During an experiment aimed at elucidating the nutritional requirements of burbot (Lota lota L.) larvae, the latter displayed a dorsal swelling of the cranium. Histological examination revealed dilated brain ventricles (hydrocephalus internus). Bacterial culture of the content of the swelling was negative. Slightly elevated copper concentrations were found in the culture water resulting in an increased larval body copper content. This is the first reported case of hydrocephalus in burbot larvae

Chiers, Koen

De Charleroy, D

Decostere, Annemie

Harzevili, AS

Meeus, W

Nevejan, Nancy

Van Den Broeck, Wim

Vanheule, Diederik

During an experiment aimed at elucidating the nutritional requirements of burbot (Lota lota L.) larvae, the latter displayed a dorsal swelling of the cranium. Histological examination revealed dilated brain ventricles (hydrocephalus internus). Bacterial culture of the content of the swelling was negative. Slightly elevated copper concentrations were found in the culture water resulting in an increased larval body copper content. This is the first reported case of hydrocephalus in burbot larvae.During an experiment aimed at elucidating the nutritional requirements of burbot (Lota lota L.) larvae, the latter displayed a dorsal swelling of the cranium. Histological examination revealed dilated brain ventricles (hydrocephalus internus). Bacterial culture of the content of the swelling was negative. Slightly elevated copper concentrations were found in the culture water resulting in an increased larval body copper content. This is the first reported case of hydrocephalus in burbot larvae.A

Vanheule, Diederik0020040573820A6699EC-F0EE-11E1-A9DE-61C894A0A6B4

Nevejan, NancyLA228020006306370000-0001-6566-460724C37076-F0EE-11E1-A9DE-61C894A0A6B4

Chiers, KoenDI058010010138100000-0002-7449-1047F4E63960-F0ED-11E1-A9DE-61C894A0A6B4

Van Den Broeck, WimDI118010009708650000-0001-6683-5658F4D510AE-F0ED-11E1-A9DE-61C894A0A6B4

Decostere, AnnemieDI050019902497378010010246210000-0001-5150-555XF4ECC762-F0ED-11E1-A9DE-61C894A0A6B4

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Hydrocephalus in burbot (Lota lota L.) larvae

