HISTOLOGICAL INTESTINAL DEVELOPMENT of AFRICAN LUNGFISH (Protopterus Aethiopicus Heckel 1851) LARVAE DURING FIRST FEEDING

HISTOLOGICAL INTESTINAL DEVELOPMENT OF AFRICAN LUNGFISH (Protopterus aethiopicus Heckel 1851) LARVAE DURING FIRST FEEDING

RONALD NTANZI Student number: 01700940

Promoters i. Prof. Dr. Gilbert Van Stappen ii. Dr.Ir. Nancy Nevejan iii. Prof. Dr. Wim Van den Broeck Supervisors I. Prof. Dr. Wim Van den Broeck II. Martin Sserwadda

A dissertation submitted to Ghent University in partial fulfilment of the requirements for the degree of Master of Science in Aquaculture

Academic year: 2017 - 2019

COPYRIGHT The author and promoters give permission to put this thesis to disposal for consultation and to copy parts of it for personal use. Any other use falls under the limitation of copyright, thus the obligation to explicitly mention the source when citing parts of this thesis.

Promoters

i) Prof.Dr. Gilbert Van Stappen ii) Dr.Ir. Nancy Nevejan

…………………………………………………………………… ………………………………………………………..

iii) Prof.Dr. Win Van den Broeck

……………………………………………………………………….

Supervisor

ii) Martin Sserwadda

……………………………………………………………

Author

Ntanzi Ronald

……………………………………………………………………..

ACKNOWLEDGEMENT

I would like to extend my sincere thanks to my promoters Prof. Gilbert Van Stappen and Dr Nancy Nevejan and supervisor Mr Martin Sserwadda of the Aquaculture Research and Development Centre (ARDC) Ghent University for the guidance rendered to me during the process of preparing this work. Special thanks also go to my third promoter Prof. Wim Van den Broeck of the department of morphology, faculty of veterinary medicine Ghent University and other members from this faculty including Prof. Pieter Cornillie and technicians Lobke De Bels and Bert De Pauw for the technical and practical guidance in preparation this work.

I would like to give gratitude to the VLIR – UOS (Vlaamse Interuniversitaire Raad – The Flemish Interuniversity council) for the financial support rendered for my studies, I am grateful, and it has been a life changing experience.

Thanks to my family members and friends who believed in me from the start and have been with me throughout the journey, your courage and guidance has brought me this far.

Lastly, I give gratitude to God for all He has done for me.

TABLE OF CONTENTS COPYRIGHT…………………………………………………………………………………………………………………………………….i ACKNOWLEDGEMENT……………………………………………………………………………………………………………………ii TABLE OF CONTENTS…………………………………………………………………………………………………………………….iii

LIST OF FIGURES…………………………………………………………………………………………………………………………….v

LIST OF TABLES………………………………………………………………………………………………………………………….…vii

LIST OF ABBREVIATIONS……………………………………………………………………………………………………………..viii

ABSTRACT…………………………………………………………………………………………………………………………………….ix

CHAPTER 1: INTRODUCTION ...... 1 CHAPTER 2: LITERATURE REVIEW ...... 5 2.1 The state of world aquaculture ...... 6 2.2 Air-breathing fishes and climate change ...... 7 2.3 Aquaculture in Africa ...... 9 2.4 Research on domestication of African lungfish ...... 10 2.5 Biology of African lungfish ...... 12 2.5.1 Taxonomy and morphology ...... 12 2.5.2 Habitat distribution of African lungfish ...... 14 2.5.3 The reproductive life cycle of African lungfish ...... 16 2.6 Freshwater fish larval feeding strategies and live feeds ...... 17 2.6.1 Larval feeding...... 17 First feeding ...... 17 Weaning ...... 18 2.6.2 Live feeds ...... 20 Rotifers ...... 20 Artemia...... 21 Microalgae ...... 21 Other live feed organisms ...... 22 2.7 Freshwater fish larval gastro-intestinal tract (GIT) ontogeny and histology ...... 23 2.7.1 General gastro-intestinal tract ontogeny ...... 23 2.7.2 Buccal cavity and oesophagus ...... 24 2.7.3 Stomach ...... 26 2.7.4 Intestines ...... 27 2.7.5 Liver and pancreas ...... 28

iii

CHAPTER 3: MATERIALS AND METHODS...... 30 3.1 Sampling and histological procedures ...... 31 3.1.1Tissue processing ...... 31 3.1.2 Tissue sectioning ...... 31 3.1.3 Staining and cover slipping ...... 31 3.1.4 Histological analysis ...... 32 3.2 3D Reconstruction ...... 32 3.2.1 Selection of slides for reconstruction ...... 32 3.2.2 2D image acquisition protocol ...... 32 3.2.3 Reconstruction procedure ...... 33 Image import and alignment of image slices ...... 33 Image segmentation ...... 33 Surface generation, editing and visualisation ...... 33 CHAPTER 4: RESULTS...... 35 4.1 Summary of histological and morphological landmarks between 17 and 26 DPH...... 36 4.2 3D Reconstruction ...... 38 4.3 Histological observations between 17 and 26 DPH ...... 40 4.3.1 Buccal cavity ...... 40 4.3.2 Pharynx ...... 41 4.3.3 Oesophagus ...... 42 4.3.4 Spiral valve ...... 44 4.3.5 Cloaca and anal opening ...... 47 4.3.6 Liver and pancreas ...... 48 CHAPTER 5: DISCUSSION ...... 51 5.1 Larval digestive ontogeny ...... 52 5.2 Morphological development ...... 52 5.3 Histological development...... 54 5.3.1 Buccal and pharyngeal cavities ...... 54 5.3.2 Oesophagus ...... 55 5.3.3 Spiral valve ...... 56 5.3.4 Cloaca ...... 59 5.3.5 Liver and pancreas ...... 59 5.4 Conclusions and recommendations ...... 60 CHAPTER 6: REFERENCES ...... 62

LIST OF FIGURES Figure 2.1.World fisheries and aquaculture production (FAO, 2018) ...... 7

Figure 2.2. The contribution of three major air-breathing fishes to the world freshwater fish production. (Data adapted from FAO,2018) ...... 8

Figure 2.3. The contribution of aquaculture to African food production between 1991 and 2016 (FAO 2018)...... 9

