Adult male morphotype variation in giant freshwater prawn (Macrobrachium rosenbergii): Genomic and experimental analysis of factors affecting the male social dominance hierarchy

The giant freshwater prawn (GFP), Macrobrachium rosenbergii (de Man, 1879) is now an international commodity and an important species in culture but one that has the disadvantage of experiencing heterogeneous individual growth (HIG) related to within-family morphotype variation. Adult male GFP differentiate into three morphotypes that develop in sequence in the population from small males (SM) through orange claw males (OC) to dominant blue claw males (BC). Understanding how genetic factors underpin this natural process and how it is mediated in the hierarchical population structure in both wild and cultured stocks is important for improving productivity and profitability of prawn farming. While currently only limited genomic information is available for any crustacean species, including GFP, a number of putative candidate genes have been identified or implicated in social interactions in other crustacean species. In the first study, a transcriptomics approach was applied to identify genes that potentially contribute to the expression of morphotype in adult males. Six different GFP tissues (abdominal muscle, hepatopancreas, stomach, claw, eyestalk and testis/ovary) were screened in a gene expression study applying a transcriptomics approach. Gene expression profiles can potentially identify candidate genes that influence determination of morphotype in adult male GFP populations with relatively high sensitivity and specificity. We identified a number of target genes, both ones reported previously in recent studies and as of now, uncharacterised genes that include: Red pigment concentrating hormone (RPCH); Adipokinetic hormone receptor; Pigment dispersing hormone precursor (PDH); Pheromone and odorant; Visual pigment like receptor; Opsin; Glycoprotein 93; and Lola protein isoform a, all that are associated with visual, olfactory, morph, behaviour, stress and pigmentation functions that will allow us to begin to understand the function of specific genes linked to GFP adult male morphotype. The overall objective was to understand the molecular mechanism(s) that control adult male morphotype determination and to develop strategies that potentially will allow control of this character to be manipulated. In the second study, we performed tank experimental trials on live animals to understand how the social dominance hierarchy in males is mediated (chemical vs visual signals). Visual and chemical cues, in particular, pheromones, are important systems by which animals can communicate. Testing visual stimuli, while restricting chemical cues and the reverse, can lead to an understanding of the influence of how different forms of communication can impact the social dominance hierarchy. Results obtained in the second trial suggest that visual cues play an important role in suppression of transition of subordinate male to dominant male morph as time to first transition of an SM individual to first or second OC male required approximately double the time (10 vs 20 days) when only visual cues were available to SM individuals. Identified candidate genes could either directly or indirectly affect behavioural processes in male GFP individuals, and differential expression of these genes could facilitate adaptation of GFP to changeable environmental conditions. To confirm this, in the final study we selected a set of candidate genes with stable expression patterns and from different tissues to be compared via RT-qPCR normalization in different GFP morphs. Results show visual and inter-male aggression behaviour genes were highly expressed in BC and SM males but were down-regulated in OC individuals. Levels of gene expression were most abundant in eyestalk tissue, followed by testis and was expressed at the lowest levels in hepatopancreas indicating eyestalk could be a major target tissue for candidate genes involved in visual display and perception associated with transition of OC to BC male phenotype. Results revealed that all signals (visual, olfactory and aggressive behaviour) were not significant for fold change indicating that overall level of gene expression in male prawns were independent of these signals, respectively. Multimodal signalling information from olfactory genes in response to visual cues in subordinate males were up-regulated only in OC males in the testis. In general, levels of gene expression associated with chemical stimuli however, did not appear to provide primary cues for transition of SM/OC to BC males. Long Wave Opsin (LW OPSIN), a vision gene associated with light, visual perception and colour vision (in particular, in response to red and blue/green light) did show suppressed gene expression in male subordinate GFP individuals compared with dominant morphs. EP, a gene involved in startle response and inter-male aggression was up-regulated in the hepatopancreas in dominant males and down-regulated in both subordinate male morphs (SM and OC), so this also could potentially be a suitable candidate gene for manipulation in an attempt to knock down or interfere with transition between adult male morphotypes. These outcomes provide a foundation for ultimately developing practices that may allow adult male morph frequencies to be modified in closed populations. Some of the differentially expressed genes (DEGs) detected in the current study likely represent novel genes of potential interest to evolutionary biologists more generally and to the community of GFP biologists in particular, as potential gene candidates that may influence social dominance and other key behavioural traits in GFP and other decapod crustaceans e.g rock shrimp (Rhynchocinetes typus) and several species of crayfishes (Procambarus sp.). While male structural polymorphisms in other decapods also involve characteristics associated with aggressiveness like claw and body size, social dominance hierarchy studies have focused more on male reproductive behaviour and alternative male mating tactics instead of evaluating dominance hierarchies between morphs. Thus, we believe that the findings and candidate genes identified here can provide a foundation for future morphotype and hierarchical structure studies to improve the efficiency of practical genetic improvement programs in farmed crustaceans, in particular for giant freshwater prawn