Ghent University Academic Bibliography

Bull. Eur. Ass. Fish Pathol., 32(3) 2012, 87* Corresponding author’s email: nancy.nevejan@ugent.beHydrocephalus in burbot (Lota lota L.) larvaeD. Vanheule1, N. Nevejan1*, K. Chiers2,  W. Meeus3, A. S. Harzevili1, W. Van den Broeck4, D. De Charleroy5 and A. Decostere41Laboratory for Aquaculture & ARC, Department of Animal Production, Faculty of Bioscience Engineering, Ghent University, Rozier 44, 9000 Gent, Belgium; 2Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium; 3Aqua-ERF, KAHO St.Lieven, Hospitaalstraat 23, 9100 Sint-Niklaas, Belgium; 4Department of Morphology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium; 5Research Institute for Nature and Forest (INBO), Kliniekstraat 25, 1070 Brussels, BelgiumAbstractDuring an experiment aimed at elucidating the nutritional requirements of burbot (Lota lota L.) larvae, the laer displayed a dorsal swelling of the cranium. Histological examination revealed dilated brain ventricles (hydrocephalus internus). Bacterial culture of the content of the swelling was negative. Slightly elevated copper concentrations were found in the culture water resulting in an increased larval body copper content. This is the ﬁrst reported case of hydrocephalus in burbot larvae.IntroductionThe burbot (Lota lota L.) is the only freshwater member of the cod family Gadidae. In several European countries, this ﬁsh species is endan-gered. The exact cause of its declining popula-tions is unknown but several factors such as pollution, global warming and destruction of the habitats play an important role (Harzevili et al., 2003). Larval rearing and weaning is needed to supply juveniles for restocking programmes and farming activities. Hitherto, lile informa-tion is available on the nutritional needs of burbot larvae, a necessary prerequisite to be able to develop commercial feeds for these larval stages. Commercial dry feeds would decrease the labour cost and need for space and avoid the introduction of infectious agents as opposed to live feed (Jensen et al., 2008). It is in this context that an experiment was set up to characterize the nutritional requirements of burbot larvae. During this experiment, a cranial swelling was noted in the burbot larvae which is described in the present study. It is possible aetiologies are listed and discussed.Materials and methodsThe experimental facilities of the Laboratory for Aquaculture & ARC (Ghent University) ob-tained 32,000 burbot larvae (14 days post hatch-ing (dph)) from the Research Institute for Nature and Forest (INBO) in Linkebeek, Belgium. They were fed Artemia nauplii since 8dph. The larvae were divided over four 90l upwelling tanks and 88, Bull. Eur. Ass. Fish Pathol., 32(3) 2012were kept under continuous lighting. The tanks were disinfected beforehand by a combination of hydrogen peroxide (210g/l) and peroxyacetic acid (55g/l). Aerated tap water was used which was supplied through copper pipe lines. The hydraulic retention time was 1.5h. The levels of ammonia and nitrite did not exceed 0.05mg/l and 0.01mg/l, respectively. The average dis-solved oxygen concentration was 7.02mg/l and was never lower than 6.67mg/l. The temperature of the water was 12.1°C±1.3°C during the ﬁrst four weeks. Then the temperature was gradu-ally increased to 14.9°C±1.3°C. The pH of the water ranged from 7.58 to 7.70. The carbonate hardness was 9°d (161.1mgCaCO3/l).In the experimental facilities, the larvae were fed plain Artemia nauplii Instar I (SepArt OF 500μm, Inve Aquaculture, Belgium) at an initial feeding intensity of 50 nauplii/larva (44 nauplii/10ml). Aer one week, the feeding intensity gradually was increased during the next three weeks to 80 nauplii/10ml. This feeding intensity was main-tained for another two weeks. The larvae of tank 1 were fed discontinuously, twice a day. The larvae of tank 2, 3 and 4 were continuously fed Artemia nauplii with a peristaltic pump. Aer two weeks, only the larvae of tank 4 received nauplii enriched with highly unsaturated fay acids (HUFA) (S.presso, INVE Aquaculture, Belgium) for the remaining live feed phase.Three weeks following their arrival, a cranial swelling was seen in a small number of burbot larvae. One week later about 20% of the popula-tion in tank 1, 2 and 3 displayed this abnormal-ity and most of the other larvae got aﬀected during the next three weeks. Initially, the swell-ing was only visible upon close examination of the larvae but gradually increased in size to become easily discernible (Figure 1). The incidence of the cranial swelling of the larvae fed enriched Artemia nauplii (tank 4) was less than 5%. Most of the aﬀected larvae died a few days later. A minority fully recovered aer some weeks with complete disappearance of the swelling. At the same time, larvae from the same batch were reared at two other institutes in Belgium (Aqua-ERF and INBO). In these larvae no ab-normalities were encountered.The content of the cranial swelling of the aﬀected larvae was sampled aseptically for bacteriologi-cal examination. Samples were inoculated on Columbia agar with 5% sheep blood (Difco, Germany) and plates were incubated at 23°C for ﬁve days. Aﬀected and clinically normal larvae were sacriﬁced using an overdose of a benzocaine solution (1g/10ml ethanol), ﬁxed in neutral buﬀered formaldehyde or Bouin’s solution for histological examination and proc-essed as described previously by Rekecki et al. (2009).Two weeks following the ﬁrst observation of the cranial swelling in the larvae, the water and whole larval bodies were analysed for their copper and lead content by means of inductively coupled plasma mass spectrometry (ICP-MS) (Chan