Diseases of marine fish and shellfish in an age of rapid climate change.

A recurring trend in evidence scrutinized over the past few decades is that disease outbreaks will become more frequent, intense, and widespread on land and in water, due to climate change. Pathogens and the diseases they inflict represent a major constraint on seafood production and yield, and by extension, food security. The risk(s) for fish and shellfish from disease is a function of pathogen characteristics, biological species identity, and the ambient environmental conditions. A changing climate can adversely influence the host and environment, while augmenting pathogen characteristics simultaneously, thereby favoring disease outbreaks. Herein, we use a series of case studies covering some of the world's most cultured aquatic species (e.g., salmonids, penaeid shrimp, and oysters), and the pathogens (viral, fungal, bacterial, and parasitic) that afflict them, to illustrate the magnitude of disease-related problems linked to climate change.

Las Bolitas Syndrome in Penaeus vannamei Hatcheries in Latin America.

In September 2023, several hatcheries in Latin America experienced significant mortality rates, up to 90%, in zoea stage 2 of Penaeus vannamei. Observations of fresh mounts revealed structures resembling lipid droplets, similar to those seen in a condition known as "las bolitas syndrome". Routine histopathological examinations identified detached cells and tissues in the digestive tracts of affected zoea, contrasting with the typical algal cell contents seen in healthy zoea. Polymerase chain reaction (PCR) testing for over 20 known shrimp pathogens indicated minimal differences between diseased and healthy batches. Both groups tested negative for acute hepatopancreatic necrosis disease (AHPND) but positive for Vibrio species and Rickettsia-like bacteria in the diseased samples. Histological analyses of the affected zoea revealed characteristic tissue degeneration in the hepatopancreas, forming spheres that eventually migrated into the upper gut, midgut, and midgut caeca, a pathology identified as bolitas syndrome (BS). Microbiological assessments revealed Vibrio species at concentrations of 106 CFU zoea/g in affected zoea, approximately two orders of magnitude higher than in healthy zoea. Bacterial isolation from both healthy and BS-affected zoea on thiosulphate-citrate-bile salts-sucrose (TCBS) agar and CHROMagar™ (Paris, France), followed by identification using API 20E, identified six strains of Vibrio alginolyticus. Despite similarities to "las bolitas syndrome" in fresh mounts, distinct histopathological differences were noted, particularly the presence of sloughed cells in the intestines and variations in hepatopancreatic lobes. This study highlights the critical need for further research to fully understand the etiology and pathology of bolitas syndrome in zoea stage 2 of P. vannamei to develop effective mitigation strategies for hatchery operations.

Effects of clove oil concentrations on blood chemistry and stress-related gene expression in Siamese fighting fish (Betta splendens) during transportation.

Siamese fishing fish (Betta splendens) or betta are usually subjected to a special method of transportation for global trade, where they are individually conveyed in plastic bags containing just enough water to cover their bodies. This study aimed to investigate the effects of transportation on their stress response by measuring hematological values, stress hormone levels, glucose levels, and stress-related gene expression. Betta fish (average body weight 1.91 ± 0.42 g; n = 30) were exposed to simulated transport in a water volume of 40 mL for 12, 24, and 48 h. Baseline levels (pre-transport) were measured prior to the experiment. The control group was transported using water without adding clove oil. Two treatment groups were transported using water with the addition of 1 and 3 mg/L concentrations of clove oil, respectively. The results revealed that transportation can be a factor that affects water quality. The pH and dissolved oxygen levels were significantly lower than baseline, while nitrite and total ammonia concentrations significantly increased. Correlating to the stress responses, significantly increasing total red blood cell counts, plasma cortisol levels, and up-regulating the expression of stress-related genes, including HSP70, GR, MR, and HIF-1α. The addition of 1 mg/L clove oil was found to reduce stress during the transport simulation, as evidenced by a reduction in these stress parameters. Conversely, increasing the concentration of clove oil to 3 mg/L significantly increased plasma cortisol after 12 h of simulated transport, and up-regulated GR, MR, and HIF-1α expression. This study revealed that the transport process can stimulates stress in betta fish but adding a concentration of 1 mg/L clove oil to the transport water could mitigate this stress response and promote animal welfare during their transportation.

