Metatranscriptome analysis reveals the putative venom toxin repertoire of the biofouling hydroid Ectopleura larynx.

Cnidarians thriving in biofouling communities on aquaculture net pens represent a significant health risk for farmed finfish due to their stinging cells. The toxins coming into contact with the fish, during net cleaning, can adversely affect their behavior, welfare, and survival, with a particularly serious health risk for the gills, causing direct tissue damage such as formation of thrombi and increasing risks of secondary infections. The hydroid Ectopleura larynx is one of the most common fouling organisms in Northern Europe. However, despite its significant economic, environmental, and operational impact on finfish aquaculture, biological information on this species is scarce and its venom composition has never been investigated. In this study, we generated a whole transcriptome of E. larynx, and identified its putative expressed venom toxin proteins (predicted toxin proteins, not functionally characterized) based on in silico transcriptome annotation mining and protein sequence analysis. The results uncovered a broad and diverse repertoire of putative toxin proteins for this hydroid species. Its toxic arsenal appears to include a wide and complex selection of toxin proteins, covering a large panel of potential biological functions that play important roles in envenomation. The putative toxins identified in this species, such as neurotoxins, GTPase toxins, metalloprotease toxins, ion channel impairing toxins, hemorrhagic toxins, serine protease toxins, phospholipase toxins, pore-forming toxins, and multifunction toxins may cause various major deleterious effects in prey, predators, and competitors. These results provide valuable new insights into the venom composition of cnidarians, and venomous marine organisms in general, and offer new opportunities for further research into novel and valuable bioactive molecules for medicine, agronomics and biotechnology.

Metabolic effects of diet containing blue mussel (Mytilus edulis) and blue mussel-fed salmon in a mouse model of obesity.

Alternative feed ingredients for farmed salmon are warranted due to increasing pressure on wild fish stocks. As locally farmed blue mussels may represent an environmentally sustainable substitute with a lower carbon footprint, we aimed to test the potential and safety of substituting fish meal with blue mussel meal in feed for Atlantic salmon. Salmon were fed diets in which fish meal was partially replaced with blue mussel meal in increments, accounting for up to 13.1 % of the ingredients. Fillets from the salmon were subsequently used to prepare obesity-promoting western diets for a 13-weeks mouse feeding trial. In a second mouse trial, we tested the effects of inclusion of up to 8% blue mussel meal directly in a meat-based western diet. Partial replacement of fish meal with blue mussel meal in fish feed preserved the n-3 polyunsaturated fatty acid (PUFA) content in salmon fillets. The observed blue mussel-induced changes in the fatty acid profiles in salmon fillets did not translate into similar changes in the livers of mice that consumed the salmon, and no clear dose-dependent responses were found. The relative levels of the marine n-3 fatty acids, EPA, and DHA were not reduced, and the n-3/n-6 PUFA ratios in livers from all salmon-fed mice were unchanged. The inclusion of blue mussel meal in a meat-based western diet led to a small, but dose-dependent increase in the n-3/n-6 PUFA ratios in mice livers. Diet-induced obesity, glucose intolerance, and hepatic steatosis were unaffected in both mice trials and no blue mussel-induced adverse effects were observed. In conclusion, our results suggest that replacing fish meal with blue mussel meal in salmon feed will not cause adverse effects in those who consume the salmon fillets.

Conserved Molecular Players Involved in Human Nose Morphogenesis Underlie Evolution of the Exaggerated Snout Phenotype in Cichlids.

Instances of repeated evolution of novel phenotypes can shed light on the conserved molecular mechanisms underlying morphological diversity. A rare example of an exaggerated soft tissue phenotype is the formation of a snout flap in fishes. This tissue flap develops from the upper lip and has evolved in one cichlid genus from Lake Malawi and one genus from Lake Tanganyika. To investigate the molecular basis of snout flap convergence, we used mRNA sequencing to compare two species with snout flap to their close relatives without snout flaps from each lake. Our analysis identified 201 genes that were repeatedly differentially expressed between species with and without snout flap in both lakes, suggesting shared pathways, even though the flaps serve different functions. Shared expressed genes are involved in proline and hydroxyproline metabolism, which have been linked to human skin and facial deformities. Additionally, we found enrichment for transcription factor binding sites at upstream regulatory sequences of differentially expressed genes. Among the enriched transcription factors were members of the FOX transcription factor family, especially foxf1 and foxa2, which showed an increased expression in the flapped snout. Both of these factors are linked to nose morphogenesis in mammals. We also found ap4 (tfap4), a transcription factor showing reduced expression in the flapped snout with an unknown role in craniofacial soft tissue development. As genes involved in cichlid snout flap development are associated with human midline facial dysmorphologies, our findings hint at the conservation of genes involved in midline patterning across distant evolutionary lineages of vertebrates, although further functional studies are required to confirm this.

Transcriptomics unravels molecular players shaping dorsal lip hypertrophy in the vacuum cleaner cichlid, Gnathochromis permaxillaris.