et al., 1998).For comparative purposes, burbot larvae from one of the other institutes exhibiting no abnor-malities were also sampled for histological, copper and lead analysis.Bull. Eur. Ass. Fish Pathol., 32(3) 2012, 89ResultsNo bacterial colonies were observed upon in-spection of the incubated agar plates.Histological examination of the aﬀected larvae revealed a dilation of the forebrain ventricles (hydrocephalus internus). The ventricles pro-truded dorsally and were only covered by the skin. The dilated ventricles were ﬁlled with protein-like material and aggregates of red blood cells occasionally present (Figure 2). Haemorrhagic zones were seen in the neural tissue. The nervous tissue itself and the skin, gills and internal organs displayed no abnor-malities, as was the case for all organs in the clinically normal burbot larvae. The concentration of copper and lead in the water of the aﬀected larvae was 4.6μg/l and below 0.2μg/l, respectively. The concentration of copper in the aﬀected larvae and the clinically normal larvae of the other institute amounted to 710μg/kg and 414μg/kg wet weight, respec-tively. DiscussionUp until now, only a few cases of hydrocephalus in ﬁsh have been reported and their aetiology discussed. Rodger and Murphy (1991) described Atlantic salmon (Salmo salar L.) alevins with a congenital Figure 1. Burbot larva (42dph) displaying a cranial swelling.90, Bull. Eur. Ass. Fish Pathol., 32(3) 2012hydrocephalus (externus). Necrotic cells were found in the meninges and on the outer surface of the optic lobes. Some of the alevins had haem-orrhages in the subarachnoidal space or in the optic lobes. Muench et al. (1997) reported hy-drocephalus internus in four week old channel catﬁsh (Ictalurus punctatus L.). Only 0.5 to 3% of the population was aﬀected. In some of the larvae a pale eosinophilic ﬂuid containing red blood cells was found in the distended fourth ventricle. One week later, the incidence dropped below 0.05%. Both anomalies were diagnosed as congenital, because no other causal agents could be detected. In our study, burbot larvae of the same batch were also reared and weaned at two other institutes. However, no similar deformities were encountered, urging us to speculate that in our case, the main aetiological factor was not genetic.It is reported that hydrocephalus in pike (Esox lucius L.) fry can be caused by two diﬀerent rhabdoviruses: Pike Fry Rhabdovirus (PFR) and Viral Haemorrhagic Septicaemia Virus (VHSV) (Speare and Frasca, 2006). Both viruses cause a generalized state of disease with high mortality. PFR causes an excess of ﬂuid accumulation in the third ventricle, including brain haemor-rhages. In the liver and kidney, necrotic lesions can be found (Muench et al., 1997). VHSV is Figure 2. Longitudinal histological section (H&E) of a deformed head of an aﬀected burbot larva (38dph). Aggregates of proteins (A) and red blood cells (B) can be detected in the cerebrospinal ﬂuid of the dilated ventricle.Bull. Eur. Ass. Fish Pathol., 32(3) 2012, 91known to cause serious problems in European rainbow trout (Oncorhynchus mykiss L.). Non-salmonid species like pike can also be infected. Hydrocephalus is the main sign (Meier et al., 1994). In the present case, a possible viral aeti-ology was excluded because no signs of a gen-eralized state of disease were noted and upon histological examination, no necrotic lesions were seen. The fact that no anomalies were observed in the larvae raised at the two other institutes supports this assumption. Indeed, the tanks in which the larvae were housed, were disinfected before use. If a virus were to have played a role, the larvae would have been car-riers upon entering the experimental facilities and one would expect all larvae from this batch (including those kept at the other institutes) to be aﬀected albeit with varying degrees due to their diﬀerent environment. In larval salmonids, hydrocephalus can also be the result of inadequate thiamine levels. These low levels can be due to a thiamine degrading factor present in prey ﬁsh and consumed by adult salmonids leading to lower thiamine levels in the eggs. Contaminants like PCBs may also play a role (Fitzsimons et al., 2001). Three similar syndromes linked with insuﬃcient thiamine levels are reported: early mortality syndrome (EMS, in salmonids in the Great Lakes), the ‘Cayuga syndrome’ (in larval landlocked Atlan-tic salmon) and M74 (in Atlantic salmon from the Baltic Sea) (Fitzsimons et al., 2001). Histo-logical investigation of these larvae revealed the presence of haemorrhages and necrotic zones in the brains (Elliot, 2005). The former could also be seen in the present larvae, but not the necrotic zones. The thiamine level in the burbot eggs was assumed to be suﬃcient, because larvae of the same origin, but reared at two other institutes did not suﬀer from similar disease signs. Bruno (2005) reported the presence of mostly unilateral cranial nodules in Atlantic salmon fry with unknown aetiology. The cerebellum was displaced dorsally, but no histological abnormalities were detected. In coho salmon (Oncorhynchus kisutch L.) fry, a focal swelling on the head due to a cranial defect was described. The aetiology is unknown, but exposure to teratogenic chemicals such as malachite green is suggested as the most likely cause. Malachite green is known to target cranial tissue. A similar sign is seen in salmon fry, with an acquired ex-ternal hydrocephalus (Speare and Frasca, 2006). The burbot larvae in the present study were not previously exposed to malachite green. Heavy metals are known to induce a wide range of deformities, including hydrocephalus. For