Coping with climate change: Phenotypic plasticity in an imperilled freshwater fish in response to elevated water temperature

1. Climate change has emerged as an increasingly important threat to freshwater systems. To cope with rapidly changing thermal regimes, freshwater fishes must either relocate or adjust through genetic adaptation and/or phenotypic plasticity. Short-term responses to elevated water temperature have been well studied in freshwater fishes; however, far less is understood about change induced by long-term exposure. Furthermore, few studies have investigated the effects of temperature on already imperilled species, which may be more sensitive to environmental change.
2. This study investigated the effects of rearing temperature on critical thermal maximum (CT_max), agitation temperature (T_ag, temperature at which fish show behavioural signs of thermal stress) and gill size in pugnose shiner, Notropis anogenus, a threatened species in Canada. Juvenile pugnose shiner were reared for 4 months across five different ecologically relevant temperatures. CT_max and T_ag were measured under normoxia and acute exposure to hypoxia to test for oxygen sensitivity of the upper thermal limits in this species.
3. CT_max increased with elevated water temperature. T_ag also increased with rearing temperature and occurred, on average, 4.3°C above acclimation temperatures. The CT_max and T_ag were lower when fish were exposed acutely to hypoxia. Interestingly, gill size (e.g. total gill filament length) increased with rearing temperature, which may increase oxygen uptake capacity and support increased metabolic demands of warmer waters.
4. Overall, pugnose shiner show plasticity in several traits in response to long-term exposure to elevated water temperature that may facilitate persistence in warmer waters. However, acute hypoxia exposure reduced thermal tolerance, stressing the importance of evaluating interactive effects of multiple stressors.
5. Identifying source populations of pugnose shiner with greater thermal tolerance or implementing captive breeding under higher temperature regimes may improve the success of re-introduction efforts in the face of climate change, but the consequences to fitness of increased thermal tolerance should be examined.

     

Differential responses of body shape to local and reach scale stream flow in two freshwater fish species

Stream flow is recognised as an important influence on the physical traits, distribution and diversity of stream fishes. However, measures of ‘stream flow’ are not consistent among studies and can refer to variables characterising microhabitat (e.g. flow velocity) or reach‐level (e.g. discharge) parameters. Because of the ambiguity associated with the term ‘stream flow’, the differential effect of these two aspects of local stream hydrology on freshwater fishes is unclear. Using two frequently sympatric freshwater fish species, Etheostoma nigrum and Pimephales notatus, we tested for correlations between population‐level body shape and two measures of stream flow, microhabitat flow velocity (m/s) and reach‐level discharge (m³·s^?1). Etheostoma nigrum body shape was correlated with flow velocity, while P. notatus body shape was correlated with mean annual discharge. The respective correlations are potentially manifestations of behavioural differences among the two species. Etheostoma nigrum is a benthic specialist rarely venturing far from its associated microhabitats. Pimephales notatus is a habitat generalist and uses many different microhabitats but is unable to escape the overall discharge pressures of a particular stream. These results demonstrate the importance of considering the spatial scale of variables when predicting phenotype–environment associations.     

Climate change and fish culture in Patagonia: present situation and perspectives

Temperature increment is one of the most visible aspects of climate change. Reservoirs in northern Patagonia are the main location for rainbow trout cage aquaculture activities in Argentina and here, surface water temperature shows an increment that despite leading towards better growth rates, also provokes lack of ovulation, atresia and a partial or total absence of spawning during the breeding season. During the early life history, low survival rates at hatching and first feeding are also observed. The increase in market size (>2 kg) has changed management practices, minimizing the difference between brood stock and individuals destined for consumption. Thus, the present situation differs deeply from carrying capacities forecasted few years ago and this analysis attempts to contribute to management policies including the idea of adaptation to climate change in the making of decisions.     

