Towards an in situ non-lethal rapid test to accurately detect the presence of the nematode parasite, Anguillicoloides crassus, in European eel, Anguilla anguilla.

Anguillicoloides crassus is an invasive nematode parasite of the critically endangered European eel, Anguilla anguilla, and possibly one of the primary drivers of eel population collapse, impacting many features of eel physiology and life history. Early detection of the parasite is vital to limit the spread of A. crassus, to assess its potential impact on spawning biomass. However accurate diagnosis of infection could only be achieved via necropsy. To support eel fisheries management we developed a rapid, non-lethal, minimally invasive and in situ DNA-based method to infer the presence of the parasite in the swim bladder. Screening of 131 wild eels was undertaken between 2017 and 2019 in Ireland and UK to validate the procedure. DNA extractions and PCR were conducted using both a Qiagen Stool kit and in situ using Whatman qualitative filter paper No1 and a miniPCR DNA Discovery-System™. Primers were specifically designed to target the cytochrome oxidase mtDNA gene region and in situ extraction and amplification takes approximately 3 h for up to 16 individuals. Our in-situ diagnostic procedure demonstrated positive predictive values at 96% and negative predictive values at 87% by comparison to necropsy data. Our method could be a valuable tool in the hands of fisheries managers to enable infection control and help protect this iconic but critically endangered species.

The impact of Anguillicoloides crassus (Nematoda) on European eel swimbladder: histopathology and relationship between neuroendocrine and immune cells.

The swimbladder functions as a hydrostatic organ in most bony fishes, including the European eel, Anguilla anguilla. Infection by the nematode Anguillicoloides crassus impairs swimbladder function, significantly compromising the success of the eel spawning migration. Swimbladders from 32 yellow eels taken from Lake Trasimeno (Central Italy) were analysed by histopathology- and electron microscopy-based techniques. Sixteen eels (50%) harboured A. crassus in their swimbladders and intensity of infection ranged from 2 to 17 adult nematodes per organ (6.9 ± 1.6, mean ± s.e.). Gross observations of heavily infected swimbladders showed opacity and histological analysis found a papillose aspect to the mucosa and hyperplasia of the lamina propria, muscularis mucosae and submucosa. Inflammation, haemorrhages, dilation of blood vessels and epithelial erosion were common in infected swimbladders. In the epithelium of parasitized swimbladders, many empty spaces and lack of apical junctional complexes were frequent among the gas gland cells. In heavily infected swimbladders, we observed hyperplasia, cellular swelling and abundant vacuolization in the apical portion of the gas gland cells. Numerous mast cells and several macrophage aggregates were noticed in the mucosal layer of infected swimbladders. We found more nervous and endocrine elements immunoreactive to a panel of six rabbit polyclonal antibodies in infected swimbladders compared to uninfected.

Encapsulation of Anguillicola crassus reduces the abundance of adult parasite stages in the European eel (Anguilla anguilla).

Encapsulation of the parasitic nematode Anguillicola crassus Kuwahara, Niimi & Hagaki is commonly observed in its native host, the Japanese eel (Anguilla japonica Temminck & Schlegel). Encapsulation has also been described in a novel host, the European eel (A. anguilla L.), and there is evidence that encapsulation frequency has increased since the introduction of A. crassus. We examined whether encapsulation of A. crassus provides an advantage to its novel host in Lake Müggelsee, NE Germany. We provide the first evidence that encapsulation was associated with reduced abundance of adult A. crassus. This pattern was consistent in samples taken 3 months apart. There was no influence of infection on the expression of the two metabolic genes studied, but the number of capsules was negatively correlated with the expression of two mhc II genes of the adaptive immune response, suggesting a reduced activation. Interestingly, eels that encapsulated A. crassus had higher abundances of two native parasites compared with non-encapsulating eels. We propose that the response of A. anguilla to infection by A. crassus may interfere with its reaction to other co-occurring parasites.

New records of philometrids (Nematoda: Philometridae) from marine fishes off Australia, including description of four new species and erection of Digitiphilometroides gen. n.