Review of current perspectives and future outlook on bacterial disease prevention through vaccination in Asian seabass (Lates calcarifer).

Asian seabass, Lates calcarifer, is an important aquatic species in mariculture. Intensive farming of this species has faced episodes of bacterial diseases, including those due to vibriosis, scale drop, and muscle necrosis disease, big belly disease, photobacteriosis, columnaris, streptococcosis, aeromoniasis, and tenacibaculosis. Vaccination is one of the most efficient, non-antibiotic, and eco-friendly strategies for protecting fish against bacterial diseases, contributing to aquaculture expansion and ensuring food security. As of now, although numerous vaccines have undergone laboratory research, only one commercially available inactivated vaccine, suitable for both immersion and injection administration, is accessible for preventing Streptococcus iniae. Several key challenges in developing vaccines for Asian seabass must be addressed, such as the current limited understanding of immunological responses to vaccines, the costs associated with vaccine production, forms, and routes of vaccine application, and how to increase the adoption of vaccines by farmers. The future of vaccine development for the Asian seabass industry, therefore, is discussed with these key critical issues in mind. The focus is on improving our understanding of Asian seabass immunity, including maternal immunity, immunocompetence, and immune responses post-vaccination, as well as developing tools to assess vaccine effectiveness. The need for an alignment of fish vaccines with state-of-the-art vaccine technologies employed in human and terrestrial animal healthcare is also discussed. This review also discusses the necessity of providing locally-produced autogenous vaccines, especially for immersion and oral vaccines, to benefit small-scale fish farmers, and the potential benefits that might be extended through changes to current husbandry practices such as the vaccination of broodstock and earlier life stages of their off-spring.

A novel vaccination strategy against Vibrio harveyi infection in Asian seabass (Lates calcarifer) with the aid of oxygen nanobubbles and chitosan.

Immersion vaccination, albeit easier to administer than immunization by injection, sometimes has challenges with antigen uptake, resulting in sub-optimal protection. In this research, a new strategy to enhance antigen uptake of a heat-inactivated Vibrio harveyi vaccine in Asian seabass (Lates calcarifer) using oxygen nanobubble-enriched water (ONB) and positively charged chitosan (CS) was explored. Antigen uptake in fish gills was assessed, as was the antibody response and vaccine efficacy of four different combinations of vaccine with ONB and CS, and two control groups. Pre-mixing of ONB and CS before introducing the vaccine, referred to as (ONB + CS) + Vac, resulted in superior antigen uptake and anti-V. harveyi antibody (IgM) production in both serum and mucus compared to other formulas. The integration of an oral booster (4.22 × 108 CFU/g, at day 21-25) within a vaccine trial experiment set out to further evaluate how survival rates post exposure to V. harveyi might be improved. Antibody responses were measured over 42 days, and vaccine efficacy was assessed through an experimental challenge with V. harveyi. The expression of immune-related genes IL1ß, TNFα, CD4, CD8, IgT and antibody levels were assessed at 1, 3, and 7-day(s) post challenge (dpc). The results revealed that antibody levels in the group (ONB + CS) + Vac were consistently higher than the other groups post immersion immunization and oral booster, along with elevated expression of immune-related genes after challenge with V. harveyi. Ultimately, this group demonstrated a significantly higher relative percent survival (RPS) of 63 % ± 10.5 %, showcasing the potential of the ONB-CS-Vac complex as a promising immersion vaccination strategy for enhancing antigen uptake, stimulating immunological responses, and improving survival of Asian seabass against vibriosis.

Review of quorum-quenching probiotics: A promising non-antibiotic-based strategy for sustainable aquaculture.

The emergence of antibiotic-resistant bacteria (ARBs) and genes (ARGs) in aquaculture underscores the urgent need for alternative veterinary strategies to combat antimicrobial resistance (AMR). These measures are vital to reduce the likelihood of entering a post-antibiotic era. Identifying environmentally friendly biotechnological solutions to prevent and treat bacterial diseases is crucial for the sustainability of aquaculture and for minimizing the use of antimicrobials, especially antibiotics. The development of probiotics with quorum-quenching (QQ) capabilities presents a promising non-antibiotic strategy for sustainable aquaculture. Recent research has demonstrated the effectiveness of QQ probiotics (QQPs) against a range of significant fish pathogens in aquaculture. QQ disrupts microbial communication (quorum sensing, QS) by inhibiting the production, replication, and detection of signalling molecules, thereby reducing bacterial virulence factors. With their targeted anti-virulence approach, QQPs have substantial promise as a potential alternative to antibiotics. The application of QQPs in aquaculture, however, is still in its early stages and requires additional research. Key challenges include determining the optimal dosage and treatment regimens, understanding the long-term effects, and integrating QQPs with other disease control methods in diverse aquaculture systems. This review scrutinizes the current literature on antibiotic usage, AMR prevalence in aquaculture, QQ mechanisms and the application of QQPs as a sustainable alternative to antibiotics.