Modelling the potential distribution of two tropical freshwater fish species under climate change scenarios

1. Climate change is causing shifts in the distribution patterns of freshwater fish at various spatio-temporal scales. Tropical freshwater fish are vulnerable, especially in areas where a high impact of climate change is predicted; thus, there is an increasing need to predict these shifts to determine conservation and adaptation strategies.
2. Ecological niche models offer a reliable way to predict the effects of climate change on species distribution. Potential shifts in the distribution of tropical fish were tested under two scenarios (4.5 – moderate and 8.5 – extreme) with three general circulation models for years 2050 and 2070 using maximum entropy software using as models two predatory species – the tropical gar Atractosteus tropicus and the giant cichlid Petenia splendida.
3. The potential distribution of both species was associated with warm and humid–sub-humid conditions. Future projections showed a higher availability of suitable areas for both species resulting from the expansion of warmer conditions in the middle and upper basins of the Central American mountain range and centre of the Yucatan Peninsula.
4. Ecological niche models of keystone or umbrella species such as A. tropicus and P. splendida could be useful to support conservation plans of protected areas. The potential distribution of both species covers areas of high suitability including six important biosphere reserves in Mexico, three protected areas in Guatemala and part of the Mesoamerican biological corridor.
5. Despite the potential expansion of the present distribution range suggested by the models, it is important to consider the biological and ecological requirements of the species and the ecological implications of these potential shifts in distribution. Both scenarios could have several implications at genetic, population, and ecosystem levels.

     

Role of exogenous glutathione in teleost fish and its effects on antioxidant defense responses in rainbow trout exposed to 3,3',4,4'-tetrachlorobiphenyl

Intraperitoneal injection of glutathione (GSH) is shown to effectively increase tissue glutathione content in two teleost species, the rainbow trout and the American eel. GSH uptake into teleost tissues is a distinct piscine feature of GSH biochemistry compared with mammalian species where little or not uptake occurs of exogenously added GSH. Enhancement and depletion of tissue GSH status was used as a tool to examine the effects of altered GSH content on other cellular antioxidants in 3,3,4,4-tetrachlorobiphenyl (TCB) exposed rainbow trout. -Tocopherol content was unchanged and thiobarbituric acid reactive substances (TBARS), an indicator of lipid peroxidation, changed little in liver and muscle of trout exposed to TCB and the various GSH treatments. However, -tocopherol levels in muscle and superoxide dismutase activities in the various tissues were significantly higher in controls and TCB-saline treated fish than in GSH deficient and lipoate supplemented TCB exposed trout. GSH status thus appears to affect some cellular antioxidant defenses in TCB exposed trout.     

Growth and feed utilization of large size rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) reared in freshwater: diet and species effects, and responses over time

Four diets differing in crude protein/crude lipid concentrations (CP/CL), 570/200, 510/220, 460/240, 430/260 (g kg^?1 dry diet) were fed to near‐satiety to rainbow trout (initial body weight, IBW = 268 g, domestic strain) and Atlantic salmon (IBW = 456 g, wild strain) for 308 days to determine the effect of diets, species and fish size on efficiency of feed, nitrogen (N) and energy utilization. Within species, weight gain, feed efficiency (FE), and energy retention efficiency (ERE, E gain/E intake) were not affected by diet (P < 0.05). N retention efficiency (NRE, N gain/N intake) increased linearly (P < 0.05) with decreasing CP/CL for both species. NRE and FE were higher for salmon compared with trout. Whole body CP of salmon was significantly (P < 0.05) higher, and lipid significantly (P < 0.05) lower, than those of trout. There was a significant (P < 0.05) linear decrease in FE as fish grew, regardless of diet and species. NRE linearly decreased (P < 0.0001) and lipid to protein deposition ratio (LD/PD) increased (P < 0.05) as trout grew. NRE linearly increased (P < 0.05) while ERE and LD/PD linearly decreased (P < 0.05) as salmon grew. Salmon appeared to rely to a greater extent on non‐protein energy‐yielding nutrients to support cost of growth than did trout.     

Cardiac,ventilatory and metabolic responses of two ecologically dissimilar species of fish to waterborne cyanide