Figure 2.4. The concept of domestication in aquaculture (Liao and Huang 2000)...... 11

Figure 2.6. Prey requirements, relative feeding cost and juvenile production cost of a 50 day juvenile seabass Dichentrarchus labrax according to weaning age (Ruyet, 1989)...... 20

Figure 2.7. Derivation, sequence, and timing of alimentary canal organs in typical larval fish (Govoni et al. 1986)...... 24

Figure 2.8. Larval ontogeny of the freshwater giant trahira Hoplias lacerdae , from 1 to 7 DPH...... 25

Figure 2.9. Main histological ontogenetical landmarks during larval development of Snakehead murrel, Channa striatus (Bilal et al., 2015)...... 29

Figure 4.1. Lateral three-dimensional view of a 17 (a) and a 26 DPH (b) lungfish larva. A = Buccal cavity, B= Pharynx, C= oesophagus, D = spiral valve, E= Cloaca, F = Liver, G =Pancreas, H =Lungs, J= Kidney and K= vertebral column. (Scale =µm) ...... 38

Figure 4.2. Three-dimensional images showing a lungfish spiral valve (A) and cloaca (B) at 17DPH (a) and 26 DPH (b)...... 39

Figure 4.3. PAS staining light microscopy cross sections of the buccal cavity (B) and pharynx (C) of the African lungfish larvae at 20 DPH (a) and 24 DPH (b, c and d)...... 42

Figure 4.4. PAS stain cross section of a 24 DPH larvae oesophagus under a light microscope...... 43

Figure 4.5. Light microscopy cross-sectional view of the spiral valve of African lungfish larvae at 17 DPH (a, b and c) and 20DPH (d) a and d are PAS stains while b and c are H&E...... 45

Figure 4.6. Cross section PAS staining light microscopy of the African lungfish larvae posterior spiral valve at 24 (a) and 26 (b & c) DPH...... 47

Figure 4.7. Light microscopy PAS staining of the cross (1) and longitudinal (2) section of a lungfish larva cloaca at 20 and 26 DPH, respectively...... 48

Figure 4.8. Cross sections of lungfish larvae liver and pancreas in PAS (a, b, c & f) and H&E (d & e) ...... …49

LIST OF TABLES Table 2.1. Morphological characteristics that distinguish Protopterus species (data from; Mlewa et al. 2010; Poll, 1961)...... 14

Table 4.1. Histological and morphological landmarks in lungfish larvae as observed between 17 and 26 DPH………………………………….…………………………………………………………………………………………………………35

vii

LIST OF ABBREVIATIONS °C……………………………………….Degrees Celsius

µm………………………………………micrometres mt/ha/yr…………………………….metric tons per hectare per year

3D……………………………….…….Three-Dimensional

AB……………………………………….Alcian blue

ARDC…………………………….…….Aquaculture Research and Development Centre

ARA…………………………………….Arachidonic Acid cm…………………………………….…centimetres

DHA………………………………………Docosahexaenoic Acid

DPH…………………….………….……Day Post Hatching

EPA……………………………………….Eicosapentaenoic Acid

FAO…………………………………….…Food and Agricultural Organisation

HUFAs……………………….………….Highly Unsaturated Fatty Acids

IFAD………………………………………International Fund for Agricultural Development

IPCC……………………………………….Intergovernmental Panel on Climate Change kg……………………………………….…Kilograms mm…………………………………………millimetres mt……………………………………….….metric tons m2………………………………….………metres squared min……………………………………….…minutes

Spp………………………………………….Species

PAS………………………………….………Periodic Acid Schiff

UNICEF…………………………………….United Nations International Children’s Emergency Fund

VLIR-IUC……………………………… Vlaamse Interuniversitaire Raad- Institutional University Cooperation

viii

ABSTRACT Understanding of the intestinal development is key to defining the appropriate nutritional and physical characteristics of feed required at every fish larval developmental stage. In the current study, the ontogeny of the digestive tract in African lungfish larvae (Protopterus aethiopicus) between 17 and 26 day post hatching (DPH) was described as a follow-up of an earlier study on larvae of the same species between 6 and 17 DPH. The study utilised larvae previously hatched from wild eggs collected from Lake Bisina in eastern Uganda and fed on a mixed diet of commercial feeds, freshly hatched Artemia nauplii and decapsulated Artemia cysts. Light microscopy and 3D simulation basing on histological sections with the help of Amira software were used to describe cellular and organ structural developments, respectively. Comparison of 3D simulation models showing larval development of a 17 and 26 DPH revealed lateral elongation of both the larvae and internal body organs with the spiral valve forming 3 spiral coils from the original laterally compressed tube. Dentition along the buccopharyngeal cavity evolved from canine like teeth protrusions to fully developed tooth plates on both sides of the cavity and the pharynx changed from a folded and wide to a narrow and straight cavity. The connective tissue composition around the oesophagus evolved from a loose and dense to predominately dense connective tissue with a high number of secretory cells. Yolk sac residues were depleted in the spiral valve and an increase in number of mucous cells was observed. Supranuclear vacuoles and lymphocytes were observed in the enterocytes and mesenchymal tissue of the spiral valve respectively from 24 DPH and these increased in number by 26 DPH. Liver lipid granules were depleted by 22 DPH while the onset of glycogen accumulation in the liver was on 24 DPH. Vacuolar liver hepatocytes had evolved from irregular to regular shaped hepatocytes by 26 DPH. A single pancreatic lobe was divided into two new lobes and the pancreatic duct was fully functional by 24 DPH. By 26 DPH, the organisation of the digestive tract in African lungfish larvae was characterised by a well- developed dentition, an increased absorptive surface, onset of extracellular protein digestion (supranuclear vacuoles), a fully functional liver (vacuolated hepatocytes and carbohydrate digestion) and presence of an exocrine and endocrine pancreas. The findings from this study will provide a basis for optimum larviculture of this potential freshwater aquaculture species through synchronisation of organ development with larval nutrition.