Immersion prime and oral boost vaccination with an inactivated Vibrio harveyi vaccine confers a specific immune response and protection in Asian seabass (Lates calcarifer).

Asian seabass (Lates calcarifer) holds significant economic value in fish farming in the Asia-Pacific region. Vibriosis caused by Vibrio harveyi (Vh) is a severe infectious disease affecting intensive farming of this species, for which prevention strategies by vaccination have been developed. This study investigated an alternative approach to injectable vaccination to prevent vibriosis in Asian seabass juveniles. The strategy begins with an immersion prime vaccination with a heat-inactivated Vh vaccine, followed by two oral booster doses administered at 14- and 28-days post-vaccination (dpv). Expression of five immune genes TNFα, IL1ß, CD4, CD8, and IgM in the head kidney and spleen, along with investigation of anti-Vh antibody response (IgM) in both systemic and mucosal systems, was conducted on a weekly basis. The efficacy of the vaccines was assessed by a laboratory challenge test at 43 dpv. The results showed that the immunized fish displayed higher levels of mRNA transcripts of the immune genes after the immersion prime and the first oral booster dose compared to the control group. The expression levels peaked at 14 and 28 dpv and then declined to baseline at 35 and 42 dpv. Serum specific IgM antibodies were detected as early as 7 dpv (the first time point investigated) and exhibited a steady increase, reaching the first peak at 21 dpv, and a second peak at 35 dpv. Although the antibody levels gradually declined over subsequent weeks, they remained significantly higher than the control group throughout the experiment. A similar antibody response pattern was also observed in the mucosal compartment. The laboratory challenge test demonstrated high protection by injection with 1.65 × 104 CFU/fish, with a relative percent of survival (RPS) of 72.22 ± 7.86 %. In conclusion, our findings highlight the potential of an immersion prime-oral booster vaccination strategy as a promising approach for preventing vibriosis in Asian seabass.

Description of Tresuncinidactylus wilmienae gen. et sp. n. (Monogenea: Gyrodactylidae), from the gills of the bulldog, Marcusenius macrolepidotus (Peters) from Lake Kariba, Zimbabwe.

The African continent has a rich diversity of fish and amphibians in its inland water systems that serve as hosts for monogeneans of seven genera of the Gyrodactylidae van Beneden et Hesse, 1832. In August 2011, eight gyrodactylid parasites were collected from the gills of two specimens of bulldog, Marcusenius macrolepidotus (Peters), from Lake Kariba, Zimbabwe. Morphometric evaluation and sequencing of 18S rDNA confirmed that the specimens represented a species of a new viviparous genus, Tresuncinidactylus wilmienae gen. et sp. n. The attachment apparatus consists of a single pair of large slender hamuli with prominently flattened roots that are connected by a simple, narrow dorsal bar. The ventral bar is small and possesses a thin lingulate membrane but no evident anterolateral processes. There are 16 marginal hooks of one morphological type, but of three different sizes, with large falculate sickles that are proportionaly equal in length to the length of their handles. The two largest pairs of marginal hooks are positioned closest to the opisthaptoral peduncle, the neighbouring two pairs of medium-sized marginal hook sickles are situated along the lateral margins of the opisthaptor. Four pairs of smallest marginal hooks are positioned along the posterior margin of the opisthaptor. The male copulatory organ consists of a muscular pouch armed with approximately 30 gracile spines. Phylogenetic analyses of partial sequences of the 18S rDNA using Maximum Likelihood and Bayesian Inference placed the new genus within the lineage of solely African genera and suggests Afrogyrodactylus Paperna, 1968, Citharodactylus Prikrylová, Shinn et Paladini, 2017 and Mormyrogyrodactylus Luus-Powell, Mashego et Khalil, 2003 as genera most closely related to the new genus.