Changes in heart rate, ventilatory activity and oxygen consumption were determined in trout (Salmo gairdneri) and brown bullhead catfish (Ictalurus nebulosus) during exposure to a steadily increasing concentration of waterborne cyanide selected to produce death in 8–9 hours for each species. The lethal cyanide concentration for the bullheads was an order of magnitude higher than for trout. Trout developed an immediate and gradually increasing bradycardia throughout the exposure period. Cyanide produced tachycardia in the bullhead followed by a gradual onset of bradycardia as the concentration of cyanide was raised. Pericardial injection of atropine (a muscarinic cholinergic antagonist) indicated that bradycardia in the trout was due initially to increased vagal tone but later due to the direct effect of cyanide on the heart. Hyperventilation in the trout persisted throughout the exposure period, although the rate and amplitude fluctuated and was variable between individual fish. During the last hour of exposure (highest cyanide concentration), ventilation was characterized by rapid, shallow breaths followed by a sudden respiratory arrest. The bullheads exhibited hyperventilation during the first 3 hours of exposure followed by a gradual, linear drop in ventilation rate and amplitude until death occurred. Cardiac and ventilatory responses in both species were attributed to stimulation of central and peripheral chemoreceptors by cyanide. Evidence is presented which suggests the initial response in the bullheads was due, at least in part, to gustatory stimulation by the cyanide. Oxygen consumption of the trout remained above pre-exposure levels for the majority of the test period. Oxygen consumption in the bullhead paralleled the changes in heart and ventilatory rates. Whole-body lactate levels of fingerlings of both species during cyanide exposure were measured to estimate the extent of anaerobiosis. Whole-body lactate levels were much greater in the bullheads than the trout, indicating a higher capacity for anaerobiosis, possibly due to a greater fuel supply. Overall, the trout responded to cyanide in a manner similar to that produced by environmental hypoxia whereas the bullheads experienced a gustatory stimulus which masked the hypoxia-like response.     

Field study indicating susceptibility differences between salmonid species and their lineages to proliferative kidney disease

Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) is the causative agent of proliferative kidney disease (PKD), which affects both wild and farmed salmonid fish. The objective of this study was to outline differences in susceptibility to PKD in different salmonid species, hybrids and breeding lineages. Susceptibility to T. bryosalmonae infection was established based on cumulative mortality, pathological findings and detection of T. bryosalmonae in the kidney using immunohistochemistry and molecular methods. Determination of pure and hybrid individuals of different species in the genus Salvelinus, and dissimilarity of rainbow trout lineages, was performed using traditional polymerase chain reaction (PCR) and microsatellite analyses. Rainbow trout displayed higher disease severity compared with brook trout and Alsatian charr. Moreover, the results indicated differences in infection susceptibility, not only among different salmonid species but also among different lineages of charr and rainbow trout. Our study indicated that some salmonid species and even different lineages of the same species are more suitable for farming under PKD pressure.     

Primary responses of rainbow trout to dietary phosphorus concentrations

A series of experiments was conducted to research sensitive indicators for phosphorus status of fish using post‐juvenile rainbow trout. Fish were fed up to 24 days with semipurified diets that varied in phosphorus content. Concentrations of glucose‐6‐phosphate, ATP, creatine phosphate, glucose, total lipids and total cholesterol in blood or skeletal muscle were relatively unchanged by the 24 days of dietary phosphorus restriction. Inorganic phosphorus and ATP levels in the blood, however, correlated significantly and positively. Inorganic phosphorus levels in plasma and urine were significantly lower in fish fed phosphorus‐deficient diets than phosphorus‐supplemented diets. Urinary phosphorus excretion peaked 6–10 h after feeding fish with diets containing potassium phosphate. Fish receiving either commercial feeds or experimental diets containing phosphorus as fish bone excreted trace amounts of phosphorus in the urine. Faecal content of phosphorus significantly increased when the diet containing potassium phosphate was supplemented with calcium carbonate. Urinary phosphorus concentration was found to be a rapid and sensitive indicator for dietary intake of phosphorus and probably phosphorus status of the fish, and had an advantage over conventional response variables in estimating dietary phosphorus requirement especially with large commercial‐size fish.     

Added value from an added chromosome: Potential of producing large fillet fish from autumn to spring with triploid rainbow trout,Oncorhynchus mykiss

