Distribution and evidence of co-infection of the two microsporidian parasites Astathelohania contejeani and Nosema austropotamobii in Austropotamobius pallipes complex in Northern and Central Italy

Abstract

Austropotamobius pallipes complex is an endangered freshwater crayfish species in Europe and the assessment of the health status of its wild populations is essential for conservation purposes. The two microsporidia Astathelohania contejeani and Nosema austropotamobii have been reported to cause in A. pallipes complex a chronic parasitic infection, known as ‘porcelain disease’, which reduces population fitness and leads the host to death. Due to the similar macroscopic signs produced, molecular biology analyses are required to unambiguously distinguish between these microsporidia. Focusing on A. pallipes complex populations located in Northern and Central Italy, the present study provides an evaluation of prevalence and distribution of A. contejeani and N. austropotamobii, and investigates the variables affecting the probability of detecting infected specimens during a survey (e.g. sex, crayfish density, longitude). Microsporidia were identified in 12 populations among the 42 monitored from 2011 to 2017, with an average prevalence of 3.12% for A. contejeani and 3.60% for N. austropotamobii, the latter being reported in a wider area than previously documented (from Lombardy to Liguria Regions). Notably, crayfish co-infected by both microsporidia were also detected in 4 populations. Moreover, it was observed that the probability of detecting a crayfish with a microsporidian infection significantly increased eastwards in the studied area, especially for N. austropotamobii. Our distribution map for microsporidiosis, combined with molecular screening, will be useful for planning breeding and translocation efforts for A. pallipes complex populations.

Keywords: detection probability; parasite; porcelain disease; white-clawed crayfish.

Graphical abstract

Figure 1.

Distribution of microsporidia, detected by molecular assays, over the evaluated areas. Pies indicate the surveyed populations: grey sections indicate the presence of Astathelohania contejeani and black sections report the presence of Nosema austropotamobii. White pies represent white-clawed populations where microsporidia were not detected. Numbers refer to the watercourses-ID according to Table 1.

Figure 2.

Macroscopic and microscopic detection of microsporidiosis in A. pallipes complex. (A) macroscopic appearance of microsporidian infection (porcelain disease) in heavily affected A. pallipes complex, whitish and opaque ventral skeletal musculature of the pleon and whitish discoloration of the chelipeds. (B–F) A. contejeani infection. (B) microscopic appearance of heavily affected skeletal muscle fibres with proliferation of A. contejeani and inflammatory reaction with haemocytic infiltration in muscle fibres (arrows), H-E 4×. (C) haemocytic infiltration with melanisation of affected muscle fibres (arrows) surrounded by other A. contejeani-infected fibres without inflammatory reaction (arrowheads), H-E 20×. (D) A. contejeani infection of the ventral nerve cord, microsporidian spores (arrow) between the pyrenophores of the ganglion (∗), affected skeletal muscle fibres (arrowhead), H-E, 10×. (E) A. contejeani infection of the cardiac musculature, affected cardiac muscle fibres (arrows), H-E 4×. (F) A. contejeani infection of hind gut (∗); the intestinal musculature appears affected (arrows), the surrounding skeletal musculature of the abdomen is also affected (arrowheads), Giemsa 4×. (G–I) N. austropotamobii infection. (G) Nosema austropotamobii severe infection of abdominal skeletal muscle fibres (arrowheads), nerve cord ganglion (∗) not affected, H-E 4×. (H) unaffected cardiac muscle fibres of N. austropotamobii affected specimen, cardiac lumen (∗), H-E 4×. (I) unaffected intestinal muscle fibres of N. austropotamobii affected specimen, infected skeletal muscle fibres (arrowheads), hind gut lumen (∗), H-E 10×. (J–L) N. austropotamobii – A. contejani co-infection. (J) Inflammatory reaction with haemocytic infiltration (arrow) in skeletal muscle fibres of co-infected N. austropotamobii – A. contejeani specimen, affected fibres with no inflammation (arrowheads) H-E 20×. (K) limited amount of microsporidian spores (arrows) in cardiac muscle fibres of co-infected N. austropotamobii – A. contejeani specimen, H-E 60×. (L) unaffected intestinal muscle fibres of co-infected N. austropotamobii – A. contejeani specimen, infected skeletal muscle fibres (arrowheads), hind gut lumen (∗), longitudinal section H-E 10×.

Figure 3.

GLMM predicted probabilities of collecting suspected individuals (see Materials and Methods for details) during a single survey along a longitudinal cline. The thick line represents the predicted value for a site, whereas the grey area represents the 95% interval of variation of sites around prediction. Increasing values in the x-axis corresponds to a West-to-East cline.

Figure 4.

GLMM predicted probabilities of infection by A. contejeani (left panel) and N. austropotamobii (right panel) along a longitudinal cline. The thick line and grey area as in Fig. 3. Increasing values in the x-axis corresponds to a West-to-East cline.