Gyrodactylus molweni sp. n. (Monogenea: Gyrodactylidae) from Chelon richardsonii (Smith, 1846) (Mugilidae) from Table Bay, South Africa.

Gyrodactylus molweni sp. n. is described from the body surface and fins of the South African mullet, Chelon richardsonii (Smith, 1846) collected from Table Bay Harbour, Cape Town and is compared to five other Gyrodactylus species described from grey mullets globally namely G. zhukovi Ling, 1963 and G. mugili Zhukov, 1970 from Planiliza haematocheila (Temminck and Schlegel, 1845); G. mugelus Rawson, 1973 from Mugil cephalus L.; G. curemae Conroy and Conroy, 1985 from Mugil curema Valenciennes, 1836 and G. xiamenensis Zang,Yang and Liu, 2001 from Planiliza macrolepis (Smith, 1846). Morphologically, G. molweni sp. n. has prominent ventral bar processes that near cover the hamulus roots, marginal sickles with large rhomboid heels, slender shafts and fine points that extend beyond the sickle toes. Gyrodactylus molweni sp. n. can, however, be readily differentiated: G. mugili and G. xiamenensis have ventral bars with small ventral processes; G. zhukovi has marginal hooks sickles with slender shafts and proportionately short points and open-faced blades; G. mugelus possesses marginal hook sickles with deep, rounded heels, forward slanting shafts and an angular, square line to the inner face of the blades. Although the length of the marginal hooks of G. curemae are similar to G. molweni sp. n., their hamuli are double the size. A GenBank BlastN search with the 931 bp sequence covering ITS1, 5.8S and ITS2 gave no close hits; the nearest species for which sequences are available is G. nipponensis Ogawa and Egusa, 1978 (identity 96.56%, 899/931 bp). The proposal of G. molweni sp. n. as a new species, therefore, is well supported by both the molecular and morphological analyses presented herein. This Gyrodactylus species is the first to be described from C. richardsonii and only the second Gyrodactylus species to be described from the marine environment off the African continent.

Geographical distribution of Gyrodactylus salaris Malmberg, 1957 (Monogenea, Gyrodactylidae).

BACKGROUND: Gyrodactylus salaris Malmberg, 1957 is an OIE (Office International des Epizooties)-listed parasitic pathogen and had until the current study been reported from 19 countries across Europe, although many of these records require confirmation. The last comprehensive evaluation regarding the distribution of G. salaris, however, was made in 2007, although some of the states identified as being G. salaris-positive were ascribed this status based on misidentifications, on partial data resulting from either morphological or molecular tests, or from records that have not been revisited since their early reporting. It is thus important to go through the reports on G. salaris to obtain a status for each country. METHODS: To provide a revised update of the G. salaris distribution, a literature review was necessary. This literature, however, was not always readily accessible and, in certain cases, the article only made superficial reference to the parasite without providing details or data to support the identification. In most cases, the original specimens were not deposited in a national collection. Additional Gyrodactylus material for the current study was obtained from selected salmonid populations with the aim to contribute to current understanding regarding the distribution of G. salaris. Additional parasite material collected for this study was processed following standard procedures for species identification in Gyrodactylus [1]. RESULTS: From the work conducted in the current study, G. salaris is reported from a further three regions in Italy, alongside three other species, and appears to occur extensively throughout central Italy without causing significant mortalities to its rainbow trout, Oncorhynchus mykiss (Walbaum), host. The analysis of archive material from G. salaris-positive farms would suggest that G. salaris has been in this country since at least 2000. Material obtained from rainbow trout from Finland and Germany are confirmed as G. salaris, supporting existing data for these countries. No specimens of G. salaris, however, were found in the additional Gyrodactylus material obtained from rainbow trout reared in Portugal and Spain. A morphologically similar species, Gyrodactylus teuchis Lautraite, Blanc, Thiery, Daniel et Vigneulle, 1999, however, was found. CONCLUSIONS: Following the present review, Gyrodactylus salaris is reported from 23 out of 50 recognised states throughout Europe; only records from 14 of these states have been confirmed by either morphology and/or by an appropriate molecular test and are considered valid, while only nine of these records have been confirmed by a combination of both methods.