BACKGROUND: Teleosts display a spectacular diversity of craniofacial adaptations that often mediates ecological specializations. A considerable amount of research has revealed molecular players underlying skeletal craniofacial morphologies, but less is known about soft craniofacial phenotypes. Here we focus on an example of lip hypertrophy in the benthivorous Lake Tangnayika cichlid, Gnathochromis permaxillaris, considered to be a morphological adaptation to extract invertebrates out of the uppermost layer of mud bottom. We investigate the molecular and regulatory basis of lip hypertrophy in G. permaxillaris using a comparative transcriptomic approach. RESULTS: We identified a gene regulatory network involved in tissue overgrowth and cellular hypertrophy, potentially associated with the formation of a locally restricted hypertrophic lip in a teleost fish species. Of particular interest were the increased expression level of apoda and fhl2, as well as reduced expression of cyp1a, gimap8, lama5 and rasal3, in the hypertrophic lip region which have been implicated in lip formation in other vertebrates. Among the predicted upstream transcription factors, we found reduced expression of foxp1 in the hypertrophic lip region, which is known to act as repressor of cell growth and proliferation, and its function has been associated with hypertrophy of upper lip in human. CONCLUSION: Our results provide a genetic foundation for future studies of molecular players shaping soft and exaggerated, but locally restricted, craniofacial morphological changes in fish and perhaps across vertebrates. In the future, we advocate integrating gene regulatory networks of various craniofacial phenotypes to understand how they collectively govern trophic and behavioural adaptations.

Gene expression profiling suggests differences in molecular mechanisms of fin elongation between cichlid species.

Comparative analyses of gene regulation inform about the molecular basis of phenotypic trait evolution. Here, we address a fin shape phenotype that evolved multiple times independently across teleost fish, including several species within the family Cichlidae. In a previous study, we proposed a gene regulatory network (GRN) involved in the formation and regeneration of conspicuous filamentous elongations adorning the unpaired fins of the Neolamprologus brichardi. Here, we tested the members of this network in the blockhead cichlid, Steatocranus casuarius, which displays conspicuously elongated dorsal and moderately elongated anal fins. Our study provided evidence for differences in the anatomy of fin elongation and suggested gene regulatory divergence between the two cichlid species. Only a subset of the 20 genes tested in S. casuarius showed the qPCR expression patterns predicted from the GRN identified in N. brichardi, and several of the gene-by-gene expression correlations differed between the two cichlid species. In comparison to N. brichardi, gene expression patterns in S. casuarius were in better (but not full) agreement with gene regulatory interactions inferred in zebrafish. Within S. casuarius, the dorsoventral asymmetry in ornament expression was accompanied by differences in gene expression patterns, including potential regulatory differentiation, between the anal and dorsal fin.

Molecular mechanisms underlying nuchal hump formation in dolphin cichlid, Cyrtocara moorii.

East African cichlid fishes represent a model to tackle adaptive changes and their connection to rapid speciation and ecological distinction. In comparison to bony craniofacial tissues, adaptive morphogenesis of soft tissues has been rarely addressed, particularly at the molecular level. The nuchal hump in cichlids fishes is one such soft-tissue and exaggerated trait that is hypothesized to play an innovative role in the adaptive radiation of cichlids fishes. It has also evolved in parallel across lakes in East Africa and Central America. Using gene expression profiling, we identified and validated a set of genes involved in nuchal hump formation in the Lake Malawi dolphin cichlid, Cyrtocara moorii. In particular, we found genes differentially expressed in the nuchal hump, which are involved in controlling cell proliferation (btg3, fosl1a and pdgfrb), cell growth (dlk1), craniofacial morphogenesis (dlx5a, mycn and tcf12), as well as regulators of growth-related signals (dpt, pappa and socs2). This is the first study to identify the set of genes associated with nuchal hump formation in cichlids. Given that the hump is a trait that evolved repeatedly in several African and American cichlid lineages, it would be interesting to see if the molecular pathways and genes triggering hump formation follow a common genetic track or if the trait evolved in parallel, with distinct mechanisms, in other cichlid adaptive radiations and even in other teleost fishes.

Maternal mRNA input of growth and stress-response-related genes in cichlids in relation to egg size and trophic specialization.

BACKGROUND: Egg size represents an important form of maternal effect determined by a complex interplay of long-term adaptation and short-term plasticity balancing egg size with brood size. Haplochromine cichlids are maternal mouthbrooders showing differential parental investment in different species, manifested in great variation in egg size, brood size and duration of maternal care. Little is known about maternally determined molecular characters of eggs in fishes and their relation to egg size and trophic specialization. Here we investigate maternal mRNA inputs of selected growth- and stress-related genes in eggs of mouthbrooding cichlid fishes adapted to different trophic niches from Lake Tanganyika, Lake Malawi, Lake Victoria and compare them to their riverine allies. RESULTS: We first identified two reference genes, atf7ip and mid1ip1, to be suitable for cross-species quantification of mRNA abundance via qRT-PCR in the cichlid eggs. Using these reference genes, we found substantial variation in maternal mRNA input for a set of candidate genes related to growth and stress response across species and lakes. We observed negative correlation of mRNA abundance between two of growth hormone receptor paralogs (ghr1 and ghr2) across all haplochromine cichlid species which also differentiate the species in the two younger lakes, Malawi and Lake Victoria, from those in Lake Tanganyika and ancestral riverine species. Furthermore, we found correlations between egg size and maternal mRNA abundance of two growth-related genes igf2 and ghr2 across the haplochromine cichlids as well as distinct clustering of the species based on their trophic specialization using maternal mRNA abundance of five genes (ghr1, ghr2, igf2, gr and sgk1). CONCLUSIONS: These findings indicate that variations in egg size in closely related cichlid species can be linked to differences in maternal RNA deposition of key growth-related genes. In addition, the cichlid species with contrasting trophic specialization deposit different levels of maternal mRNAs in their eggs for particular growth-related genes; however, it is unclear whether such differences contribute to differential morphogenesis at later stages of development. Our results provide first insights into this aspect of gene activation, as a basis for future studies targeting their role during ecomorphological specialization and adaptive radiation.