CHAPTER 1: INTRODUCTION

African lungfish (Protopterus) are air-breathing Dipnoi fishes endemic and thriving in Africa’s lakes, rivers, swamps and marshes (Mlewa et al. 2010; Icardo et al. 2010; Otuogbai et al. 2004). The African lungfish belongs to the class Osteichthyes and subclass Sarcopterygii containing the marbled lungfish Protopterus aethiopicus (Heckel 1851) and three other species (Mlewa et al. 2010). African lungfish adapts to extreme tropical climate conditions by undergoing aestivation characterised by reduced metabolism, supressed osmoregulation and accumulation of metabolites (Icardo et al. 2010). Aquaculture production, especially in sub- Saharan Africa, contributes relatively low to the total fish production, thus diversification of cultured species away from the traditional Tilapia and African catfish (Clarias gariepinus) would improve the economic, social and ecological assurance of aquaculture systems (Defaux & Hjort, 2012; Harvey et al. 2017). Amidst climate change effects on tropical aquatic ecosystems coupled with the decreasing numbers of fish from the wild, the culture of air- breathing indigenous fishes like African lungfish is to play a major role in future of aquaculture on the continent, given its resilience to drought and stressful water quality conditions (Graham, 1997; Chapman et al. 2002; Helfman et al. 2009; Walakira et al. 2012; Harrod, 2016). Currently, African lungfish from the wild provides a lot of income to women in African communities involved in harvesting, processing and marketing of fried, smoked, whole gutted and fish based soup as observed around the lake Victoria basin (Sayer et al. 2018). Previous trials on the culture of African lungfish have involved the collection of larvae and juveniles from the wild, but these have proved to be unreliable in quality, availability and timing, thus a proper larval production and rearing strategy is needed to enable closing of the production cycle for African lungfish (Liao & Huang, 2000; Walakira et al. 2012).

The larval stage is one of the most critical stages in fish life history associated structural and functional development of tissues and organs, which later determines the success of fish larviculture (Kjørsvik et al. 2004; Bilal et al. 2015). The successful development of the digestive tract during this stage is essential for the survival of the fish because it enables capture, ingestion, digestion and absorption of food (Hanke et al. 1994; Kjørsvik et al. 2004). Fish larvae can capture food particles at first feeding though the digestive tract at this stage requires further developmental changes to realise full functionality (Govoni et al. 1986; Hanke et al. 1994). Beyond the onset of first feeding, development and differentiation will then depend on both nutrient provision and environmental factors, thus starvation at this stage affects the

morphological development and may degenerate the digestive tract and trunk muscles (Gisbert & Sarasquete, 2008).

Studies of histological intestinal morphology have been carried out in a number of marine fish species (Hamlin et al. 2000; Mai et al. 2005; Zambonino-Infante et al. 2014). However, few studies have been done on freshwater fish (Pradhan et al. 2012) with exception of species including the catfish (Kumar etal. 2019; Nyang’ate Onura et al. 2018; Gisbert et al. 2014; Verreth et al. 1992) , snakehead (Bilal et al. 2015) and knifefish (Mitra et al. 2015) among others. These studies acknowledged the importance of observing larval digestive tract changes in development and nutrient absorption and their importance in identification and the elimination of bottlenecks in larval rearing and weaning. However, despite the potential of African lungfish as a future aquaculture candidate amidst the rapid climate change, the need for aquaculture diversification and decreasing wild stocks (Harvey et al. 2017;Walakira et al. 2012;Chapman et al. 2002), there has been a knowledge gap on its histological intestinal morphology until recent.

Under the framework of VLIR-IUC (Belgium) project with Mountains of the Moon university (Uganda), tests on larviculture of African lungfish with different feeds are underway with larval histology as one of the parameters being assessed. A histological study has been done before on larvae from 6 to 17 days post hatch (DPH). The study utilised larvae that had previously been hatched at the Aquaculture Research and Development Centre (ARDC), Kajjansi, Uganda in a flow-through system at 27°C using borehole water. The larvae had been hatched from egg nests obtained from lake Bisina in eastern Uganda and disinfected before hatching after 3 days. They were provided a mixed diet of commercial feed made by ARDC, freshly hatched nauplii and decapsulated cysts of Great Salt Lake Artemia franciscana, (Kellogg 1906) from Ocean Nutrition (Belgium) since no information on proper starter diets for African lungfish was available. A study on the organogenesis of the digestive tract in African lungfish from 6 to 17 (DPH) revealed the evolution from an initial simple undifferentiated digestive tract to an almost fully differentiated digestive tract with yolk sac reserves still present at the end of the study (17 DPH), proving a possibility of endo-exogenous feeding (Sserwadda et al. under review).

As a follow-up to the above study, the current study was conducted to investigate the histological intestinal development between 17 and 26 DPH. Using light microscopy and 3D simulation, the study was to provide an account of the ontogenic development within the first feeding days. Employing histomorphological techniques to describe the development at the cellular level and 3D simulation to describe organ structural developments that occurred between 17 and 26 DPH, the study was to generate background information to be used in the optimisation of larviculture of African lungfish through synchronisation of larval organ development and maturation with feeding protocol and rearing practices.

CHAPTER 2:

LITERATURE REVIEW

2.1 The state of world aquaculture The sustainability of world protein supply is to stand one of the greatest tests with the ever- increasing world population threatening future food security (United Nations, 2017). The world population is predicted to increase by over 2.3 billion in the next 30 years and 3.6 billion people 80 years to come (United Nations, 2017). Most of this increase is expected in the developing world (United Nations, 2017). This threatens the United Nation`s sustainable development goal to end hunger and provide safe nutrition worldwide by 2030. This threat is justified by the increasing child malnutrition cases more so in sub-Saharan Africa and South America where one in nine children is malnourished and the number of undernourished people being 821 million worldwide as of 2017 (FAO, IFAD, UNICEF, 2018). At a mean body requirement of 0.66 g/kg/day, proteins are one of the major essential nutritional requirements of a healthy body with animal proteins having a near perfect utilization (United Nations, 2002). Fish represents 16.6% of animal protein supply and 6.5% of all protein supply for human consumption (FAO, 2012). This in addition to the essential micronutrients and polyunsaturated omega-3 fatty acids highlights the importance of fish as a tool to fight malnutrition in the third world, given that aquatic proteins are more healthful and affordable to humans (FAO, 2012; Liao & Huang, 2000).