The following six species of the Philometridae (Nematoda: Dracunculoidea) were recorded from marine fishes off the northern coast of Australia in 2015 and 2016: Philometra arafurensis sp. n. and Philometra papillicaudata sp. n. from the ovary and the tissue behind the gills, respectively, of the emperor red snapper Lutjanus sebae (Cuvier); Philometra mawsonae sp. n. and Dentiphilometra malabarici sp. n. from the ovary and the tissue behind the gills, respectively, of the Malabar blood snapper Lutjanus malabaricus (Bloch et Schneider); Philometra sp. from the ovary of the goldbanded jobfish Pristipomoides multidens (Day) (Perciformes: all Lutjanidae); and Digitiphilometroides marinus (Moravec et de Buron, 2009) comb. n. from the body cavity of the cobia Rachycentron canadum (Linnaeus) (Perciformes: Rachycentridae). Digitiphilometroides gen. n. is established based on the presence of unique digital cuticular ornamentations on the female body. New gonad-infecting species, P. arafurensis and P. mawsonae, are characterised mainly by the length of spicules (252-264 µm and 351-435 µm, respectively) and the structure of the gubernaculum, whereas P. papillicaudata is characterised mainly by the body length (70 mm) of gravid female, extent of the oesophageal gland, size of caudal projections and the location in the host. Dentiphilometra malabarici differs from congeners mainly in the arrangement of circumoral teeth (in a single row), extent of the oesophageal gland and the absence of sclerotised teeth or protuberances on the oesophageal lobes in the mouth. Digitiphilometroides marinus has not previously been reported from fishes in Australian waters.

Transcript levels of members of the SLC2 and SLC5 families of glucose transport proteins in eel swimbladder tissue: the influence of silvering and the influence of a nematode infection.

The rate of glucose metabolism has been shown to be correlated to glucose uptake in swimbladder gas gland cells. Therefore, it is assumed that in the European eel silvering, i.e., the preparation of the eel for the spawning migration to the Sargasso Sea, coincides with an enhanced capacity for glucose uptake. To test this hypothesis expression of all known glucose transport proteins has been assessed at the transcript level in yellow and in silver eels, and we also included Anguillicola crassus infected swimbladders. Glucose uptake by rete mirabile endothelial cells could be crucial for the countercurrent exchange capacity of the rete. Therefore, this tissue was also included in our analysis. The results revealed expression of ten different members of the slc2 family of glucose transporters, of four slc5 family members, and of kiaa1919 in gas gland tissue. Glucose transporters of the slc2 family were expressed at very high level, and slc2a1b made up about 80% of all slc2 family members, irrespective of the developmental state or the infection status of the eel. Overall, the slc5 family contributed to only about 8% of all detected glucose transport transcripts in gas gland tissue, and the slc2 family to more than 85%. In rete capillaries, the contribution of sodium-dependent glucose transporters was significantly higher, leaving only 66% for the slc2 family of glucose transporters. Neither silvering nor the infection status had a significant effect on the expression of glucose transporters in swimbladder gas gland tissue, suggesting that glucose metabolism of eel gas gland cells may not be related to transcriptional changes of glucose transport proteins.

Anthropogenic spreading of anguillid herpesvirus 1 by stocking of infected farmed European eels, Anguilla anguilla (L.), in the Schlei fjord in northern Germany.

The Schlei fjord in northern Germany is the recipient water of a comprehensive eel, Anguilla anguilla (L.), stocking programme. Since 2015, stocked eels become alizarin red S marked, but to date no control mechanism is implemented in this stock enhancement measure to prevent anthropogenic spreading of diseases. Consequentially, it was possible that farmed stocking cohorts of 2015 and 2016 (in total ca. 1040 kg) were subsequently tested positive for anguillid herpesvirus 1 (AngHV 1). For this study, 100 eels [total length (TL) 24.3-72.9 cm, age ca. 1-6 years] were caught in 2016 and investigated with regard to AngHV 1 infection, parasite load (Anguillicoloides crassus) and body conditions. 68% of the eels were found to be virus positive while larger specimens were more often infected. In addition, a fitted generalized linear model (area under the curve = 0.741) demonstrated that an increase in individual TL is accompanied with an increased risk of clinically relevant virus loads. Anguillicoloides crassus turned out to be an important stressor for eels, because parasite and virus load revealed a significant positive correlation. The results of this study evidently show the urgent need of a disease containment strategy for eel stocking programmes.

The effect of foraging and ontogeny on the prevalence and intensity of the invasive parasite Anguillicola crassus in the European eel Anguilla anguilla.

