Updating specific PCR primer for detection of Cryptocaryon irritans from reared Larimichthys polyactis

Artificial breeding of small yellow croaker (Larimichthys polyactis) was recently achieved, providing a bright future for its commercial farming. In May 2019, a disease outbreak occurred among small yellow croakers in an aquaculture farm near Xiangshan Bay, charactering by white spots spotted on the surface of fish skin, gills and fins. The parasite was preliminarily identified as Cryptocaryon irritans based on morphological feature of the parasite and the symptoms on fish. However, the previously published specific primer pairs failed to confirm the existence of C. iriitans. Six nucleotides mismatches were discovered after mapping specific forward primer back to targeted gene. Therefore, an updated PCR specific primer was developed within the 9th highly variable region of 18S rRNA gene and conserved in all C. irritans sequences available in GenBank database. The specificity was verified in silico by Primer-BLAST against GenBank nucleotide. Laboratory cultured ciliates (Mesanophrys, Pseudokeronopsis and Uronema) as well as natural microbial community samples collected from sea water and river water was used as negative control to verify the specificity of the primer in situ. Besides, tank transfer method was used to evaluate the treatment of the parasite infection. By tank transfer method, 2.00 ± 0.61 out of 10 fish that already sever infected were successfully survived after 8 days treatment, meanwhile the control group died out at d 6. More loss to the treatment group during first five days was observed and may attribute to the combined effect from infection and stress the recent domesticated fish suffered during rotation. Therefore, tank transfer method was also effective to prevent small yellow croaker from further infection, however the loss of the small yellow croaker suffered from stress during rotation also needs to be carefully concerned. In conclusion, this study reported the first diagnose of C. irritans infection on small yellow croaker, provided updated specific primer to detect C. irritans infection on fish body and reported the effect of tank transfer on small yellow croaker treatment.