Journal of Fish Diseases 2015 doi:10.1111/jfd.12352 Effects of Loma morhua (Microsporidia) infection on the cardiorespiratory performance of Atlantic cod Gadus morhua (L). M D Powell1 and A K Gamperl2 1 Norwegian Institute for Water Research, Bergen, Norway 2 Department of Ocean Sciences, Memorial University of Newfoundland, St. John’s, NF, Canada Abstract Introduction The microsporidian Loma morhua infects Atlantic Microsporidial diseases pose significant challenges cod (Gadus morhua) in the wild and in culture and to the development of marine fish aquaculture, spe- results in the formation of xenomas within the gill cifically in the North Atlantic (Murchelano, filaments, heart and spleen. Given the importance Despres-Patanjo & Ziskowski 1986; Bricknell, of the two former organs to metabolic capacity and Bron & Bowden 2006; Kahn 2009), the North thermal tolerance, the cardiorespiratory perfor- Pacific (Brown, Kent & Adamson 2010) and more mance of cod with a naturally acquired infection of recently the Red Sea (Abdel-Ghaffar et al. 2011). Loma was measured during an acute temperature Of particular significance is the infection of gadoid À increase (2 °Ch 1)from10°C to the fish’s criti- fishes [e.g. Atlantic cod (Gadus morhua)] with the cal thermal maximum (CTMax). In addition, oxy- microsporidian Loma morhua; this species is gen consumption and swimming performance were recently identified separately from Loma branchialis measured during two successive critical swimming (Brown et al. 2010). Microsporidian xenomas are ° speed (Ucrit)testsat10 C. While Loma infection characterized by their distinct morphology, with had a negative impact on cod cardiac function at those produced by Loma sp. having a well-defined warm temperatures, and on metabolic capacity in and characteristic thick granular amorphous wall both the CTMax and Ucrit tests (i.e. a reduction of and various developmental stages of the parasite 30–40%), it appears that the Atlantic cod can lar- (Lom & Dykova 2005). It is believed from data on gely compensate for these Loma-induced cardiore- related species (Loma salmonae) that the na€ıve host spiratory limitations. For example, (i) CTMax ingests spores that enter the host through the gut, Æ ° ~ À1 (21.0 0.3 C) and Ucrit ( 1.75 BL s ) were that the sporoplasm is injected into a host cell that very comparable to those reported in previous stud- migrates to the heart and enters a merogony-like ies using uninfected fish from the same founder phase and finally that the parasite utilizes macro- population; and (ii) our data suggest that tissue phage-mediated transport to the gill where endo- oxygen extraction, and potentially the capacity for thelial and pillar cells hypertrophy to form anaerobic metabolism, is enhanced in fish infected xenomas (Sanchez, Speare & Markham 2000; San- with this microsporidian. chez et al. 2001a; Rodriguez-Tovar et al. 2003). However, L. morhua xenomas regularly occur in Keywords: cardiac performance, critical thermal the heart as well as in the gills and other organs maximum, gill pathology, heart pathology, metab- (Murchelano et al. 1986). Infection with Loma olism, oxygen consumption. leads to a reduced body condition, a decrease in energy stores (liver somatic index), and mild anae- Correspondence M D Powell, Norwegian Institute for Water mia and leukaemia (Khan 2005). In salmonids, Research, Thormøhlens gate, 53D Bergen 5006 Norway such as rainbow (Oncorhynchus mykiss) and brook (e-mail: [email protected]) Ó 2015 John Wiley & Sons Ltd 1 Journal of Fish Diseases 2015 M D Powell & A K Gamperl Effects of Loma morhua infection trout (Salvelinus fontinalis) that are infected with and the Norwegian Animal Welfare Authority Loma salmonae, decreases in specific growth rate are (FDU). The Atlantic cod G. morhua L. used in correlated with increases in routine and maximum this study (mean body mass Æ SEM, metabolic rate, and reductions in routine but not 509.1 Æ 12.3; total length 37.1 Æ 0.2 cm; maximum metabolic rate, respectively (Powell et al. N = 42) were raised and maintained at the Dr. 2005): the metabolic cost of the disease is largely Joe Brown Aquatic Research Building (JBARB; attributed to changes in branchial O2 permeability Ocean Sciences Centre, Memorial University of (Powell, Speare & Becker 2006). Newfoundland) in a 3000-L tank supplied with The thermal biology of Atlantic cod has aerated sea water at 10–11 °C for at least received considerable attention over the past dec- 6 months prior to experimentation. The fish were ade (e.g. see Drinkwater 2005; Gollock et al. fed daily with a commercial cod diet (EWOS) 2006; Perez-Casanova et al. 2008a,b; Hori et al. under a photoperiod of 8 hours light: 16 hours 2012). Although cod prefer cooler waters (8– dark. 15 °C; Pettersen & Steffansen 2003), local tem- peratures may fluctuate significantly on a seasonal Critical thermal maximum experiment and diurnal basis, and cod in the wild and in cul- ture may not be able to escape acutely elevated Surgical