# Reciprocal Hybridization Between Herbivorous and Carnivorous Sub-Cold-Water Fish Reveals Divergent Intestinal Characteristics and Microbiome Assembly

## TL;DR

This study shows how hybridizing two types of cold-water fish affects their gut structure, enzyme activity, and gut microbes, depending on which parent is which.

## Contribution

The study reveals how reciprocal hybridization influences intestinal traits and microbiome assembly in sub-cold-water fish.

## Key findings

- Orthogonal hybrids combined the muscular gut of the mother with high lipase activity of the father and enriched Lactococcus.
- Reciprocal hybrids showed mismatched gut structure and function with low enzyme activity and environmental bacteria dominance.
- Host genetics strongly influence microbiome in the foregut and midgut, while diet drives hindgut convergence.

## Abstract

Hybridization is a fundamental approach in aquaculture breeding, yet the specific effects of reciprocal crossing on the intestinal phenotype of sub-cold-water fish remain under-explored. This study characterized the intestinal morphology, digestive enzyme profiles, and microbiome composition in the hybrid offspring of a carnivorous species (Schizothrax davidi) and an herbivorous species (Schizothorax prenanti). We observed that the direction of hybridization resulted in distinct trait combinations. The orthogonal hybrids (paternal carnivore × maternal herbivore) exhibited a mosaic phenotype, combining the muscular gut structure of the mother with the high lipase activity of the father, accompanied by an enrichment of Lactococcus. In contrast, the reciprocal hybrids displayed a different structural–physiological pattern, characterized by extensive fold development but lower amylase activity, and a microbiome composition with a higher proportion of environmental taxa. These findings provide a detailed description of how hybridization differentially remodels the digestive tract and microbial assembly in sub-cold-water fish, highlighting the importance of crossing direction in shaping biological traits.

Hybridization is pivotal for germplasm innovation, yet how reciprocal crossing regulates digestive characteristics in sub-cold-water fish remains unclear. This study systematically compared differences in intestinal morphology, physiological function, and microbial community assembly among herbivorous Schizothorax prenanti, carnivorous S. davidi, and their reciprocal hybrids using histological analysis, digestive enzyme assays, and 16S rRNA sequencing. Results indicated that parental intestinal characteristics were highly consistent with their feeding habits. Orthogonal hybrids exhibited a mosaic phenotype, combining the maternal muscular gut structure with high paternal-like lipase activity, and were characterized by an enrichment of the potential probiotic Lactococcus. In contrast, reciprocal hybrids presented a mismatch between morphology and function: despite developed hindgut folds, key digestive enzyme activities were low, and the gut microbiota was dominated by environmental bacteria such as Methylobacterium. Our findings indicate a spatially dependent assembly dynamic: the host genetic background strongly drives microbiome divergence in the anterior segments (foregut and midgut), whereas the long-term administration of a standardized diet ultimately promotes structural convergence in the hindgut. The orthogonal cross yielded a phenotype characterized by an apparent co-occurrence of specific host enzymes and distinct microbiota, suggesting an inferred physiological potential for lipid digestion that requires further multi-omics validation. These findings provide preliminary insights into the associations between genetic background and intestinal traits, providing a theoretical basis for the targeted breeding of Schizothorax species.