Infection patterns of the invasive Anguillicola crassus nematode were investigated in a population of the European eel Anguilla anguilla where parasite invasion is very recent, Loch Lomond, Scotland. Intensity levels of the parasite were associated with differences in fish ontogeny and trophic ecology. Although eels foraged on both fish and invertebrates, individuals which were smaller and fed on invertebrates (>70% contribution to diet) were found to contain a greater number of swim bladder parasites compared to larger eel with a predominance of fish (>60% contribution) in their diet. Within affected fish, a significant negative relationship was found between fish length and parasite intensity, with smaller individuals having higher parasite intensity than larger individuals. This study indicates that food intake and infection risk are linked in this recently infected host-parasite system. From a management perspective increasing our understanding of how infection intensity and repeated exposure is linked to resource use in an ecosystem is important for the future management of this endangered species in Europe.

Comparative imaging of European eels (Anguilla anguilla) for the evaluation of swimbladder nematode (Anguillicoloides crassus) infestation.

This study compares diagnostic imaging tools in detecting the parasitic swimbladder nematode Anguillicoloides crassus in Anguilla anguilla (L.) and focuses on ultrasound in an attempt to develop a non-destructive, field diagnostic test. Ultrasound use could allow the parasite to be diagnosed without decreasing the number of critically endangered European eels through post-mortem. In the preliminary study, eels were examined with computed radiography, computed tomography, magnetic resonance imaging, 14 MHz high-end ultrasound and 5 MHz low-end portable ultrasound, and the results were compared with post-mortem findings. This ultrasound scanning technique did not produce any promising results. A second batch of eels was examined using the same high-end and low-end ultrasounds, but employing a different scanning technique and comparing the results with post-mortem. This second study, scanning along the midline from below, allowed for the detection of anomalies associated with moderately infected animals. None of the eels used in this study were severely infected; thus, no conclusions can be made regarding the use of ultrasound in those animals. Overall, it was found that none of the techniques were useful in diagnosing mildly infected individuals; therefore, no single diagnostic imaging tool is sensitive enough to replace post-mortem for definite diagnosis.

A new species of philometrid parasite (Nematoda, Philometridae) and histopathological lesions in juvenile whitemouth croakers, Micropogonias furnieri (Desmarest).

A new species of nematode parasite, Philometroides tahieli sp. nov. is described. The juvenile whitemouth croaker, Micropogonias furnieri Desmarest, was collected during the spring and summer of the years 2008-2010 from estuarial environments of Argentina. During investigations of the parasite fauna a nematode encapsulated in the musculature of the operculum was found. The nematodes were removed from the host's muscle tissue and slide-mounted in lactophenol solution to clarify the specimens. Opercular muscle with the parasite was processed for histopathological examination. Philometroides tahieli sp. nov. can be identified by the location of gravid females in the host, presence of anterior oesophageal bulb, bosses on the surface of the body, presence of transversal mounds and 14 cephalic papillae in two circular rows. The pathogenicity of the parasite is low in the natural environment, but lesions are consistent with a chronic process. The appearance of caseous necrosis suggests the presence of a locally acting substance. Philometroides tahieli sp. nov. is the second species of this genera described from brackish waters, and since the discovery of Philometroides maplestoni in 1928, is the first species of this genus recorded for South America.

The Hsp70 response of Anguillicola species to host-specific stressors.

The present study is based on infection experiments of two different swim bladder parasite species, Anguillicola crassus Kuwahara et al., 1974 and Anguillicola novaezelandiae Moravec and Taraschewski, 1988, which were experimentally transferred to the two eel species Anguilla anguilla Linnaeus, 1758 and Anguilla japonica Temmink and Schlegel, 1846, respectively. The host-parasite groups were selected due to their different grades of mutual adaptation. The main aim of this study was to analyze the stress responses within the parasites, which were confronted with different hosts, i.e. with different stressors related to the respective host. For this purpose, mean intensities, recovery rates, larvae output, and levels of synthesized heat shock proteins (Hsp70) were determined in nematodes of each infection group. Increased stress responses were detected in the endemic system of A. crassus parasitizing A. japonica and A. crassus in its recently acquired host A. anguilla, which seems to be associated with the immune response of the particular host species and the expenditure of energy on producing larvae. A. novaezelandiae showed overall weak activities in its unknown host species A. japonica, with the lowest recovery rate of all examined groups neither featuring elevated Hsp responses, nor a high mean intensity, nor any reproductive output. On the contrary, in A. anguilla, the parasite reached higher recovery rates, mean intensities, and reproductive output, but no increased Hsp70 levels could be detected. The four considered factors proved partially interdependent, whereas few results did not follow a clear pattern.