procedures. Fish were individually netted water temperatures as high as 20 °C (Gollock and anaesthetized in sea water containing À et al. 2006; Righton et al. 2010). Given that it is 100 mg L 1 MS-222 (tricaine methane sulpho- widely recognized that limitations in metabolic nate) until ventilatory movements ceased. The fish scope and cardiac output are a key determinant of were then weighed and measured before being the upper thermal limits of fishes (Farrell 2002; transferred to a surgical table where their gills Farrell et al. 2009; Gamperl, Swafford & Rodnick were constantly irrigated with oxygenated, and 2011; Keen & Gamperl 2012; Sandblom et al. chilled, sea water containing MS-222 À 2014), that Loma infections impact fish metabo- (0.5 mg L 1). To allow for the direct measure- lism (Powell et al. 2005) and that this parasite ment of cardiac function (cardiac output Q; heart forms xenomas in the cod heart and gills, it might rate fH; and stroke volume Hsv), a 2PS Tran- be expected that infection with this microsporidi- sonicTM blood flow probe (Transonic Systems) was an constrains the upper temperature that infected implanted around the ventral aorta using a cod can tolerate and/or reduces the capacity of method modified from Thorarensen, Gallaugher individuals to perform other important physiologi- & Farrell (1996) and Gollock et al. (2006). In cally (and metabolically) demanding functions brief, the left operculum and underlying gills were such as swimming. Thus, we measured the follow- elevated and secured in this position with umbili- ing: (i) the cardiorespiratory physiology (oxygen cal tape, before a small 5–7 mm incision was consumption and cardiac performance) of adult, made at the base of the junction between the sec- 10 °C acclimated, cod with varying severities of ond and third gill arches. The ventral aorta was L. morhua infection when acutely exposed to then located and cleared of the surrounding con- increasing temperatures up to their critical thermal nective tissue by careful dissection, and the flow maximum (CTmax); and (ii) oxygen consumption probe was placed around the ventral aorta anterior in this same population of cod when they were to the intact pericardium. Finally, the cable from subjected to two consecutive critical swimming the flow probe was secured to the skin using 1-0 speed (Ucrit) tests. silk at positions immediately posterior to the oper- culum, just above the lateral line behind the left pectoral fin, and in between the first and second Materials and methods dorsal fin. A PE50 (Clay Adams Inc.) cannula, with a 45-degree bend approximately 2 cm Experimental animals from its tip, was also sutured at the upper All experiments were carried out in accordance edge of the opercular cavity for measurements with the guidelines of the Canadian Council on of ventilation frequency. This cannula was then Animal Care and approved by the Institutional secured to the skin with 1-0 silk sutures using Animal Care committee of Memorial University the same attachment points as for the flow of Newfoundland (protocol number 11–25 KG) probe cable. Ó 2015 John Wiley & Sons Ltd 2 Journal of Fish Diseases 2015 M D Powell & A K Gamperl Effects of Loma morhua infection Following surgery, the fish were transferred to transducer. This transducer was calibrated daily 6.5-L tubular clear acrylic respirometers supplied against a static water column. Signals from the with flush and recirculating pumps (EHEIM Transonic flow meter and pressure transducers GmbH & Co. KG) and left to recover overnight were amplified and filtered using a data acquisi- (typically 20–24 h) with the pumps in the ‘flush’ tion system (MP100A-CE; BIOPAC Systems, mode. These respirometers were submerged in a Inc.) and universal interface module (UIM100C; ‘water table’ supplied with temperature-controlled BIOPAC Systems Inc.) connected to a laptop â (10 °C), and aerated, sea water from a large computer running AcqKnowledge software (ver- (~300 L) reservoir; the temperature in the reser- sion 3.8.2; BIOPAC Systems, Inc.). As Transonic voir controlled using a custom-designed heater/ flow probes are temperature sensitive, and the chiller (Technical Services, Memorial University characteristic of this relationship varies between of Newfoundland). probes, the temperature–flow relationship for each probe was measured at different flow rates (5– À Experimental protocol. To begin the experiment, 25 mL min 1) over the temperature range used cardiac output Q, ventilation frequency (Vf) and in the experiment using Transonic calibration oxygen consumption (MO2) were measured at tubing, a high precision peristaltic pump, a foam- 10.0 °C. The fish were then challenged with an lined chamber, and saline with a haematocrit of À increase in temperature of 2 °Ch 1 until loss of 15–20%. These relationships were then used to equilibrium [i.e. they reached their critical thermal correct in vivo flow rates (Q) at various tempera- maximum (CTMax)], with cardiorespiratory tures.