lead, the maximum acceptable toxic concen-tration for trout fry is 14.6μg/l (Davies et al., 1976). This is far beyond the level found during this study.For copper, chronic toxicity concentrations for freshwater ﬁsh vary between 2μg/l and 14μg/l (Brix et al., 2001). The copper concentration in the water of the present rearing unit was 4.6μg/l. The copper concentration in the larval tissue was 710μg/kg wet weight aer three weeks of exposure and with a dry weight content of 15%, the calculated body copper content thus amounts to 4.7μg/g dry weight. Copper expo-sure of rainbow trout fry to 0.19μg/l, 4.6μg/l and 9.5μg/l for 20 days resulted in a body copper concentration of 4.42μg/g, 5.82μg/g and 6μg/g dry weight, respectively (Marr et al., 1996; Hansen et al., 2002). All exposures resulted 92, Bull. Eur. Ass. Fish Pathol., 32(3) 2012in reduced growth. Deformations were only seen at higher concentrations. Incubation of common carp (Cyprinus carpio L.) eggs in water containing 0.2mg/l copper induced mostly ver-tebral malformations in the larvae, while only some larvae displayed craniofacial deformi-ties (Jezierka et al., 2000). Copper is known to disrupt osmoregulation by inﬂuencing the Na+/K+-adenosine triphosphate pump and the cupric ion replaces the calcium ion in the tight para-cellular junctions of the gill cells, leading to in-creased cell membrane permeability (Zahner et al., 2006). Whether a similar phenomenon took place in the brain of the burbot larva leading to ﬂuid accumulation is unknown. Hence, we cannot conﬁrm nor exclude the slightly elevated copper level of 4.6μg/l having a causative role in the noted hydrocephalus. Important to keep in mind is that burbot is known to be a very sensitive species to polluting agents. Industrial pollution of the aquatic environment is indeed indicated as a reason of the declining popula-tions (Polinski et al., 2010). When the larvae were fed Artemia nauplii enriched with HUFA’s, the prevalence of the cranial deformity was markedly lower. Supple-menting HUFA’s can improve the larval quality and increase their stress resistance, as was seen in several other marine ﬁsh species (Sorgeloos et al., 2001; Wilson, 2009; Vizcaino-Ochoa et al., 2010). However, the exact role of HUFA’s in these observations remains to be elucidated.AcknowledgementThis study was partly ﬁnanced by the European Commission - European Regional Development Fund (ERDF) – Interreg IV A Flanders – The Netherlands: AquaVlan; the Flemish govern-ment and the Province of Oost-Vlaanderen. Christian Puevils and Delphine Ameye are thanked for their excellent technical assist-ance. Annelies Declercq is thanked for her help with the sampling of the larvae. Prof. Dr. Paul Simoens and Prof. Dr. Pieter Cornillie are acknowledged for critically reviewing the manuscript. Prof. Dr. Dominique Adriaens and Anamaria Rekecki are thanked for their help with the interpretation of the histology.ReferencesBrix KV, DeForest DK and Adams WJ (2001). Assessing acute and chronic copper risks to freshwater aquatic life using species sensitivity distributions for different taxonomic groups. Environmental Toxicology and Chemistry 20(8), 1846-1856.Bruno DW (1997). Cranial nodules in farmed Atlantic salmon, Salmo salar L., fry. Journal of Applied Ichthyology 13, 47-48. Chan S, Gerson B and Subramaniam S (1998). The role of copper, molybdenum, selenium, and zinc in nutrition and health. Clinics in Laboratory Medicine 18(4), 673-685.Davies PH, Goel JJP, Sinley JR and Smith NF (1976). Acute and chronic toxicity of lead to rainbow trout Salmo gairdneri, in hard and so water. Water Research 10(3), 199-206.Elliot D (2005). Early mortality syndrome in Coho salmon (Oncorhynchus kisutch) and Chinook salmon (Oncorhynchus tshawytscha) in the Great Lakes. Histopathology Workshop: Reproductive and Early Life Stages Pathology. 12th International Conference European Association of Fish Pathologists. Copenhagen 2005.Fitzsimons JD, Vandenbyllaardt L and Brown SB (2001). The use of thiamine and thiamine antagonists to investigate the etiology of early mortality syndrome in lake trout (Salvelinus namaycush). Aquatic Toxicology 52, 229-239.Hansen JA, Lipton J, Welsh PG, Morris J, Cacela D and Suedkamp MJ (2002). Relationship Bull. Eur. Ass. Fish Pathol., 32(3) 2012, 93between exposure duration, tissue residues, growth, and mortality in rainbow trout (Oncorhynchus mykiss) juveniles sub-chronically exposed to copper. Aquatic Toxicology 58 (3-4), 175-188.Harzevili AS, De Charleroy D, Auwerx J, Vught I, Van Slycken J, Dhert P and Sorgeloos P (2003). Larval rearing of burbot (Lota lota L.) using Brachionus calyciﬂorus rotifer as starter food. Journal of Applied Ichthyology 19, 84-87.Jensen RN, Ireland SC, Siple JT, Williams SR and Cain KD (2008). 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The eﬀect of dietary docosahexaenoic acid (DHA) on growth, survival and pigmentation of California halibut Paralichthys californicus larvae (Ayres, 1810) Aquaculture 302, 228-234.Wilson R (2009). Dietary eﬀects of n-3 highly unsaturated fay acid levels on egg and larval quality, and the fay acid composition of the eggs of Chilean ﬂounder Paralichthys adspersus broodstock. Aquaculture Research 40, 1400-1409.Zahner HM, VanGenderen EJ, Tomasso JR and Klaine SJ (2006). Whole-body sodium concentration in larval fathead minnows (Pimephales promelas) during and following copper exposure. Environmental Toxicology and Chemistry 25(6), 1635-1639.

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Hydrocephalus in burbot (Lota lota L.) larvae

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Ghent University Academic Bibliography