Influence of adult Anguillicoloides crassus load in European eels swimbladder on macrophage response.

Anguillicoloides crassus has become one of the most important threats to the European eel (Anguilla anguilla). Adult parasites colonize the swimbladder leading to an impaired functioning of this organ. The infection is also responsible for an increased in the stress level of infected eels, that could produce an altered immune response as well. Differences in parasite loads and effects in the European and Japanese eel (Anguilla japonica) have been described. We have studied the influence of the number of adult parasites present in the swimbladder of wild eels on the macrophage response (phagocytosis and respiratory burst) as part of the first immune response to pathogens. Our results show an increased phagocytozed bacterial survival 24 h post-infection in macrophages of eels infected with more than ten adult parasites compared to macrophages from eels infected with less than those ten adult parasites. Respiratory burst results also showed a less efficient response in macrophages from eels infected with more than ten adult parasites, although in this case results were not found to be significant.

Infection ecology of Philometra ovata (Nematoda: Philometridae) in a wild European minnow (Phoxinus phoxinus) population in Finland.

Seasonal life cycle of body cavity dwelling (BCD) Philometra ovata (Nematoda: Philometridae) has been reported in southern and central European countries, but its swim bladder dwelling (SBD) stage and northern populations have remained unstudied. In this study, we investigated the seasonal life cycle and infection ecology of P. ovata in both swim bladder and body cavity in the European minnow (Phoxinus phoxinus) in Finland. The larval P. ovata infected the swim bladder of minnows mainly in August. Female SBD P. ovata emigrated to body cavity mostly in September, grew to their full size by the end of the next June, and evacuated from minnows in July. In addition, female SBD P. ovata retarding their development and staying in swim bladder were found commonly in minnows, thus the mean monthly prevalence (6·7 ± 3·9%) and mean intensity (1·4 ± 0·8) of BCD P. ovata was lower than that of SBD P. ovata (37·8 ± 15·1% and 2·0 ± 1·5, respectively). Finally, despite the large size of BCD individuals, infection of P. ovata did not impair body condition and relative gonad size of minnows, but increased the mortality and caused physical damages in their hosts during the evacuation period.

Effects of Anguillicola novaezelandiae on the levels of cortisol and hsp70 in the European eel.

The nematodes Anguillicola novaezelandiae and Anguillicola crassus are both alien parasites of the European eel with severe adverse effects on their new host. Both species differ in terms of their invasiveness and their severity of harmful effects on the European eel. The purpose of this study was to determine under laboratory conditions whether stages of A. novaezelandiae induce stress in European eels (Anguilla anguilla) and if these levels differ from stress levels induced by A. crassus. We analysed levels of plasma cortisol and hepatic hsp70 of eels experimentally infected with A. novaezelandiae and compared them to uninfected eels as well as to eels experimentally infected with A. crassus. Larval stages of A. novaezelandiae induced higher levels of plasma cortisol compared to uninfected controls, while adult parasites increased the levels of hepatic hsp70 above those of uninfected controls. The eels' cortisol response is induced by larval stages of A. novaezelandiae, while adult stages elevate levels of hepatic hsp70. Levels of stress induced by A. novaezelandiae are comparable to those induced by A. crassus.

Two new gonad-infecting species of Philometra Costa, 1845 (Nematoda: Philometridae) from marine fishes off the northern coast of Australia.

Based on light and electron microscopical studies, two new gonad-infecting species of Philometra Costa, 1845 (Nematoda: Philometridae) are described from the ovary of marine perciform fishes off the northern coast of Australia (near Darwin): Philometra carangis n. sp. from the bluespotted trevally Caranx bucculentus Alleyne & Macleay (Carangidae) and P. carponotati n. sp. from the Spanish flag snapper Lutjanus carponotatus (Richardson) (Lutjanidae). Philometra carangis is mainly characterised by the length of the spicules (153-189 µm), the presence of a distinct dorsal protuberance consisting of two dorsolateral lamellar parts separated from each other by a smooth median field, a V-shaped mound on the male caudal extremity, a pair of large post-cloacal papillae and the body length of the males (3.22-4.15 mm). Philometra carponotati is distinguished from other congeneric species parasitising lutjanids by the length of the spicules and gubernaculum (225-252 and 99-117 µm, respectively), the absence of a dorsal protuberance on the distal lamellar part of the gubernaculum, the presence of a U-shaped mound on the male caudal extremity, a pair of large post-cloacal papillae and the body length of the male (3.74-4.31 mm). Besides the recently established Philometra zabidii Moravec & Diggles, 2014 (based on a single female), these two newly described nematodes are the only nominal gonad-infecting species of Philometra known to parasitise marine fishes in Australian waters.