Efficacy of an inactivated whole-cell injection vaccine for nile tilapia, Oreochromis niloticus (L), against multiple isolates of Francisella noatunensis subsp. orientalis from diverse geographical regions.

Francisellosis, induced by Francisella noatunensis subsp. orientalis (Fno), is an emerging bacterial disease representing a major threat to the global tilapia industry. There are no commercialised vaccines presently available against francisellosis for use in farmed tilapia, and the only available therapeutic practices used in the field are either the prolonged use of antibiotics or increasing water temperature. Recently, an autogenous whole cell-adjuvanted injectable vaccine was developed that gave 100% relative percent survival (RPS) in tilapia challenged with a homologous isolate of Fno. In this study, we evaluated the efficacy of this vaccine against challenge with heterologous Fno isolates. Healthy Nile tilapia, Oreochromis niloticus (∼15 g) were injected intraperitoneally (i.p.) with the vaccine, adjuvant-alone or phosphate buffer saline (PBS) followed by an i.p. challenge with three Fno isolates from geographically distinct locations. The vaccine provided significant protection in all groups of vaccinated tilapia, with a significantly higher RPS of 82.3% obtained against homologous challenge, compared to 69.8% and 65.9% with the heterologous challenges. Protection correlated with significantly higher specific antibody responses, and western blot analysis demonstrated cross-isolate antigenicity with fish sera post-vaccination and post-challenge. Moreover, a significantly lower bacterial burden was detected by qPCR in conjunction with significantly greater expression of IgM, IL-1 ß, TNF-α and MHCII, 72 h post-vaccination (hpv) in spleen samples from vaccinated tilapia compared to fish injected with adjuvant-alone and PBS. The Fno vaccine described in this study may provide a starting point for development a broad-spectrum highly protective vaccine against francisellosis in tilapia.

Streptococcus agalactiae infection kills red tilapia with chronic Francisella noatunensis infection more rapidly than the fish without the infection.

In this study we examined the effect that a Francisella noatunensis (Fno) infection had on hybrid red tilapia (Oreochromis niloticus × Oreochromis mossambicus) subsquently infected with Streptococcus agalactiae. A variety of hemato-immunological parameters (haematocrit, total red blood cell count, mean corpuscular volume, total white blood and differential cell counts, total plasma protein, plasma lysozyme and plasma peroxidase activities, and respiratory burst and phagocytic activities of head-kidney macrophages) were measured in hybrid red tilapia that had been previously exposed to an Fno outbreak in a tilapia grow-out farm. The head-kidneys of these apparently healthy survivors, when checked by PCR were found to be Fno-positive with hemato-immunological parameters that were similar to fish without an a priori infection. The only exception was the percentage lymphocyte count in the peripheral blood, which was slightly, but significantly, lower in the Fno-infected fish, compared to those without the infection. When experimentally infected with S. agalactiae, the Fno-infected fish died more rapidly and at a significantly higher rate than fish without the infection. During the challenge, the hemato-immunological parameters of both groups of fish were very similar, although the Fno-infected fish, challanged with S. agalactiae expressed significantly higher plasma lysozyme and peroxidase activities, and their head kidney macrophages had significantly higher respiratory burst activity compared to non-Fno-infected fish challanged with S. agalactiae. The only two parameters for which Fno-infected fish showed significantly lower expressions than that of their non-infected counterparts were haematocrit and total red blood cell count. The cause of the rapidity and higher rates of mortality observed in the Fno-infected fish when challenged with S. agalactiae is unknown; but it may be due to a reduced erythropoiesis capability within the head-kidney because of the presence of Fno.

Correction: Predicting the Potential for Natural Recovery of Atlantic Salmon (Salmo salar L.) Populations following the Introduction of Gyrodactylus salaris Malmberg, 1957 (Monogenea).

[This corrects the article DOI: 10.1371/journal.pone.0169168.].

Description of Citharodactylus gagei n. gen. et n. sp. (Monogenea: Gyrodactylidae) from the moon fish, Citharinus citharus (Geoffroy Saint-Hilaire), from Lake Turkana.