Over the last two decades, aquaculture has emerged to become the world’s fastest-growing food industry contributing greatly to the global animal protein consumption (Troell et al. 2014). A total of 598 aquatic species were being farmed by 2016 equating to a 26.7% increase over the previous decade contributing 46.8% to global production of aquatic organisms and representing a 45% rise over the previous 6 years (FAO, 2018).

Global aquaculture had a fast growth between the 1980s and 1990s realizing 10.8% and 9.5% growth respectively. However, this dropped moderately to an average of 5.8% between 2001 and 2016 though aquaculture has still remained a leading food production sector with fastest growth evident in Africa (FAO, 2018). Global aquaculture production has been growing at an annual rate of 3.2% over the last 50 years reaching 110.2 million tons in 2016 with a first sell value of 243.5 billion USD representing 54.8% of the world’s aquatic production. This reflected a 33% growth in production and 34.2% growth in first sale value over two years from 2014 to 2016 (Ababouch et al. 2016; FAO, 2018). This growth has greatly reduced the pressure on the

capture fisheries which have remained constant despite the increasing human population for the last four decades (Fig.2.1).

Figure 2.1.World fisheries and aquaculture production (FAO, 2018)

The total fish supply is expected to rise by 7% between 2016 and 2030 with aquaculture having an equal share as capture fisheries but contributing over 60% to the total fish supply for human consumption (World Bank, 2013). However, the overall aquaculture growth rate is projected to continuously slowdown from the 1980’s peak (World Bank, 2013).

2.2 Air-breathing fishes and climate change The surface temperature of the earth has been increasing by a range of 0.75 to 0.85 °C over the past century and this rate has greatly increased since the 1970s (Trenberth & Josey, 2007; Intergovernmental Panel on Climate Change (IPCC), 2014). This increase and other climate change impacts not only extend to freshwater ecosystems but have individual effects on freshwater organisms including fish, thus posing a threat to the future of aquaculture more so in tropical regions of the world (Harrod, 2016). Terrestrial regions have been reported to warm faster than the oceans amidst a projected global temperature rise of 6.4°C by 2100

(Meehl et al. 2007). Under the high temperatures, the dissolved oxygen level in the water drastically reduces which affects most of the fishes, except the air-breathing fishes with the ability to supplement gill respiration with atmospheric oxygen (Helfman et al. 2009).

Air-breathing fishes have proved to tolerate low oxygen, drought, and poor water quality, thus making good species for high density, low maintenance aquaculture in warmer climates like Africa which makes their aquaculture possible in these areas (Graham, 1997; Helfman et al. 2009).

Air-breathing arose independently at various times in evolution of Osteichthyes, producing fish that remain in water all the time (aquatic breathers) with some supplementing their respiration (facultative air breathers) while others must have access to air (obligate air breathers). Lungfishes are among the most specialized air breathers and closest relatives of the tetrapods (Graham, 1997; Helfman et al. 2009; Ishimatsu, 2012). Other air-breathing fishes include catfishes like Clarias gariepinus and Pangasius spp, swamp eels, snakeheads, mudskippers and eel gobbies (Ishimatsu, 2012), which all together contribute 9% to world’s freshwater fish production (Fig. 2.2).

Tilapia spp 2% 3% Carp spp 74% 1% 9% Other freshwater fishes Panga Catfishes 5% Torpedo shaped 15% Catfishes Snakehead

Figure 2.2. The contribution of three major air-breathing fishes to the world freshwater fish production. (Data adapted from FAO,2018) ( Panga spp includes all Pangasius farmed species, Carp spp includes all the Carp species, Tilapia spp includes all tilapia species and Torpedo shaped catfishes includes all other farmed catfishes that are not Pangasius.

2.3 Aquaculture in Africa Africa has 48 countries and 5 island nations most of which have some aquaculture practices, though mostly at a low scale with over half of the countries producing less than 100 metric tons annually (Machena & Moehl, 2000). In 2016, 10% of Africa’s population was engaged in fish related economic activities with 1.6% (3 million people) engaged directly in aquaculture, which reflected a 79% increase between 1995 and 2016 (FAO, 2018). This has made aquaculture a great contributor to Africa’s food production over the years, making 18% of total food production by 2016 (Fig. 2.3).

Aquaculture Capture Aquaculture percentage share Figure 2.3. The contribution of aquaculture to African food production between 1991 and 2016 (FAO 2018). Africa’s total aquaculture production was 1982 metric tons in 2016 with Egypt the leading producer contributing 69%, followed by Nigeria with 15% and Sub-Saharan Africa without Nigeria producing 14%, while the rest of North Africa without Egypt produced only 2% (FAO, 2018).

Tilapia and African catfish (Clarias gariepinus) are the major contributors to African aquaculture with yields of 10 to 15 mt/ha/yr and 20 mt/ha/yr, respectively (FAO, 2018).

However, mussels, oysters, abalone and seaweed are also important in aquaculture of countries like Tanzania and South Africa (FAO, 2018; Machena & Moehl, 2000).

The potential of aquaculture in Africa still remains very high despite the under-utilization of resources (water and land), more so in Sub-Saharan Africa amidst the inexpensive labour, high fish demand and year-round favourable climate (Machena & Moehl, 2000). There is a need to refocus the direction of aquaculture in Africa, concentrating on main issues including widening production systems, increasing production intensities and efficiency, developing management technologies for indigenous species with high local demand, emphasizing on marketing and processing of high value end products, promoting policy research on production in response to dynamic macroeconomic policies and empowerment of the private sector (Jamu & Ayinla , 2003).

2.4 Research on domestication of African lungfish Domestication is a process in which an animal becomes generation after generation more adapted to captive conditions. This practice started around 13000 years ago becoming an important development in human history ( Liao & Huang, 2000; Diamond, 2002; Teletchea, 2015). The domestication of fish species is however recent, thus most farmed species only slightly changed from their wild counterparts, with 90% of the world aquaculture still being dependent on wild stock and only Atlantic salmon (Salmo salar) having 100% total production based on selectively bred stocks (Gjedrem, 2012; Teletchea, 2015).

In relation to fish, domestication involves closing the life cycle in captivity independent of wild sources for eggs, larvae, juveniles or breeders, and this involves broodstock management followed by the rearing of larvae and juveniles (Liao & Huang, 2000; Teletchea, 2015) (Fig. 2.4).

