Morphological diversity of Paramoeba perurans trophozoites and their interaction with Atlantic salmon, Salmo salar L., gills

Abstract

Amoebic gill disease (AGD) caused by the ectoparasite Paramoeba perurans affects several cultured marine fish species worldwide. In this study, the morphology and ultrastructure of P. perurans in vitro and in vivo was investigated using scanning and transmission electron microscopy (SEM and TEM, respectively). Amoebae cultures contained several different morphologies ranging from a distinct rounded cell structure and polymorphic cells with pseudopodia of different lengths and shapes. SEM studies of the gills of AGD-affected Atlantic salmon, Salmo salar L., revealed the presence of enlarged swellings in affected gill filaments and fusion of adjacent lamellae. Spherical amoebae appeared to embed within the epithelium, and subsequently leave hemispherical indentations with visible fenestrations in the basolateral surface following their departure. These fenestrated structures corresponded to the presence of pseudopodia which could be seen by TEM to penetrate into the epithelium. The membrane-membrane interface contained an amorphous and slightly fibrous matrix. This suggests the existence of cellular glycocalyces and a role for extracellular products in mediating pathological changes in amoebic gill disease.

Generation of Paramoeba perurans clonal cultures using flow cytometry and confirmation of virulence

“…Nielsen et al . () reported the presence of an amorphous matrix at the P. perurans –host membrane interface which they suggested indicated a role for extracellular products in generating pathology. Comparison of average gill score and average amoebae values, represented by P. perurans qPCR detection values normalized for Salmo salar ELF, representing the amount of mucous sampled, showed a significantly positive relationship between gill score and number of amoebae detected on the gills.…”

Aquatic Parasite Cultures and Their Applications

“…infecting Lates calcarifer [70] and Paramoebae spp. infecting Salmo salar [71]) or when a single parasite species exhibits several different morphologies [56,72]. Parasite cultures provide a means to determine species diversity (e.g.…”

Effect of repeated exposure to AQUI‐S® on the viability and growth of Neoparamoeba perurans

“…In this study, the amoebae were classed as "non-viable" primarily due to the lack of uptake of the Neutral Red vital stain (Repetto, Del Peso, & Zurita, 2008), but morphology was also taken into consideration. Amoebae in which no stain was seen all held the same spherical morphology (Figure 1), characteristic of in vitro cultures with a suboptimal subculturing schedule, suggesting this morphology is a response to overcrowding, lack of nutrients or environmental stressors (Lima, Taylor, & Cook, 2017;Wiik-Nielsen et al, 2016). As the cultures in this study were washed regularly at 4-day intervals, and percentage of non-viable amoebae were higher in cultures with lower amoebae numbers (Figures 2 and 4), it is unlikely that the As previously highlighted, the suspended amoebae in metomidate and MS-222 flasks were in contact with their anaesthetics for substantially longer than required for in vivo anaesthetization.…”

“…This infers that with repeated exposure to AQUI‐S ® , an increased proportion of amoeba remain attached to their substrate; however, current AGD literature offers no hypothesis as to why this effect may be seen. During in vivo challenge experiments, an artificially elongated duration of attachment, during which parasitic amoeba could theoretically spend more time colonizing the gill substrate (Wiik‐Nielsen et al., ) than completing the natural emigration to the surrounding sea water, may lead to an increased level of disease progression and therefore an elevated immune response, which may not be comparable to the speed of disease progression found in the field. Nonetheless, with reported loss of virulence seen in cultured N. perurans possibly due to lack of attachment to gills (Bridle, Davenport, Crosbie, Polinski, & Nowak, ), increased attachment due to the use of isoeugenol‐based anaesthetics may help mitigate this problem, if similar attachment processes are involved.…”

“…AQUI-S â -treated amoebae show rapid growth in total population in vitro from day 0 to 8 followed by a steady decrease in population as seen in the control (Figure 2). When comparing the suspended and attached population data (Figure 3 -Nielsen et al, 2016) than completing the natural emigration to the surrounding sea water, may lead to an increased level of disease progression and therefore an elevated immune response, which may not be comparable to the speed of disease progression found in the field. Nonetheless, with reported loss of virulence seen in cultured N. perurans possibly due to lack of attachment to gills (Bridle, Davenport, Crosbie, Polinski, & Nowak, 2015), increased attachment due to the use of isoeugenolbased anaesthetics may help mitigate this problem, if similar attachment processes are involved.…”