A new genus and species of monogenean belonging to the Gyrodactylidae, Citharodactylus gagei n. gen. et n. sp. (Plathyhelminthes, Monogenea), is described from the gills of the moon fish, Citharinus citharus (Geoffroy Saint-Hilaire), a characiform fish collected from Lake Turkana in northern Kenya. The new viviparous genus can be readily distinguished from the six other gyrodactylid genera recorded from Africa and from the other viviparous genera within the Gyrodactylidae based on the morphology of the male copulatory organ (MCO), which consists of a muscular ovate organ with an opening onto the tegument through which the narrow tapered end of a sclerotised curved cone-shaped structure protrudes. The tegumental opening of the MCO is surrounded by a collar of short spines. Sequencing of the nuclear ribosomal DNA internal transcribed spacers 1 and 2, the 5.8S and the 18S rDNA genes and a comparison with the gyrodactylid species listed in GenBank confirmed the specimens are unique and do not match with any existing entry. When phylogenies for each genomic region were conducted (i.e. 0.064 gamma-corrected pairwise genetic distance based on a alignment of 1750 bp of the 1857 bp long 18S rDNA gene), the most similar match was that of Afrogyrodactylus sp. [= A. girgifae (Folia Parasitol 61:529-536, 2014)] from Brycinus nurse (Rüppell). The proposed name of the new parasite is Citharodactylus n. gen. which represents the seventh gyrodactylid genus to be found in Africa and the 25th viviparous genus and the 32nd genus to be added to the Gyrodactylidae.

Predicting the Potential for Natural Recovery of Atlantic Salmon (Salmo salar L.) Populations following the Introduction of Gyrodactylus salaris Malmberg, 1957 (Monogenea).

Gyrodactylus salaris (Monogenea, Platyhelminthes) is a notifiable freshwater pathogen responsible for causing catastrophic damage to wild Atlantic salmon stocks, most notably in Norway. In some strains of Baltic salmon (e.g., from the river Neva) however, the impact is greatly reduced due to some form of innate resistance that regulates parasite numbers, resulting in fewer host mortalities. Gyrodactylus salaris is known from 17 European states; its status in a further 35 states remains unknown; the UK, the Republic of Ireland and certain watersheds in Finland are free of the parasite. Thus, the parasite poses a serious threat if it emerges in Atlantic salmon rearing regions throughout Europe. At present, infections are generally controlled via extreme measures such as the treatment of entire river catchments with the biocide rotenone, in order to remove all hosts, before restocking with the original genetic stock. The use of rotenone in this way in EU countries is unlikely as it would be in contravention of the Water Framework Directive. Not only are such treatments economically and environmentally costly, they also eradicate the potential for any host/parasite evolutionary process to occur. Based on previous studies, UK salmon stocks have been shown to be highly susceptible to infection, analogous to Norwegian stocks. The present study investigates the impact of a G. salaris outbreak within a naïve salmon population in order to determine long-term consequences of infection and the likelihood of coexistence. Simulation of the salmon/ G. salaris system was carried out via a deterministic mathematical modelling approach to examine the dynamics of host-pathogen interactions. Results indicated that in order for highly susceptible Atlantic strains to evolve a resistance, both a moderate-strong deceleratingly costly trade-off on birth rate and a lower overall cost of the immune response are required. The present study provides insights into the potential long term impact of G. salaris if introduced into G. salaris-free territories and suggests that in the absence of external controls salmon populations are likely to recover to high densities nearing 90% of that observed pre-infection.

To each his own: no evidence of gyrodactylid parasite host switches from invasive poeciliid fishes to Goodea atripinnis Jordan (Cyprinodontiformes: Goodeidae), the most dominant endemic freshwater goodeid fish in the Mexican Highlands.

