Characterisation and aetiology of jelly-like gonad condition (JGC) in the common carp, Cyprinus carpio (L)

Fisheries is the fifth largest food production industry, worth more than $2.2 billion to the Australian economy every year. However, the aquatic ecosystems that support these fisheries are very vulnerable to threats such as invasion by aquatic pest species, which can negatively impact native biodiversity and increase risks to allied industries such as recreational fisheries and aquaculture. The common carp (Cyprinus carpio) is extensively farmed in Asia, as well as in Europe and Middle East. Whilst it is a popular angling fish in Europe, it is considered a significant pest in many countries including North America, Canada, New Zealand and Australia. Overall, carp cause significant impact on aquatic ecosystems by increasing turbidity, changing nutrient composition, altering the macrophyte and macroinvertebrate composition and can quickly become the dominant species after introduction. Immediate control of carp in Australia is required as its distribution has exceeded an area of one million km\(^2\). As a species with high reproductive capability, both aquaculture and pest management of carp could benefit from greater knowledge about the control of reproductive capability. This study aimed to examine a naturally occurring ‘sterile’ condition observed during the routine carp management program in Lake Sorell, Tasmania, Australia where the fish develops a testicular condition without any external symptoms. Occurrence of gonad abnormality in wild fish is rare but has been reported in a number of species including carp. However, the gonadal condition observed in Lake Sorell has many unique characteristics that make it distinguishable from all of the previously studied cases. Morphologically the affected gonad appears ‘jelly like’ and therefore described as Jelly-like Gonad Condition (JGC). The overarching objective of the study was to describe the JGC condition, specifically: 1) determine its characteristics, including prevalence, morphology and histopathology; 2) test relatedness of the condition to select biotic and abiotic factors, and; 3) investigate the expression signature to discern a genetic role in its development. Objectives were addressed by analyses of catch data, gross morphology, histology, milt quality, hormone levels and next generation total transcriptome profiling of JGC and normal carp testes. Gross examination of the gonad revealed multi-nodular fluid filled, clear or semi opaque blisters in JGC affected testicular tissue. Investigation of over 6000 fish indicated the JGC is male specific i.e. it does not occur in female gonadal tissue, with increasing frequency that was related to maturity of the fish. Parallel examinations in the golden galaxiid, short fin eel and brown trout that inhabit the same lacustrine system indicate that the condition is absent in all three species, suggesting a species specificity of the condition. Morphological analysis using ImageJ suggested four distinguishable severity stages—early (stage 1), mild (stage 2), medium (stage 3) and severe (stage 4). Additionally, histology with differential and fluorescent TUNEL staining for each severity stage revealed the early stages (stages 1 and 2) of JGC were marked by an abnormally high number of Sertoli cells (11 fold) coinciding with arrest of spermatogenesis whereas late stages were marked by apoptotic cells. Collectively, data suggested that this fish could be an excellent model for understanding Sertoli cell malignancy and testicular cell apoptosis. Regression modelling using 4,594 fish caught between 2013-2017 indicated a significant relationship of the JGC occurrence to body weight, fork length and fishing year. Oneway ANOVA followed by Tukey’s HSD was used to confirm the accelerated growth of JGC fish compared to normal male, female and immature fish. However, no relationship of the condition was associated with month of capture, fish strain (i.e. scale type) or capture location. Growth analysis of mark-recaptured fish indicated that this enhanced growth is triggered post onset of JGC. Quantitative and qualitative assessment of JGC and normal milt using spermatocrit and Bengal rose staining followed by light and phase contrast microscopy was undertaken. Briefly, quantity and quality of spermatozoa dropped drastically with the advancing condition. Significantly, a higher number of sperm abnormalities (e.g. coiled tail sperm, broken tail sperm) was associated with advanced JGC. Similarly, analysis of the normal and JGC milt using computer assisted sperm analysis (CASA) indicated that motility, activity time and velocity parameters (e.g. VCL, VAP, VSL) drop significantly in the advanced condition. Collectively data suggested that JGC fish are near sterile, particularly in the advanced conditions. Hormonal analysis using ELISA confirmed the level of circulating 11-Keto-testosterone (11-KT) in JGC fish was significantly (p<0.05) lower than in unaffected males. However, the Luteinising hormone (LH) level in primary JGC fish (Stage 1-3) was similar to unaffected males and females, yet significantly higher in Stage 4. While a lower level of 11-KT supports the dysfunctionality of the testis, a higher level of LH in extremely affected JGC fish indicates the feedback regulation of LH by 11-KT. Transcriptome analyses using next generation total RNA sequencing (RNAseq) followed by differential expression analysis revealed that 7,129 genes are differentially expressed between JGC and control testis, of which 40 genes were found to have central roles in development and/ or regulation of the condition. BLAST analyses of the 40 candidate genes indicated that many (n=26) of these are novel to date. Pathway analysis of the differentially expressed genes revealed that 130 pathways including chromosome separation, cell morphogenesis, nucleic acid activity, spermatogenesis, gonad development, steroid production and apoptosis were significantly altered in the JGC testis. However, no immune pathway/s were enriched. Collectively, RNAseq analysis followed by the pathway investigation of the differentially expressed genes discovered the key pathways that are responsible for the large cellular changes in JGC tissue observed in histology. It appears that this sterile condition is unique but shares key features with human nongerm cell testicular cancer. For example, the initial uncontrolled proliferation of Sertoli cells with upregulation of many cancer markers (e.g. cx43, fbox32, sall4) suggests that the condition is likely triggered by a malignant pathway. Observed non-activation of immune system process with microbiological investigation supports the hypothesis of non-pathogenic origin. Although, few cases of Sertoli cell malignancy have been reported previously, observed mass apoptosis of all testicular cell resulting in a sterile animal is unique making JGC carp a potential model to study testicular non-germ cell cancer, apoptosis, sterility and related reproductive processes. Moreover, high prevalence of this naturally occurring condition should permit comparative evaluation between individuals allowing rigorous testing, including therapies. In conclusion, this study suggests that JGC carp are near sterile and exhibit enhanced somatic growth compared with unaffected cohorts. JGC carp can be used as sterile Judas fish in carp control programs such as the one in Tasmania and may have excellent potential for use in aquaculture. Moreover, future characterization of the ‘novel’ genes (n=26) associated with the condition may lead to the discovery of unique master regulator of testicular cancer, Sertoli cell specific marker, sterility or fertility that are highly relevant to human health. The sterility associated gene/s could also provide opportunities for developing species specific genetic carp eradication options.

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