Figure 2.4. The concept of domestication in aquaculture (Liao and Huang 2000).

Broodstock management involves culturing of high-quality breeders, inducing maturation and spawning, collection of gametes and eggs followed by successful incubation (Liao & Huang, 2000). Larval and juvenile rearing requires developed farming systems, feeding practices, and health management as a necessity for completing the cycle in captivity (Liao & Huang, 2000). Various conditions are to be considered before the domestication of a fish species, including growth aspects, economic value, docility, the complexity of life cycle, adaptation to artificial feeds, stress resistance, maintenance of genetic variability under domestication and others (Liao & Huang, 2000).

African lungfish is a good candidate for aquaculture in Sub-Saharan Africa because of its high economic value, resistance to stress, being an indigenous species in Africa and adaptability to artificial feeds (Walakira et al. 2012).

There has been an evident decline in the wild stocks of African lungfish from its major habitats like Lake Victoria where a 93% reduction in bottom trawling catch and 96% reduction in trawling by commercial vessels in the Mwaza and Speke gulfs of Tanzania, respectively, was realised between 1973 and 1990 (Witte et al. 2002). This decline coupled with the need for more cultured indigenous freshwater species to meet the world protein demands highlights the need for the domestication of African lungfish in Sub-Saharan Africa (Liao & Huang, 2000).

Research into the feeding biology of African lungfish in its natural habitats has of recent been carried out with a goal of conservation and later on domestication (Otuogbai et al. 2004; Walakira et al. 2014). Over the years indigenous farmers and fishermen have collected seed and juveniles from the wild between July and November and mixed results have been obtained upon culture depending on culture methods and feeding (Walakira et al, 2012). A survey carried out in Uganda proved that 56% of fish farmers were willing to invest in lungfish culture due to the availability of a local and regional market. However, production technologies remained a hindrance to foster their interests (Walakira et al. 2012).

Polyculture of 1000 African lungfish (P. aethiopicus) juveniles collected from the wild and bought at 0.2 to 0.5 US$ each (15-20 cm total length) with 400 mirror carp (Cyprinus carpio) and 800 tilapia (Oreochromis niloticus) fingerlings in 400m2 ponds fed on fish meal mixed with maize bran yielded 1-3 Kg African lungfish after 1.5 years with a survival of 40% (Walakira et al. 2012). Attempts to raise wild juveniles (15-30 cm) in Uganda in 1 m deep excavated holes having 40 cm diameter, while feeding them on fisheries by-catch, tilapia (O. niloticus) fry, snails, waste food and grasshoppers, yielded 70 cm long lungfish after a year with the low survival attributed to cannibalism and burrowing affecting final harvest (Walakira et al. 2012). Male and juvenile lungfish live in swamps near lakes and tend to end up in nearby fish ponds in rainy season flooding, causing low survivals at harvest of such ponds due to their predatory activities on the fish cultured in these ponds (Greenwood, 1958; Walakira et al. 2012).

2.5 Biology of African lungfish 2.5.1 Taxonomy and morphology African lungfish (Protopteridae ) are air-breathing fishes belonging to the class Sarcopterygii, subclass Dipnoi which also contains the Australian lungfish (Ceratodontidae) and South America lungfish (Lepidosirenidae) (Johanson, 2010). The African lungfish and the South American lungfish belong to the order Lepidosirenoiformes and are distantly related the Australian lungfish Neoceratodus fosteri (Kreft 1870) in the order Ceratodontiformes which are the closest relatives of the tetrapods (Brinkmann et al. 2004; Takezaki, 2017). The lungfish (Dipnoi) together with the coelacanth (Latimeria) are reported to be the only living representatives of the Sarcopterygii (Graham, 1997). The African lungfish (Protopterus) has

four species, namely the West African lungfish Protopterus annectens (Owen 1893), the marbled lungfish Protopterus aethiopicus (Heckel 1851) (Graham, 1997), the slender lungfish Protopterus dolloi (Boulenger 1909) and the gilled lungfish Protopterus amphibius (Peters 1844) as the only extant species (Boulenger, 1870; Mlewa et al. 2010).

African lungfish possess elongated bodies with filamentous pectoral and pelvic fins and are also characterized by the fusion of the dorsal, anal and caudal fins to form a diphycercal tail (Bemis & Lauder, 1986) (Fig. 2.5).

Figure 2.5. Diagrammatic representation of Protopterus aethiopicus (Eccles, 1992).

Protopterus larvae have external gills, a characteristic they share with Lepidosiren larvae, which is missing in Neoceratodus larvae. This larval characteristic (external gills on each side of the head) is lost before the end the juvenile stage for the case of Protopterus aethiopicus and P. dolloi but retained by P. amphibius and P. annectens (Greenwood, 1986) (Table 2.1).

Table 2.1. Morphological characteristics that distinguish Protopterus species (data from Mlewa et al. 2010; Poll, 1961). SPECIES NUMBER OF HL/AL (%) DL/AL(%) EXTERNAL RIBS GILLS

P. DOLLOI 45-55 16.2-19.6 63.7-66 Absent in adult

P. AETHIOPICUS 37-39 24-29.6 62.1-67.1 Absent in adult

P. ANNECTENS 32-37 22.8-28.2 31-57.5 Present in adult

P. AMPHIBIUS 27-30 33.2 45-56 Present in adult

HL= Head lengths, AL=distance from snout to anterior base of the anal fin, DL = distance from snout to anterior base of the dorsal fin.

Protopterus have 5-gill arches unlike Lepidosiren with 4 but lack the hypervascularized pelvic fins found in male Lepidosiren at spawning. However, they both lack the large scales, flipper- like pectoral and pelvic fins and single rather than paired lungs which occur in the Neoceratodus (Kemp, 1986; Nelson et al. 2016).

2.5.2 Habitat distribution of African lungfish The African lungfish are distributed in the tropical fresh waters of Africa where they occupy the lentic (still) waters including swamps, shallow pools, deep lakes and lotic (flowing) waters including streams and rivers as predators. P. annectens and P. dolloi inhabit West, central and southern water bodies of Africa including the rivers Congo, Zambezi, Limpopo and their associated water bodies while P. aethiopicus and P. amphibius are inhabiting East and South Central Africa (Graham, 1997; Berra, 2001; Helfman et al. 2009; Mlewa et al. 2010).