BACKGROUND: Goodeid topminnows are live-bearing fishes endemic to the Mexican Highlands (Mesa Central, MC). Unfortunately, in the MC, environmental degradation and introduced species have pushed several goodeid species to the brink of extinction. Invasive fishes can introduce exotic parasites, and the most abundant goodeid, blackfin goodea Goodea atripinnis Jordan, is parasitised by six exotic helminths. Poeciliids are widely dispersed invasive fishes, which exert negative ecological effects on goodeids. Poeciliids host several species of the monogenean genus Gyrodactylus von Nordmann, 1832, including pathogenic, invasive parasites. Here, we looked for evidence of Gyrodactylus species switching hosts from poeciliids to goodeids. METHODS: Fish were collected in rivers draining the MC into both sides of the continental divide. Hosts were screened for gyrodactylid parasites in localities where G. atripinnis and poeciliids occurred sympatrically. Gyrodactylus specimens were characterised morphologically (attachment apparatus) and molecularly (internal transcribed spacer region, ITS). A Bayesian phylogenetic tree using ITS sequences established relationships between gyrodactylids collected from goodeid fishes and those from parasites infecting poeciliids. RESULTS: Gyrodactylids were collected from G. atripinnis in six localities on both sides of the watershed where exotic poeciliids occurred sympatrically. Morphological and molecular analyses indicated the presence of four undescribed species of Gyrodactylus infecting this goodeid host. Gyrodactylus tomahuac n. sp., the most abundant and geographically widespread species, is described here. The other three Gyrodactylus spp. are not described, but their ITS sequences are used as molecular data presented here, are the only available for gyrodactylids infecting goodeid fishes. Morphological and molecular data suggest that two distinct groups of gyrodactylids infect goodeids, one of which shares a common ancestor with gyrodactylids parasitizing poeciliids. CONCLUSIONS: No evidence was found of gyrodactylids switching hosts from invasive poeciliids to endemic goodeids, nor vice versa. Moreover, considering that G. atripinnis is known to host both Gyrodactylus lamothei Mendoza-Palmero, Sereno-Uribe & Salgado-Maldonado, 2009 and Gyrodactylus mexicanus Mendoza-Palmero, Sereno-Uribe & Salgado-Maldonado, 2009, with the addition of G. tomahuac n. sp. and the three undescribed Gyrodactylus spp. reported, at least six gyrodactylids may infect this host. This would make monogeneans the second most abundant parasite group infecting G. atripinnis, which to date is known to harbour 22 helminth species: nine digeneans, five nematodes, four cestodes, three monogeneans and one acanthocephalan.

The effects of feeding ß-glucan to Pangasianodon hypophthalmus on immune gene expression and resistance to Edwardsiella ictaluri.

Pangasianodon hypophthalmus (striped catfish) is an important aquaculture species and intensification of farming has increased disease problems, particularly Edwardsiella ictaluri. The effects of feeding ß-glucans on immune gene expression and resistance to E. ictaluri in P. hypophthalmus were explored. Fish were fed 0.1% fungal-derived ß-glucan or 0.1% commercial yeast-derived ß-glucan or a basal control diet without glucan. After 14 days of feeding, the mRNA expression of immune genes (transferrin, C-reactive protein, precerebellin-like protein, Complement C3 and factor B, 2a MHC class II and interleukin-1 beta) in liver, kidney and spleen were determined. Following this fish from each of the three diet treatment groups were infected with E. ictaluri and further gene expression measured 24 h post-infection (h.p.i.), while the remaining fish were monitored over 2 weeks for mortalities. Cumulative percentage mortality at 14 days post-infection (d.p.i.) was less in ß-glucan fed fish compared to controls. There was no difference in gene expression between dietary groups after feeding for 14 days, but there was a clear difference between infected and uninfected fish at 24 h.p.i., and based on principal component analysis ß-glucans stimulated the overall expression of immune genes in the liver, kidney and spleen at 24 h.p.i.

Histopathology and the inflammatory response of European perch, Perca fluviatilis muscle infected with Eustrongylides sp. (Nematoda).

