Aspects of the digestive physiology of larvae of the North African catfish, Clarias gariepinus (Burchell 1822), during early development

Abstract

Aspects of the digestive physiology of larvae of the North African catfish, Clarias gariepinus (Burchell, 1822), during early development The development of a cost-effective off-the-shelf micro-particulate diet with a nutritionally optimal formulation for larval North African catfish, Clarias gariepinus (Burchell, 1822) is one of the most important requirements for the aquaculture sector in Uganda. North African catfish contribute ~50% of Uganda’s production of farmed fish, both in terms of tonnage and economic value, however, studies conducted thus far to develop a micro-diet have taken no account of the digestive physiology of the fish itself, but rather have dwelt more on the composition of the diet and its apparent performance in terms of fish growth. Under these conditions, however, large mortalities are experienced during early development. Only a few studies have been conducted concerning the digestive physiology of the larvae or have characterised aspects of the key digestive enzymes produced by this species. Hence a major mismatch has existed between the general approach to creating micro-diets and the level of knowledge of the functional development of the sensory and digestive system in larval fish, including their theoretical digestive capabilities. This study sought to characterise the ontogeny of key digestive enzymes in North African catfish larvae from hatching to 21 days post-hatch (dph) with special emphasis on the period 2-4 dph where live feed is the more accepted diet for the fish larvae. In real-world terms, however, extensive use of live-feed makes farming extremely labour-intensive and it is costly to operate a catfish hatchery providing a consistent product and at the same time generate profit for farmers. The work described in this thesis was conducted at the University of Stirling’s Tropical Aquarium, a facility of the Institute of Aquaculture, which houses a population of North African catfish introduced into the facility over 30 years ago and assumed to be, but not fully verified as, Clarias gariepinus Burchell 1822. In order to ensure that the results of the research presented in the thesis could be effectively applied in Uganda, the identity of this species needed to be confirmed. From a morphological and meristic characterisation, in addition to use of a molecular taxonomic classification, the species held at the Institute of Aquaculture and employed for the present study was confirmed to be Clarias gariepinus, (Burchell, 1822) rather than a hybrid with, Clarias anguillaris which is a situation that is common in African populations. Studies were undertaken to determine the point at which when exogenous feeding should be commenced and to establish when fish could be weaned on to an inert formulated diet in order to improve growth and survival during larval rearing. From the growth studies conducted, findings revealed that at 2Dph a mixed diet of (live) Artemia and (inert) Coppens diet gave better growth than both Coppens and Artemia fed alone, which gave similar growth rates. The histomorphology of both adult and larval C. gariepinus was examined in order to determine the course of development of digestive structures in the larva. In addition, new in situ labelling procedures were developed to highlight the localisation of expression of transcripts for key digestive enzymes comprising two alkaline proteolytic enzymes, namely chymotrypsin and trypsin, one acidic proteolytic enzyme, pepsin, and one chitinolytic enzyme, gastric chitinase. Larval development followed a time-course similar to that described by other researchers with the pancreas being discrete in the larval stages but being more dispersed throughout the intestinal region in the adults. The stomach was present by 4 to 5 Dph, supporting a suggestion that alkaline protease digestion dominates early in development, switching to a more acidic digestive mode following development of the stomach. In situ labelling protocols showed trypsin and chymotrypsin to be localised in the pancreas by as early as 2 Dph, while pepsin and gastric chitinase were localised in the stomach. More specifically, pepsin was localised to oxynticopeptic cells and gastric chitinase to the gastric pits with the former being present by 4Dph and the latter being present by 6Dph. Quantitative real-time PCR (qRT-PCR) was employed to investigate the timing and level of transcript expression for trypsin, chymotrypsin, pepsin and gastric chitinase. First expression was observed, perhaps unsurprisingly, to occur largely at a time when the tissue representing the site of production was first observable by histological examination and expression increased from this time. Trypsin and chymotrypsin expression were detectable from 1Dph, with clear expression by 2Dph, coinciding with the appearance of the first anlage of the pancreas in histology. Pepsin and gastric chitinase mRNA was detected at 3Dph and was highly expressed from 4dph onwards. This coincided with the appearance of the first anlage of the stomach in histology. Important new resources for North African catfish, including the first assembled and annotated transcriptome are now available for this species. These were created from a mixed tissue / stage transcriptome library. Using this transcriptome resource as a basis, an oligo-microarray was constructed, which allowed broad-scale transcriptome analysis to be conducted on samples from early larvae fed using two diet regimes. During development on the standard diet, a number of pathways were observed to be over-represented in terms of transcript expression between 1 Dph and 4 Dph. These included protein digestion and absorption, bile secretion, complement and coagulation cascades, glycolysis, gluconeogenesis, PPAR signalling pathways, and ribosomal pathways. Together these pathways denoted changes in elements of growth, protein production, digestion, the immune system and synaptic transmission inter alia. From the research presented in this thesis, it is clear that the key digestive enzymes studied here are rapidly switched on during the first few days of development and first feeding. Different dietary regimes had important effects on expression of digestive enzymes and knock-on effects for digestion, growth, and immune system development, which may have important consequences for larval health and survival under aquaculture conditions. The results of this work have the potential to assist the Ugandan feed industry in that they can be used as a baseline to make improved diets for early weaning in larval fish, using increased knowledge of which enzymes are involved in larval digestion and when they are produced by the fish. Clues about the course of development of larval immunity, particularly innate immunity, may help to provide strategies to mitigate the high losses experienced by farmers in early development and may assist in the management of high intensity catfish hatcheries. The findings of the work presented in this thesis can help to better formulate inert feeds and optimise timing of introduction to larval stages to maximise growth and survival. Public transcriptomic resources produced in the course of this work can be used by other researchers as a basis for larger broad-scale transcriptomic investigations using microarray, RNAseq or allied technologies to characterise different states including disease, nutrition, physiology and development which could be used to e.g. improve diets, develop vaccines, or increase production values such as growth characteristics.