P. aethiopicus has been naturally occurring in the upper tributaries of rivers including Congo, Nile and their associated water bodies, the major East African rift valley lakes like Tanganyika, George, Edward, Albert and other major lakes including Kyoga and Victoria (Greenwood, 1958; Mlewa et al. 2006). P. aethiopicus has however been successfully introduced in other water bodies, where it has successfully established itself, with the major introduction being lake Baringo in Kenya in the 1970s (Greenwood, 1958; Mlewa et al. 2006). Many of its natural habitats in the Victoria basin have been affected by the introduction of invasive Actinopterygii

predators, thus reducing its survival and affecting the species distribution in its natural habitats (Helfman et al. 2009; Walakira et al. 2014).

P. annectens has been reported to exhibit discontinuous distribution in river Congo and its associated water bodies , unconnected water bodies in North West Africa, Nile and Zambezi rivers which gives a hint about the African lungfish species speciation and how it can be explained by the habitat distribution of these different lungfish populations (Mlewa et al. 2010)(Fig. 2.6).

Figure 2.6. The current distribution of modern species and subspecies of Protopterus in Africa (Otero, 2011).

2.5.3 The reproductive life cycle of African lungfish Protopterus and Lepidosiren genus exhibit the same parental care behaviour while Neoceratodus genus provides no parental care but produces sticky eggs which adhere to aquatic vegetation during their development. This supports the close relationship between Protopterus and Lepidosiren as compared to Neoceratodus genus (Kemp, 1986).

There is a lack of recent information on the reproduction biology of the African lungfish (Protopterus species) with spawning nests of P. amphibius not described (Mlewa et al. 2010). In P. aethiopicus, the males construct tunnel-like nests in shallow weedy areas where they guard the eggs and larvae (Mlewa et al. 2010). P. aethiopicus males make varying nest structures, but generally with tunnels having multiple entrances (submerged or exposed depending on the water level), excavated from a soft substrate of mud and vegetation, with eggs and larvae at the expanded base of the tunnel (Mlewa et al. 2010). Four different types of nests for P. aethiopicus were observed on the shores of Lake Victoria in Uganda with various forms and environment, but all in calm waters with low dissolved oxygen (less than 3ppm) and temperatures between 17.8 and 25 °C (Greenwood, 1958).

Males usually emerge from aestivation burrow with rising water levels to excavate breeding burrows in the mud of the temporary wet season swamps and mashes for P. annectens (Johnels & Svensson, 1954). P. aethiopicus and P. dolloi nest burrows are situated in permanent marshes in papyrus banks of about 3 m water depth, with water lilies and swamp grasses of 1 m deep and around 20 cm below the water surface (Greenwood, 1958, 1986). P. dolloi in Uganda has been documented to reproduce in pools with permanent water which are used for reproduction in rainy season and refugee by the males in the dry season. The males safeguard the eggs while the females forage offshore after spawning and reach the pools from open waters through submerged vegetation ( Greenwood, 1958; Brein, 1959; Bouillon, 1961). Exposed nests however also exist in shallow waters with no aquatic vegetation (Greenwood, 1986). There have been reports of year-round reproduction of P. aethiopicus in Lake Baringo, Kenya as juveniles (36-48 cm total length) were sampled throughout the year (Mlewa et al. 2006). Males of P. annectens and P. aethiopicus have been reported to spawn with various females during the same breeding season. This was reported by Bouillon (1961) and Greenwood (1958), who found both eggs and larvae of different stages in the same tunnel.

Hatching of P. aethiopicus eggs takes two weeks and a female of around 90 cm can produce around 5000 eggs while other reports have shown an increase from 4000 to 16000 eggs over a range of 2 to 10 kg, representing a mass exponent for the fecundity of 0.65 (Greenwood, 1958; Mlewa et al. 2006). The fecundity of African lungfish (1.6 eggs /g of mature female) may seem lower as compared to African carp, Labeo senegalensis with 1.79 eggs /g of mature female, African catfish (Clarias gariepinus) with 25.6 eggs /g of mature female and tilapia (O. niloticus) with 5.5 eggs/g of mature female. But this low level of fecundity is compensated by the high survival ensured by guarding males, the early tolerance of embryo and larvae to low dissolved oxygen levels. The anoxic conditions in the breeding nests scare away predators of both larvae and the guarding males ( Greenwood, 1958; Mlewa et al. 2010; Hossan et al. 2013; Abobi et al. 2015; Absalom et al. 2017).

Females of P. aethiopicus provide no parental care and are less available for fishing and sex ratio stock assessments, this is becuase they migrate inshore after attaining maturity yet most lungfish fishery is focused on swamps and marshes close to the shores (Greenwood, 1958; Mlewa, 2006). During excessive anoxic conditions, males of P. aethiopicus and P. annectens increase dissolved oxygen in the breeding tunnels by tail lashing, a vigorous mixing of surface water in the tunnel nest with air, using the posterior part of the body, this reduces predation of larvae by surface dwelling predators through reduced need for surface aerial respiration (Greenwood, 1986).

2.6 Freshwater fish larval feeding strategies and live feeds 2.6.1 Larval feeding First feeding Newly hatched freshwater fish larvae have a high nutrient demand due to their immature digestive system as live feeds have proved to be able to supply the required nutrients to the first feeding fish larvae (Sales, 2011).

Fish larvae feed on a variety of live feeds, however, zooplankton organisms are the most important live feeds for fish larvae (Davis et al. 2018). Rotifers (Brachionus spp.) and brine

shrimp (Artemia spp.) nauplii are the major live feed organisms used in freshwater fish larviculture, although other live organisms including microalgae, copepods and cladocerans also contribute to larval fish feeding (Sales, 2011).

Feeding based on live feeds is, however, expensive in terms of equipment and manpower needed for production. Thus live feeds should be limited to a short time after hatching to ensure economic viability amidst high growth and survival (Ruyet, 1989; Wang et al. 2009).

Some freshwater fish like catfish are able to first feed on manufactured feeds and thus have no need for live feeds during first feeding (Carter, 2015). An appropriate larval feeding regime can have various types of live feed and formulated feeds offered together or sequentially, which should satisfy the nutritional requirements right from the time the fish open their mouth to feed in order to reduce mortality and increase survival (Sales, 2011).

