‐17F gene expression in the intestinal tissue were observed with 1.0 g garlic supplemented diet where IL‐8 and IL‐1β expression levels were relatively static. The dietary supplementation of garlic, therefore, could be effective in the prevention of S. iniae infection in fish.     

Histopathology of experimental Streptococcus sp. infection in tilapia, Oroochromis niloticus (L.), and channel catfish, Ictafurus punctatus (Ratinesque)

Nile tilapia, Oreochromis niloticus (L.), and channel catfish, Ictalurus punctatus (Rafinesque), were experimentally infected by immersion with three isolates (Lake, DL8O5 and MS91452) of Streptococcus sp. from diseased fish. To enhance infection, the lateral body surface of each fish was scraped prior to bacterial exposure. The Lake and DL8O5 isolates caused exophthalmia, ocular opacity and ocular haemorrhage in some tilapia. Histopathology of these fish revealed; meningitis; polyserositis of heart, liver, spleen, ovary and kidney; splenitis; ovaritis; and myocarditis. Isolate MS91452 induced only mild granulomas in spleen, kidney and ovary of tilapia. The Lake and DL8O5 isolates induced endophthalitis, Channel catfish infected with the Lake and DL805 isolates developed similar eye lesions to tilapia. Histologic lesions caused by all three isolates in channel catfish consisted of meningoencephalitis, mild myocarditis, splenitis and ovaritis, but these lesions were not as severe as in Nile tilapia.     

Protection against heterologous Streptococcus iniae isolates using a modified bacterin vaccine in Nile tilapia,Oreochromis niloticus (L.)

Streptococcus iniae is a significant pathogen impacting aquaculture production worldwide. The objectives of this study were to determine whether a developed modified S. iniae (ARS-98-60) bacterin vaccine is efficacious in Nile tilapia, Oreochromis niloticus (L.), against challenge with heterologous isolates from diverse geographical locations and to evaluate protein and antigenic variability among the isolates tested. Two groups of tilapia (approximately 5 g) were intraperitoneally (IP) vaccinated with 100 μL of the vaccine or sham vaccinated with 100 μL of sterile tryptic soy broth and held for 28 days. Fish were challenged with each isolate by IP injection of 2–3 × 10⁷ CFU per fish using calcein to mark fish prior to cohabitation for challenge. The results demonstrated significant protection against all challenge isolates, and relative percent survivals ranged from 79% to 100%. SDS–PAGE analysis of whole-cell lysate proteins from the S. iniae isolates demonstrated similar protein profiles between 10 and 31 kDa and variation in profiles between 35 and 100 kDa. Western blot analysis using antiserum from vaccinated fish (ARS-98-60) demonstrated shared immunogenic proteins among all isolates in the molecular mass range of 22–35 kDa and high molecular mass material >150 kDa. The results suggest that the developed S. iniae vaccine has broad ranging protection among isolates exhibiting different protein profiles.     

Growth performance and feed utilization of Nile tilapia Oreochromis niloticus (Linnaeus, 1758) and tilapia galilae Sarotherodon galilaeus (Linnaeus, 1758) fingerlings fed plant protein-based diets

This study was designed to determine the effect of complete substitution of fish meal (FM) by three plant protein sources including extruded soybean meal (SBM), extruded full‐fat soybean (FFSB) and corn gluten meal (CGM) on growth and feed utilization of Nile tilapia Oreochromis niloticus and tilapia galilae Sarothrodon galilaeus. Four isonitrogenous of crude protein (ca. 28.0%) and isocaloric (ca. 19 MJ kg⁻¹) experimental diets were formulated. The control diet (diet 1) was prepared with FM as the main protein sources. Diets 2–4, each FM control diet, were completely substituted with SBM (diet 2), FFSB (diet 3) and CGM (diet 4). l ‐lysine and dl ‐methionine were added to plant protein diets to cover the nutritional requirements of tilapia. Each treatment was allocated to three net pens and fed for 17 weeks. Nile tilapia fed the control diet showed significantly higher (P≤0.05) values for final body weight (FBW), feed intake (FI), weight gain (WG) and specific growth rate (SGR), whereas fish fed the diet with CGM achieved the lowest values. Tilapia galilae fed SBM diet recorded the highest (P≤0.05) values for growth performance. Better feed conversion ratio (FCR) for both Oreochromis niloticus and Sarothrodon galilaeus was observed when fish were fed SBM diet, whereas the worse FCR was recorded for FFSB diet. Feed utilization parameters including protein productive value (PPV), fat retention (FR) and energy retention (ER) showed significant differences (P≤0.05) for both the species fed different dietary protein sources. The present results suggest that, for Nile tilapia, both SBM and FFSB supplemented with dl ‐methionine and l ‐lysine can completely replace dietary FM. Meanwhile, S. galilaeus fed SBM diet exhibited comparable growth and feed utilization with those fish fed a fish‐meal‐based diet.     

Evaluation of visible implant elastomer tags for tagging juvenile gilthead seabream (Sparus auratus L.); effects on growth, mortality, handling time and tag loss

The use of the fluorescent visible implant elastomer (VIE) tagging system in juvenile gilthead seabream, Sparus auratus L., between 7 and 18 g was examined. Four different colours (red, green, orange and yellow), three body positions (dorsal, lateral and caudal) and two orientations (horizontal and vertical) were tested. The mean tag application time for each fish was 15.7±0.32 s. There was no mortality associated with the method of tagging. The most visible tag colour was red. Injection orientation had a significant effect on length, width, fragmentation and fluorescent intensity of the tag. Horizontal tagging is recommended because of high fluorescent intensity, low fragmentation and for double tagging. There were no differences in growth between untagged controls and the VIE‐tagged fish.     

A comparison of the effect of three common tagging methods on the survival of the galatheid Munida rugosa (Fabricius, 1775)

Three tagging methods – T-bar tags, streamer tags and visual implant elastomer (VIE) – were tested on Munida rugosa and survival was monitored for 8 weeks. The VIE technique appeared not to affect survival (95% survival after 60 d). T-bar tags did not significantly affect short-term survival either, but there was a gradual increase in mortality (52% after 60 d) associated with the presence of black necrotic tissue around the tag wound, suggesting delayed mortality due to infection. Streamer tags as applied in the study are not suitable for this species, since short-term mortality was high (38% survival after 10 d). In conclusion, VIE was found to be the best type of tag to use on M. rugosa, although it is not as visible to the casual observer as the other types, which could be an important consideration if recapture reports from fishers are an important element of a mark-recapture study.     

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.