BACKGROUND: The European perch, Perca fluviatilis L. is a common paratenic host of dioctophymatid nematodes belonging to the genus Eustrongylides. In this host, once infected oligochaetes, which serve as the first intermediate host, are ingested, Eustrongylides migrates through the intestine and is frequently encountered within the musculature, free within the body cavity, or encapsulated on the viscera. The current study details the first Italian record of Eustrongylides sp. with larvae reported in the muscle of P. fluviatilis. METHODS: Uninfected and nematode-infected muscle tissues of perch were fixed and prepared for histological evaluation and electron microscopy. Some sections were subjected to an indirect immunohistochemical method using anti-PCNA, anti-piscidin 3 and anti-piscidin 4 antibodies. RESULTS: A total of 510 P. fluviatilis (TL range 15-25 cm) from Lake Trasimeno, Perugia were post-mortemed; 31 individuals had encysted nematode larvae within their musculature (1-2 worms fish(-1)). Histologically, larvae were surrounded by a capsule with an evident acute inflammatory reaction. Muscle degeneration and necrosis extending throughout the sarcoplasm, sarcolemmal basal lamina, endomysial connective tissue cells and capillaries was frequently observed. Within the encapsulating reaction, macrophage aggregates (MAs) were seen. Immunohistochemical staining with the proliferating cell nuclear antigen (PCNA) revealed numerous PCNA-positive cells within the thickness of the capsule and in the immediate vicinity surrounding Eustrongylides sp. larvae (i.e. fibroblasts and satellite cells), suggesting a host response had been initiated to repair the nematode-damaged muscle. Mast cells (MCs) staining positively for piscidin 3, were demonstrated for the first time in response to a muscle-infecting nematode. The piscidin 3 positive MC's were seen principally in the periphery of the capsule surrounding the Eustrongylides sp. larva. CONCLUSIONS: A host tissue response to Eustrongylides sp. larvae infecting the musculature of P. fluviatilis was observed. Numerous fibroblasts, MAs and MCs were seen throughout the thick fibroconnectival layer of the capsule enclosing larvae. PCNA positive cells within the capsule suggest that host repair of nematode damaged muscle does occur, while the presence of the antimicrobial peptide piscidin 3 is shown for the first time. This is first report of Eustrongylides sp. in an Italian population of P. fluviatilis.

Zoothamnium duplicatum infestation of cultured horseshoe crabs (Limulus polyphemus).

An outbreak of the sessile peritrich Zoothamnium duplicatum in a pilot, commercial-scale Limulus polyphemus hatchery resulted in the loss of ∼96% (40,000) second/third instar larvae over a 61day period. peritrich growth was heavy, leading to mechanical obstruction of the gills and physical damage. The peritrichs were controlled without resultant loss of juvenile crabs by administering 10ppm chlorine in freshwater for 1h and the addition of aquarium grade sand; a medium into which the crabs could burrow and facilitate cleaning of the carapace. Peritrich identity was confirmed from a partial SSU rDNA contiguous sequence of 1343bp (99.7% similarity to Z. duplicatum).

Reservoir hosts for Gyrodactylus salaris may play a more significant role in epidemics than previously thought.

BACKGROUND: Gyrodactylus salaris Malmberg, 1957 has had a devastating impact on wild Norwegian stocks of Atlantic salmon Salmo salar L., and it is the only Office International des Epizooties (OIE) listed parasitic pathogen of fish. The UK is presently recognised as G. salaris-free, and management plans for its containment and control are currently based on Scandinavian studies. The current study investigates the susceptibility of British salmonids to G. salaris, and determines whether, given the host isolation since the last glaciation and potential genetic differences, the populations under test would exhibit different levels of susceptibility, as illustrated by the parasite infection trajectory over time, from their Scandinavian counterparts. METHODS: Populations of S. salar, brown trout Salmo trutta L., and grayling Thymallus thymallus (L.), raised from wild stock in UK government hatcheries, were flown to Norway and experimentally challenged with a known pathogenic strain of G. salaris. Each fish was lightly anaesthetised and marked with a unique tattoo for individual parasite counting. A single Norwegian population of S. salar from the River Lærdalselva was used as a control. Parasite numbers were assessed every seven days until day 48 and then every 14 days. RESULTS: Gyrodactylus salaris regularly leads to high mortalities on infected juveniles S. salar. The number of G. salaris on British S. salar rose exponentially until the experiment was terminated at 33 days due to fish welfare concerns. The numbers of parasites on S. trutta and T. thymallus increased sharply, reaching a peak of infection on days 12 and 19 post-infection respectively, before declining to a constant low level of infection until the termination of the experiment at 110 days. CONCLUSIONS: The ability of S. trutta and T. thymallus to carry an infection for long periods increases the window of exposure for these two hosts and the potential transfer of G. salaris to other susceptible hosts. This study demonstrates that G. salaris can persist on S. trutta for longer periods than previously thought, and that the role that S. trutta could play in disseminating G. salaris needs to be considered carefully and factored into management plans and epidemics across Europe.