Besides a recent study by Sserwadda et al. (under review), no previous study has been reported on the larval rearing of African lungfish. However, various attempts have been done with juvenile African lungfish collected from the wild in many parts of Uganda by local fishermen and fish farmers. Walakira et al. (2014) cultured juvenile African lungfish collected from the wild in tanks feeding on fish pellets from silver cyprinids Rastrineobola argentea (68.3% crude protein and pellet size of 4-5 mm diameter). He reported that juveniles gradually accepted commercial diets and average body weight increased with dietary protein increase. Indigenous knowledge has also been used in wild caught juvenile culture (15-20 cm), these were fed on maize bran mixed with fish meal, tilapia and African catfish fry, grasshoppers, snails and food trash. This led to low survival (less than 40%) and cannibalism (Walakira et al. 2012). A study on the use of live feeds for the growth of African lungfish larvae has not been reported but it would provide a broader view into the larviculture of this species.

Weaning Weaning refers to the transition from feeding on live prey to feeding on manufactured feeds. The process is more complicated as compared with shifting from one manufactured feed to another and is often associated with high mortality and slow growth (Stoss et al., 2004; Carter, 2015).

Weaning strategies differ from species to species, with some fish larvae being liable to co- feeding, i.e. the combined feeding of live and artificial feeds, while others can be weaned abruptly to artificial diets or to another type of live diet (Giacometti et al. 2009; Kumar et al 2014; Carter, 2015). Various weaning strategies can be applied to the same species depending on the availability of feeds and the profitability of the production (Fig. 2.5). Co-feeding gives animals time to adapt in behaviour and physiology to new feeds providing suitable feeds across a range of sizes and developmental stages (Carter, 2015).

Figure 2.5. Different larval feeding (T1, T2 and T3) and weaning strategies (T4 and T5) for pengba (Osteobrama belangeri) larvae up to 25 DPH (Z=Zooplankton, D=Dry feed, GNOC= Groundnut oil cake & SE=Soya egg custard (Kumar et al. 2017). Freshwater fish larvae have all the necessary digestive enzymes at the onset of exogenous feeding, unlike marine fish larvae which acquire digestive enzymes activation by autolysis or uptake of zymogens from live feed organisms (Kolkovski, 2001). By the onset of exogenous feeding, freshwater fish larvae of white fishes Coregonous mareana and C. afferensis together with grayling fish Thymallus thymallus and burbot Lota lota were capable of protein, lipid and carbohydrate digestion due to proteolytic, lipolytic and carbohydrate enzyme splitting activities already taking place (Lahnsteiner, 2017). However, without exogenous digestive enzymes in micro-diets for first feeding marine larvae of gilthead seabream Sparus aurata, a 30% reduction in micro-diet assimilability was realised (Kolkovski et al. 1993). This explains the need for more live feed in marine fish larvae compared to freshwater fish larvae due to their role in activation of larvae endogenous digestive enzymes. Freshwater fish larvae have also been reported to be more adaptable to early weaning as compared to marine water fish

larvae, this is due to the bigger size of the larva that is more able to take up formulated feeds (Zitzow & Millard, 1988; Ruyet, 1989; Soltan, 2015). However, this is not always the case as butter catfish (O. bimaculatus) larvae showed poor survival when weaned as early as 7 DPH (Kumar et al. 2014). Early weaning (20 DPH instead of 40 DPH) in marine fish seabass Dichentrarchus labrax larvae, on the other hand, reduced feeding costs by the time fish larvae transformed to 50-day juveniles (Fig. 2.6).

Figure 2.6. Prey requirements, relative feeding cost and juvenile production cost of a 50 day juvenile seabass Dichentrarchus labrax according to weaning age (Ruyet, 1989). 2.6.2 Live feeds Rotifers Rotifers are microscopic aquatic organisms from the phylum Rotifera (Davis et al. 2018). The major rotifer species used as live feeds are Brachionus plicatilis and Brachionus rotundiformis. These have been cultured under both batch and continuous methods, with both methods used to require close to three times the larval rearing tank volume for rotifer and algae culture (algae to feed rotifers) which used to make hatchery operation costs high (Maruyama et al. 1997). However, this has been solved by new technologies like algae pastes with high algae concentrations which can be frozen for up to a year thus reducing rotifer production costs as these look to be cheaper and easily accesible (Davis et al. 2018). Brachionus spp. are very small, slow swimming and euryhaline filter feeders with a prolific reproduction rate and cultured by hatcheries using their parthenogenetic reproduction mode throughout the year

(Dhont et al. 2013; Carter, 2015; Davis et al. 2018). The use of the readily available yeast, and commercial products derived from this, to replace microalgae enables mass production of rotifers for larviculture (Peña-Aguado et al. 2005).

Rotifers usually lack sufficient highly saturated fatty acids (HUFA) for use for marine fish larvae but enrichment can be done since they are non-selective feeders, using algal concentrates from Tetraselmis spp. and Isochrysis galbana and other commericial enrichments high in both DHA and EPA to increase their HUFA content ( Davis et al. 2018; Dhert et al. 2001). Larvae can start feeding on rotifers immediately when the mouthparts develop (between 2 and 5 DPH), feeding on 3 to 10 rotifers/l and going up to 30 DPH depending on the fish species (Davis et al. 2018).

Artemia Brine shrimp (Artemia spp.) nauplii are widely used as live feeds in freshwater fish larviculture mainly due to their high nutrient content, large size which provides more biomass to the larvae, and convenience in use: i.e. they can be hatched within 24 h from dormant cysts which can be stored for a long time (Dhont et al. 2013; Thanh et al. 2013). Artemia is harvested from specific salt lakes where they grow naturally but they can also be cultured in artificial ponds and bio-floc systems, unselectively feeding on microalgae, bacteria and detritus (Fernández, 2001).

The umbrella stage of Artemia, is the first stage emerging from the cyst, is smaller than the nauplii and immobile, thus it can as well be used for fish larvae with a small mouth like cobia (Rachycentron canadum) as a replacement for rotifers (Dhont et al. 2013; Van Can et al. 2010). The use of Artemia as a live feed has increased over the years despite its increasing prices, unpredictability and high variability (Dhont et al. 2013). Artemia enrichment involves feeding Artemia on algae or commercial products, generally emulsions, to improve its nutritional content (Carter, 2015).