Three‐summers‐old all‐female triploid and diploid rainbow trout were compared after one on‐growing season in sea net cages. Slaughter traits of round weight, gutted weight, fillet weight, carcass% and fillet% were measured at three times in November 2017, January and April 2018. The triploid group had lower daily growth coefficient mean (4.25) and higher feed conversion ratio (1.18) than diploids (4.48 and 1.05, respectively) during on‐growing (June–November). In November, no difference of means was found between mature or immature diploids and triploids for any of the weight traits when the effect of vertebrae defects was statistically removed. However, the triploids had attained higher means than mature or immature diploids in gutted and fillet weight by January, suggesting that the loss of muscle mass during early winter was lower in triploids. Sexually maturing diploids (46%) had lower slaughter yield means compared to triploids or immature diploids at each measurement time, and these differences also increased during overwintering. Instead, the means of yield traits remained similar between the triploid and immature diploid groups through the winter. Likewise, fillet redness remained at equally high level in triploids and immature diploids, whereas in maturing diploids this attribute decreased substantially during overwintering. The triploid group had a higher incidence of vertebral defects (12.0%) than diploids (5.3%). The present results demonstrate the potential of triploid trout in producing large‐sized (>2 kg) fillet fish until spring markets. However, more detailed investigations are needed, particularly regarding the animal health and growth efficiency in triploids, relative to their diploid counterparts.     

New salmonid hosts for Tenacibaculum species: Expansion of tenacibaculosis in Chilean aquaculture

The success and sustainability of Chilean aquaculture largely depends on the control of endemic and emerging pathogens, including several species of the genus Tenacibaculum. Tenacibaculum dicentrarchi and “Tenacibaculum finnmarkense” have been detected and confirmed in Chilean Atlantic salmon (Salmo salar). However, no outbreaks of tenacibaculosis in rainbow trout (Oncorhynchus mykiss) or coho salmon (Oncorhynchus kisutch) have been reported, either in Chile or globally. The aims of this study were to determine whether the mortalities recorded for rainbow trout and coho salmon from five marine fish farms located in the Los Lagos, Aysén, and Magallanes Regions could be caused by Tenacibaculum spp. The diseased fish exhibited cutaneous haemorrhages, tail and peduncle rots, and damage on the mouth and tongue. Microbiological analysis of infected external tissues yielded 13 bacterial isolates. The isolates were identified as members of the genus Tenacibaculum through biochemical analysis (e.g. Gram-stain negative, straight rods, filamentous cells and motile by gliding), but differences existed in biochemical results, making species-level identification through biomolecular tools essential. The 16S rRNA analysis found that the majority of isolates were more closely related to “T. finnmarkense” than T. dicentrarchi, while the phylogenetic trees resulting from multilocus sequence data recovered the four main clades (clades I to IV) identified by Olsen et al. (2017, Veterinary Microbiology, 205, 39). This is the first documented occurrence of clinical tenacibaculosis in farmed rainbow trout and coho salmon globally, and it extends the known host distribution of this pathogen in Chile. Moreover, we confirm the presence of Tenacibaculum species in the Chilean Patagonia. These findings highlight the importance of establishing preventative measures to minimize the spread of this disease within the Chilean marine aquaculture industry, as well as the need for monitoring initiatives worldwide in these farmed fish species.     

Replacement of fish meal with two fermented soybean meals in diets for rainbow trout (Oncorhynchus mykiss)

The present study investigated the effect of fish meal (FM) replacement with fermented soybean meal (FSM) on growth and feed utilization of rainbow trout. Two FSM products, FSM1 (more fermentation with more small peptide and acid than FSM2) and FSM2 were used to replace 20%, 40% and 60% of FM in control diet (250 g/kg FM), respectively (FSM1‐20, FSM1‐40, FSM1‐60, FSM2‐20, FSM2‐40 and FSM2‐60). Then the seven diets were fed to rainbow trout (18.1 g) for 8 weeks. Weight gain (WG), feed conversion ratio (FCR) and digestibility of crude protein and dry matter showed no significant difference among the groups of FSM1‐20, FSM1‐40, FSM2‐20, FSM2‐40 and the control, but WG significantly decreased and FCR increased when 60% FM was replaced by both FSMs (p < .05). The replacement of 40%, 60% FM resulted in lower villus height than the control (p < .05), and intestinal protease activity was lower in FSM2‐40, FSM2‐60 and FSM1‐60 groups than the control group (p < .05). In addition, the activity of alkaline phosphatase and superoxide dismutase increased with increasing levels of FSM (p < .05). In conclusion, dietary fish meal could be replaced by 40% with both FSMs without adverse effects on growth and feed utilization of rainbow trout based on an eight weeks feeding trial.     