New record of Philometra species from the marine edible fish Terapon jarbua collected from the Sindh, Arabian Sea, Pakistan.

Diseases in fish due to helminth parasites, especially Philometra species, are the primary worry in aquaculture. Philometra are responsible for health problem in fishes they directly affect fish growth and population parameters. A comprehensive survey was conducted involving the examination of the marine fish species Terapon jarbua, gathered from the coastal waters of Sindh, Pakistan In this research different Philometra species from marine fish Terapon jarbua during 2021 and 2022. Philometra nematodes, belonging to the family Philometridae, are common parasitic organisms inhabiting both marine and freshwater environments. Their prevalence, particularly when existing in high numbers within host organisms, can lead to severe and potentially lethal consequences. Employing light microscopy techniques, diverse species of Philometra were identified, including Philometra teraponi, P. jarbuai, P. arabiai, P. karachii, and P. awarii, localized primarily within the ovaries of the host fish. A total of 140 fish samples were examined and 76 were infected. The intensity of infected fish was 54.28%. The identification process encompassed meticulous analysis of crucial parameters, such as body size, esophagus length, positioning of the nerve ring, dimensions of the ventriculus, and ligament size. Intriguingly, the parasites were found in varying contexts; while some were free within the ovaries, others were embedded within tissues, inducing severe muscular dystrophy. This research presents novel findings of Philometra nematodes in the marine waters of Pakistan, extending their host and geographical distribution records. Future studies are needed to better evaluate and describe the dynamics and the epidemiology of Philometra infection in wild and cultured fish species.

Evolutionary divergence of the swim bladder nematode Anguillicola crassus after colonization of a novel host, Anguilla anguilla.

BACKGROUND: Anguillicola crassus, a swim bladder nematode naturally parasitizing the Japanese eel, was introduced about 30 years ago from East Asia into Europe where it colonized almost all populations of the European eel. We conducted a common garden experiment under a reciprocal transfer design infecting both European and Japanese eels with populations of A. crassus from Germany, Poland and Taiwan. We tested, whether differences in infectivity, developmental dynamics and reproductive output between the European and Asian parasite populations occur while harboured in the specimens of native and colonized eel host, and if these differences are genetically based or are plastic responses to the new environment. RESULTS: Under common garden conditions an evolutionary change in the both European parasite populations of A. crassus compared with their Taiwanese conspecifics was observed for infectivity and developmental dynamics, but not for reproductive output. When infecting the European eel, current European populations of the parasite were less infective and developed faster than their Taiwanese conspecifics. In the reciprocally infected Japanese eel the genetically induced differences between the parasite strains were less apparent than in the European eel but higher infectivity, faster development and higher larval mortality of the European parasite populations could be inferred. CONCLUSIONS: The differences in infectivity and developmental dynamics between European and Taiwanese populations of A. crassus found in our study suggest rapid genetic divergence of this parasite after a successful host switch in Europe.

Can differences in life cycle explain differences in invasiveness? - A study on Anguillicola novaezelandiae in the European eel.

Anguillicola crassus is the most invasive species of its genus and it is a successful colonizer of different eel species worldwide. It is so far the only species of the genus Anguillicola whose life cycle has been studied completely. To analyse whether differences in life cycle may explain differences in invasiveness, we infected European eels with Anguillicola novaezelandiae under laboratory conditions. Anguillicola novaezelandiae shows a synchronized development in the European eel. Eggs with second-stage larvae appeared 120 days after infection. No density-dependent effect in parasite development could be found for A. novaezelandiae. The present study shows that the life cycle of A. novaezelandiae differs on final host level compared with A. crassus in ways which result in a less successful invasion of new host species.

A synthesis of our current knowledge of philometrid nematodes, a group of increasingly important fish parasites.

