Post Mortem Muscle Softness in the Spotted Seatrout Cynoscion nebulosus: Effect of the Myxozoan parasite Kudoa inornata

Candice Alge, Eric McElroy, and Isaure de Buron Department of Biology, College of Charleston, Charleston, SC 29424

Abstract
Kudoa inornata is a myxozoan that infects the skeletal muscles of the spotted seatrout, Cynoscion nebulosus. A previous study in our laboratory indicated that infected wild fish displayed increased post mortem flesh softness when compared to non-infected fish (mariculture raised). We hypothesized that infection by K. inornata was at the origin of the difference in flesh softness observed. Plasmodium density, plasmodium area, and spore density were determined from biopsies of seatrout previously tested for muscle softness (3 biopsies per fish, N=33). Results indicated that spore density was positively correlated with plasmodium density and area. Although muscle softness was not correlated with spore and plasmodia densities, data suggest that the larger the plasmodia, the softer the muscle is. Hence, the older the infection in a fish, the higher the post mortem muscle softness may be. Since old infection may occur in larger (older) fish, this may be of concern to anglers.

Methods and Materials

Materials 11 fish were examined 3 biopsies per fish (Fig. 5)- each was weighed Each biopsy was taken at a different post mortem time point: 24, 72, and 144 hours Plasmodium Density Standardized squashes of each biopsies were made Squashes were observed via light microscopy and counted (Fig. 6) Plasmodium Area Pictures of first 10 plasmodia per biopsy were taken Area was determined using NIH program Image J (Fig. 7) Spore density Biopsies were trypsinized Spores were counted via hemocytometer (Fig. 8) Total number of spores per biopsy was inferred

Results
A significant positive correlation was found between spore density and plasmodium density, R2= 0.34, p<0.003 (Fig. 8). A significant positive correlation was found between spore density and plasmodium area, R2=0.36, p<0.0002 (Fig. 9). A positive correlation between plasmodium area and muscle softness at 24 hours post mortem was found to be on the verge of significance, R2= 0.28, p=0.059 (Fig. 10).

Introduction
Kudoa inornata Spores of K. inornata (Fig.1) are organized into plasmodia (Fig. 2). The plasmodium infects the skeletal muscle of Cynoscion nebulosus1 (the spotted seatrout) (Fig. 3). Previous studies show that Kudoa inornata is associated with post mortem muscle softness2 (Fig. 4 ).

Fig. 9 Log spore density and Log plasmodium density

Hypothesis Post mortem muscle softness of infected muscle is associated with spore density, plasmodium density, and plasmodium area.

Fig. 5 Biopsies taken from seatrout filet Fig. 10 Log spore density and Log plasmodium area 5m Fig. 1 Spores of Kudoa inornata in muscle squash Fig. 2 Plasmodium in skeletal muscle

Fig. 11 Log plasmodium area compared to log force at 24, 72, and 144 hours. Fig. 3 Cynoscion nebulosus Fig. 6 Skeletal muscle squash with plasmodium under light microscopy Fig. 7 Plasmodium outlined to determine area Individual spores

Conclusions
Muscle softness is observed in post-mortem infected muscle. Spore density is correlated to both plasmodium density and plasmodium area. Number of spores, plasmodia do not seem to be associated with post-mortem muscle softness but plasmodium size may be.

Figure 4. Maximum force readings over time for infected and uninfected fish Parasite Effect: F1,60 = 5.4, p =0.023 Time Effect: F2,327 = 32.6, p < 0.001 (Sunquist et. al, 2013)

Fig. 8 Spores on a hemocytometer

Analysis of replicate samples need to be observed to determine if plasmodium area at 24 hours post mortem is associated with muscle softness.

Contact
Candice Alge Department of Biology, College of Charleston Email: candicealge@gmail.com Website:

References
1. Dykov, I., de Buron , I., Fiala, I., & Roumillat, W. A. (2009). Kudoa inornata sp. n. (Myxosporea: Multivalvulida) from the skeletal muscles of Cynoscion nebulosus (Teleostei: Sciaenidae). FOLIA PARASITOLOGICA, 91-98.

2. Sunquist,L., McElroy, E., & de Buron, I. (2013) Temperature mediated myoliquefaction: The effect of a myxozoan parasite Kudoa inornata on the spotted seatrout (Cynoscion nebulosus), College of Charleston, SC