Relationship between stream temperature, thermal refugia and rainbow trout Oncorhynchus mykiss abundance in arid-land streams in the northwestern United States

Abstract – Warm stream temperatures may effectively limit the distribution and abundance of Pacific salmon Oncorhynchus spp. in streams. The role of cold thermal refugia created by upwelling groundwater in mediating this effect has been hypothesized but not quantitatively described. Between June 21 and September 15, 1994, rainbow trout O. mykiss abundance within 12 northeast Oregon (USA) stream reaches was inversely correlated with mean ambient maximum stream temperatures ( r =−0.7, P <0.05). Some rainbow trout used thermal refugia (1–10 m² surface area) that were on average 3–8°C colder than ambient stream temperatures. Within the warmest reaches, high ambient stream temperatures (>22°C) persisted from mid-June through August, and on average 10–40% of rainbow trout were observed within thermal refugia during periods of midday maximum stream temperatures. Frequency of cold-water patches within reaches was not significantly associated with rainbow trout density after accounting for the influence of ambient stream temperature ( P =0.06; extra sum of squares F -test). Given prolonged high ambient stream temperatures in some reaches, the thermal refugia available in the streams we examined may be too small and too infrequent to sustain high densities of rainbow trout. However, these refugia could allow some rainbow trout to persist, although at low densities, in warm stream reaches.     

The influence of dietary fish oil replacement with mixture of vegetable oils on reproductive performance,immune responses and dynamic of fatty acids during embryogenesis in Oncorhynchus mykiss

A 90‐day feeding trial was carried out to examine the influence of fish oil (FO) substitution with blends of vegetable oils (VOs) on reproductive efficiency of female brooders and fluctuation in fatty acid (FA) profile of embryos in Oncorhynchus mykiss. A basal diet was formulated in which 20% (80FO/20VO), 50% (50FO/50VO), 75% (25FO/75VO) and 100% (100VO) of FO were replaced by mixture of VO. Reproductive performance of brooders was not affected by drastic alternations in FA profile of diets. The level of saturated and monounsaturated FAs (MUFAs) significantly increased, whereas the levels of long‐chain polyunsaturated FAs (LC‐PUFAs), mainly docosahexaenoic acid, profoundly decreased during embryogenesis. The concentrations of MUFA and n?6 PUFA increased in the eggs with increasing the incorporation of VO mixture in diets; however, the concentration of LC‐PUFA and n?3/n?6 PUFA ratio decreased. Haematological parameters and humoral immune responses including total immunoglobulin content, lysozyme and alternative complement pathway activities in brooders fed with the experimental diets did not statically different. In summary, incorporating mixture of various VO sources especially linseed and sunflower oils as good sources of α‐linolenic and linoleic acids, respectively, along with low levels of residual fat from fish meal in diet suggested a good strategy for providing the appropriate essential FA requirements of O. mykiss brooders for their successful reproduction.     

Physiological and behavioural responses to hypoxia in an invasive freshwater fish species and a native competitor

The spread of invasive species is one of the major environmental concerns which can have negative effects on biodiversity. While several life history traits have been identified as being important for increasing the invasiveness of introduced species, the physiological factors that allow certain species to become successful invaders remain poorly understood. It has been speculated that good invaders are thriving in disturbed environments. In unfavourable conditions, as during hypoxic events, invasive species might be better adapted in their physiological and behavioural responses towards this stressor. We compared physiological and behavioural traits between two freshwater fish species: the European bullhead (Cottus gobio), an invasive fish species in Scotland, and its native competitor the stone loach (Barbatula barbatula) over different dissolved oxygen concentrations (DO). Contrary to expectations, bullhead displayed a reduced hypoxia tolerance as compared to stone loach, indicated by a higher threshold (P_crit) for the maintenance of standard metabolism. Avoidance behaviour during progressive hypoxia was similar between bullhead and stone loach. When given a choice between an open normoxic zone and a shelter located in hypoxia, both species spent most of their time hiding under the shelter in hypoxic conditions (bullhead: 100%; stone loach: 93.93%–99.73%), although stone loach showed brief excursions into normoxic conditions under 25% DO level. These results suggest that stone loach might be more resistant to hypoxia as compared to bullhead, and thus that increased hypoxia tolerance is likely not a trait by which bullhead have been able to expand their range within the United Kingdom.     