Members of the Philometridae represent the most important group of dracunculoid nematodes parasitizing fishes. In his monograph treating the Dracunculoidea, Moravec (2006) reported a total of 11 genera and 105 species of philometrids parasitizing freshwater, brackish-water and marine fishes. However, during the last six years (2007-2012), an additional 42 new species of Philometridae have been described, representing a 40% increase of the number of nominal species. Most of these species (30) belong to Philometra Costa, 1845, mainly represented by parasites of marine fishes, a few others (8) to Philometroides Yamaguti, 1935, and a single one to each of the following genera: Caranginema Moravec, Montoya-Mendoza et Salgado-Maldonado, 2008, Dentiphilometra Moravec et Wang, 2002, Dentirumai Quiazon et Moravec, 2013* and Spirophilometra Parukhin, 1971. Moreover, three new genera, Afrophilometra Moravec, Charo-Karisa et Jirku, 2009, Caranginema and Dentirumai, were erected. Representatives of seven genera, Afrophilometra, Buckleyella Rasheed, 1963, Caranginema, Dentiphilometra, Dentirumai, Paraphilometroides Moravec et Shaharom-Harrison, 1989 and Rumai Travassos, 1960, were studied using scanning electron microscopy (SEM) for the first time. Thirteen known but poorly described philometrid species were redescribed and, in some species of Caranginema and Philometra, previously unknown conspecific males were discovered and described. The male surface ultrastructure studied by SEM provided new taxonomically important features for species distinction. Gene sequencing was used in several recent studies and advanced our understanding of phylogenetic interrelationships among representatives of seven genera (Afrophilometra, Alinema Rasheed, 1963, Caranginema, Nilonema Khalil, 1960, Philometra, Philometroides and Rumai) and of the extent of the biodiversity of philometrids. New data were obtained on the biology and pathogenicity of several species of Nilonema, Philometra, Philometroides and Rumai. The need to carry out surveys in order to find males and to use SEM and gene sequencing to identify philometrids is emphasized. Appropriate quantitative methods to determine the impact of philometrids in ovarian tissue on host fecundity are recommended. Further detailed studies on philometrids would be significant not only from the theoretical viewpoint, but also because of their practical implications. A list of philometrid nematode species by continents is provided.

Two philometrids (Nematoda: Philometridae) infecting the tigertooth croaker Otolithes ruber (Bloch & Schneider) (Teleostei: Sciaenidae) off Iran, including erection of a new genus.

Two philometrid nematodes (Philometridae) are described from the marine perciform fish (tigertooth croaker) Otolithes ruber (Bloch & Schneider) (Sciaenidae) from off Iran: Clavinemoides annulatus n. g., n. sp. based on female specimens from the caudal fin of fish from the Persian Gulf and Philometra otolithi Moravec & Manoharan, 2013 from subgravid and nongravid females collected from the ovary of fishes in the Sea of Oman and the Persian Gulf. The monotypic genus Clavinemoides n. g. is mainly characterised by a triangular oral aperture, a markedly large anterior oesophageal bulb, a distinctly annulated body and numerous fine elevated transverse cuticular bands. The finding of P. otolithi in Iranian waters represents a new geographical record of this parasite species. An updated key to the genera of the Philometrinae Baylis & Daubney, 1926 is provided.

An evaluation of indices of gross pathology associated with the nematode Anguillicoloides crassus in eels.

This study compares two alternative indices for quantifying the gross pathology of the swimbladder of eels, Anguilla anguilla (L.), infected with the nematode Anguillicoloides crassus. Two observers recorded twice the scores obtained by the two indices on the same set of 71 wild caught eels (from elver to silver eels, French Mediterranean lagoons). The Length Ratio Index (LRI), performed better than the Swimbladder Degenerative Index (SDI), in three of four predefined criteria of decision. First, the LRI better correlated with an estimate of the swimbladder volume reduction, a functional consequence of the infection (representativeness). Also, the LRI was less prone to subjectivity (inter-observer variability) and more precise (intra-observer variability), although less easy to generate (time needed for measurement/assessment). Using a sub-sample of 32 unaffected eels (showing minor if any swimbladder damage and no living worms at autopsy), we ascertained a linear relationship between the swimbladder length and the total body length, a prerequisite of isometric growth, to definitively accept the new ratio index as a valid alternative to the SDI. Also, because the LRI can be recorded on live specimens with radio-imagery (non-invasive method), we recommend its use, and provide a graph of correspondence between the SDI scores, the LRI scores and the estimated proportion of gas loss in the swimbladder.