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

BackgroundTeleosts 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.ResultsWe 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.ConclusionOur 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.Peer reviewe

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

Reference databases, primer choice, and assay sensitivity for environmental metabarcoding: Lessons learnt from a re‐evaluation of an eDNA fish assessment in the Volga headwaters

Biodiversity monitoring via environmental DNA, particularly metabarcoding, isevolving into a powerful assessment tool for riverine systems. However, formetabarcoding to be fully integrated into standardized monitoring programmes, somecurrent challenges concerning sampling design, laboratory workflow, and data analy-sis need to be overcome. Here, we review some of these major challenges and poten-tial solutions. We further illustrate three potential pitfalls, namely the choice ofsuitable metabarcoding primers, the necessity of complete reference databases, andvarying assay sensitivities, by a reappraisal of our-own recently carried outmetabarcoding study in the Volga headwaters. TaqMan qPCRs had detected catfish(Silurus glanis) and European eel (Anguilla anguilla), whereas metabarcoding had not, inthe same samples. Furthermore, after extending the genetic reference database by12 additional species and re-analysing the metabarcoding data, we additionallydetected the Siberian spiny loach (Cobitis sibirica) and Ukrainian brook lamprey(Eudontomyzon mariae) and reassigned the operational taxonomic units previouslyassigned toMisgurnus fossilistoCobitis sibirica. In silico analysis of metabarcodingprimer efficiencies revealed considerable variability among primer pairs and amongtarget species, which could lead to strong primer bias and potential false-negatives inmetabarcoding studies if not properly compensated for. These results highlight someof the pitfalls of eDNA-metabarcoding as a means of monitoring fish biodiversity inlarge rivers, which need to be considered in order to fully unleash the full potential ofthese approaches for freshwater biodiversity monitori

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

Lecaudey LA, Netzer R, Wibberg D, Busche T, Bloecher N. Metatranscriptome analysis reveals the putative venom toxin repertoire of the biofouling hydroid Ectopleura larynx. Toxicon. 2024;237: 107556.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. Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved

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

Fish diversity assessment in the headwaters of the Volga River using environmental DNA metabarcoding

The headwaters of the Volga River exhibit large reaches with near‐pristine conditions, and therefore long‐term biodiversity monitoring of this catchment can provide rare and valuable information on a European lowland river. More specifically, freshwater fish species assemblages are a good indicator of ecosystem status, as they are particularly sensitive to environmental changes and hydromorphological alterations. Historical records show that the fish fauna of the Upper Volga has changed over time, both in species composition and in abundance. The construction of the Volga–Kama cascade (a series of large dams) has specifically affected the migration of diadromous species. Environmental DNA metabarcoding offers a non‐invasive approach to determine the number of species in an aquatic ecosystem, as well as their identity and distribution. This approach is especially useful for fish fauna surveys along large rivers and long‐term biomonitoring, with the advantage of having no impact on the species and their habitats. To infer the current fish species diversity and the spatial distribution of each species in the free‐flowing section of the Upper Volga River, as well as in selected tributaries, an environmental DNA metabarcoding approach was applied, using three mitochondrial DNA markers. This method allowed the positive identification of 23 fish species and their respective distributions in the headwaters of the Volga. This assessment provides a valuable example of the application of environmental DNA metabarcoding in a large river system, and constitutes a starting point for future investigations and long‐term biomonitoring in the Upper Volga system. In addition, the results can also serve as a reference for fish diversity assessments of other large European lowland rivers, and can guide future conservation and management measures in the headwaters of the Volga

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.Peer reviewe

Comparative transcriptomics reveals candidate carotenoid color genes in an East African cichlid fish

Background Carotenoids contribute significantly to animal body coloration, including the spectacular color pattern diversity among fishes. Fish, as other animals, derive carotenoids from their diet. Following uptake, transport and metabolic conversion, carotenoids allocated to body coloration are deposited in the chromatophore cells of the integument. The genes involved in these processes are largely unknown. Using RNA-Sequencing, we tested for differential gene expression between carotenoid-colored and white skin regions of a cichlid fish, Tropheus duboisi "Maswa", to identify genes associated with carotenoid-based integumentary coloration. To control for positional gene expression differences that were independent of the presence/absence of carotenoid coloration, we conducted the same analyses in a closely related population, in which both body regions are white. Results A larger number of genes (n = 50) showed higher expression in the yellow compared to the white skin tissue than vice versa (n = 9). Of particular interest was the elevated expression level of bco2a in the white skin samples, as the enzyme encoded by this gene catalyzes the cleavage of carotenoids into colorless derivatives. The set of genes with higher expression levels in the yellow region included genes involved in xanthophore formation (e.g., pax7 and sox10), intracellular pigment mobilization (e.g., tubb, vim, kif5b), as well as uptake (e.g., scarb1) and storage (e.g., plin6) of carotenoids, and metabolic conversion of lipids and retinoids (e.g., dgat2, pnpla2, akr1b1, dhrs). Triglyceride concentrations were similar in the yellow and white skin regions. Extracts of integumentary carotenoids contained zeaxanthin, lutein and beta-cryptoxanthin as well as unidentified carotenoid structures. Conclusion Our results suggest a role of carotenoid cleavage by Bco2 in fish integumentary coloration, analogous to previous findings in birds. The elevated expression of genes in carotenoid-rich skin regions with functions in retinol and lipid metabolism supports hypotheses concerning analogies and shared mechanisms between these metabolic pathways. Overlaps in the sets of differentially expressed genes (including dgat2, bscl2, faxdc2 and retsatl) between the present study and previous, comparable studies in other fish species provide useful hints to potential carotenoid color candidate genes.Peer reviewe