Use of fluidized bed biofilter and immobilized Rhodopseudomonas palustris for ammonia removal and fish health maintenance in a recirculation aquaculture system

Intensive recirculating aquaculture relies on biofilters to sustain satisfactory water quality in the system. Fluidized bed and immobilized cell technologies were used to remove ammonia from the water and maintain fish health. A high‐rate nitrifying fluidized bed biofilter combined with valveless filter was designed for use in a recirculation aquaculture system (RAS). The suspended solids produced during fish culture could automatically be removed using a valveless filter. Natural porosity with fitting proportion, steady fluidization and expanding rate was chosen as the fluidized carrier. The technology of bacterial separation and cultivation was used. The immobilized Rhodopseudomonas palustris (R. palustris) produced through a biotechnologically embedding medium is suitable for fish and could help prevent diseases. Nitrification was promoted through the selective rearing of nitrobacteria in a fluidized bed biofilter. Water quality was improved using fluidized bed biofilter and immobilized R. palustris in the RAS. In addition, the proposed system was able to reduce costs. Maximum fish load was 45 ± 3 kg m^?3 in the closed recirculating water fish culture system, and water use was reduced by 80–90%. The total ammonia nitrogen removal rate of the technology was 80–95%, and nitrite N removal rate was above 80%.     

Utilization of ascorbate-2-sulfate in fish

Although most vertebrate animals synthesize L-ascorbic acid (C₁), some animal species lack the ability to produce L-gulonolactone oxidase and are thus dependent upon a dietary source of vitamin C. Fish are unique among this latter group in that they store a chemically stable form of vitamin C and appear to metabolize this compound differently from other vitamin C-requiring organisms. Ascorbate-2-sulfate (C₂) contributes to total body stores of ascorbate, but the commonly used assays for ascorbate concentrations in tissues and body fluids do not generally measure C₂. An HPLC assay distinguishes between and measures both C₁ and C₂. Modification of the less exact but commonly used DNPH method can provide adequate data to estimate total vitamers C, C₁, and (by difference) C₂. Since vitamin C is a required component of feed for salmonids, catfish, eels, shrimp and carp, use of C₂ in feed formulation would provide a bioavailable form of ascorbate which is heat and water stable at pH 4–13.     

Influence of spoilage and processing temperature on the quality of marine fish protein sources for salmonids

Press cake meals were prepared from previously frozen herring immediately following thawing and after storage for 8 or 12 days at 2-5°C. Each of the raw sources of herring was subjected to two processing temperatures, 75°C and 100°C, during meal preparation. Also, protein hydrolysates were prepared using ocean perch when fresh or after storage at 2-5°C for 4 or 8 days. Subsequently, each of the three hydrolysates was dried at 85°C or 93°C. In two separate experiments, each of the herring press cake meals and dried perch protein hydrolysates was blended with a reference diet in a 30:70 ratio (test protein source: reference diet). All diets contained 5 g kg^?1 chromic oxide as an indigestible marker. The reference diet and all test diets were provided to satiation to chinook salmon in salt water and rainbow trout in fresh water, with digestibility of organic matter, protein and energy measured by difference. Digestibility of protein was also measured by the pH-stat and dilute pepsin solubility in vitro techniques. The results indicated that variation in processing temperature to a maximum of 100°C had little effect on digestibility of marine fish protein sources. By contrast, raw material storage for 8 days or more at 2-5°C prior to processing was found to reduce organic matter digestibility and sometimes nitrogen digestibility in salmonids. In vitro measures of digestibility were of little help in predicting the nutritive value of the test protein sources. Cadaverine level in herring press cake meal was shown to be a good indicator of spoilage in the raw material.     

Chondroitin AC lyase activity is related to virulence of fish pathogenic Flavobacterium columnare

The virulence of eight Flavobacterium columnare strains was studied to find correlations between several virulence-related factors and virulence. Virulence was tested in vivo using rainbow trout, Oncorhynchus mykiss (Walbaum). Suggested virulence-related factors such as production of the degradative enzyme chondroitin lyase, plasmid occurrence and adhesion capability were studied in vitro. Infection with the four most virulent strains resulted in 95-100% mortality within 114 h. Chondroitin lyase activity was found to be significantly related to the virulence of the strains at 25 degrees C and it was also shown to be temperature-dependent, being higher at 25 degrees C than at 20 degrees C. Virulence was not plasmid associated. The adhesion capability of the strains in vitro varied substantially when tested on crude mucus-coated slides and no statistical relationship between adhesion and virulence was found using this method.