Microalgae Microalgae are not only used to feed zooplankton live prey but are also import as sole larval feed in larviculture due to their mass culture potential, cell size and digestibility (Tredici et al. 2009). A lot of algae species used in aquaculture have been reported to have 25-50 % protein and high Highly Unsaturated Fatty Acids (HUFAs), including Docosahexaenoic Acid (DHA) ,

Eicosapentaenoic Acid (EPA) and Arachidonic Acid (ARA), which are essential for larval development (Tredici et al. 2009; Dhont et al. 2013). Various species of freshwater microalgae are used in fish hatcheries including Monoraphidium minitum, Chlorella spp, Screnedesmus abundans and others (Isik et al. 1999). Some algae species are deficient in HUFAs but in some species like Chlorella, enrichment to provide these essential nutrients has been done by incorporating n-3 HUFAs in their cells through supplying of hydrolysed with fish oil from Tuna (Hayashi et al. 2001).

Other live feed organisms Copepods in nature constitute a major diet in early life stages of many fish species and have a superior nutrient composition in terms of HUFAs compared to Artemia and rotifers, in addition to a better-balanced triglyceride and phospholipid class composition. However, large scale reliable culture techniques remain a major bottleneck to copepod use as live feed in aquaculture (Davis et al. 2018; Dhont et al. 2013). Nauplii stage from copepods of order Cyclopoida is an important live feed in freshwater aquaculture mainly for common carp Cyprinus carpio (Szlauer & Szlauer, 1980).

Cladocerans (water fleas) are microscopic crustaceans mainly of freshwater origin with a few marine species, thriving in warm temperate climates where they are important zooplankton in the food web (Davis et al. 2018). Cladocerans like Daphnia spp and Moina spp are already being explored as live prey for aquaculture due to their good nutrient content, economic feasibility for mass culture, the slow movement making them easily visible by fish larvae and high content of digestive enzymes (Peña-Aguado et al. 2005; Gogoi et al. 2016).

Ciliates are single-celled eukaryotes that can be cultured as live feeds in aquaculture. Laboratory experiments have shown that ciliates survive a high range of environmental physico-parameters, have a higher amino acid content and swim slower as compared to copepods. Mass culture of ciliates for live feed use in larviculture has however not been realized of recent (Davis et al. 2018).

2.7 Freshwater fish larval gastro-intestinal tract (GIT) ontogeny and histology 2.7.1 General gastro-intestinal tract ontogeny The primordium (clustering of cells from which a part of an organ develops ) of the digestive system in fish appears during embryonic development (Zambonino-Infante et al. 2008). The digestive system morphogenesis and differentiation in fish also varies from species to species depending on the type of egg cleavage (Zambonino-Infante et al. 2008). In bony fishes, all organs including the digestive/gastro-intestinal tract differentiate in the embryonic shield and the yolk sac is later surrounded by periblast. In meroblastic eggs, a thick endoderm string on the yolk sac represents the primordium of the gastrointestinal tract (Zambonino-Infante et al. 2008).

The gastro-intestinal tract undergoes a rapid development from the simple undifferentiated, straight incipient gut of the yolk sac larvae lined with pseudostratified or cuboidal epithelia having basal and central nuclei and distinct apical short microvilli, to the complex segmented adult gastro-intestinal tract (Govoni et al. 1986; Zambonino-Infante et al. 2008; Bilal, 2012). This is in contrast to the gradual development in other body organs due to the immediate need to shift to exogenous feeding after yolk sac absorption (Govoni et al. 1986; Infante et al. 2008; Bilal, 2012; Moghadam et al. 2014; Gagnat et al. 2016). Gradual ontogenetical changes that occur as fish larvae increase size lead to differences in digestive requirements since the gastro-intestinal tract is functional at first feeding though structurally less complex as compared to the adults (Govoni et al. 1986).

The yolk-sac reserve is not always completely depleted in most fishes at the start of exogenous feeding, providing for a period of mixed nutrition. However, the first exogenous feed determines the rate of somatic growth, survival, and tolerance to stress (Gagnat et al. 2016; Kemp, 1981). In giant trahira Hoplias larcerdae, the yolk sac starts to reduce in size from 5 DPH and is there is near complete yolk-sac absorption by 7 DPH (Gomes et al. 2010) (Fig. 2.8). After completion of yolk-sac absorption, the incipient gut which has been appearing as an undifferentiated tube becomes segmented by valves to form the buccal pharynx, foregut, midgut, and hindgut ( Govoni et al. 1986; O’Connell, 1981) (Fig. 2.7).

Figure 2.7. Derivation, sequence, and timing of alimentary canal organs in typical larval fish (Govoni et al. 1986).

Development of the stomach and pyloric caeca from the posterior foregut, of the anterior intestines from the midgut and of the posterior intestines from the hindgut occurs after transformation (Govoni et al. 1986; O’Connell, 1981; Moghadam et al. 2014) (Fig.2.8). The liver and pancreas together with their respective juices are present at hatching though only functional at yolk-sac absorption (O’Connell, 1981).

2.7.2 Buccal cavity and oesophagus The buccal cavity is open at hatching in most larval fish with a posterior section filled with sac platelets. At the time of yolk sac absorption, it is always well developed and lined with squamous epithelium, mucous cells, and taste buds in preparation for exogenous digestion. The oesophagus at 1 DPH (Day Post Hatching) is always not differentiated but has a developed columnar epithelium with cells having supra nuclei vacuoles having acidophilic yolk inclusions (Govoni et al. 1986; Pradhan et al. 2012; Asgari et al. 2014). The start of exogenous feeding is related to the opening of the mouth and the associated development in the buccal cavity, and may range from hours to around 6 DPH depending on the feeding nature of the fish (Marimuthu & Haniffa, 2007; Pradhan et al. 2012; Nyang’ate Onura et al. 2018). In freshwater

angelfish Pteropyllum scalare, the mouth is only open at around 4 DPH while in giant trahira Hoplias lacerdae it opens at 3DPH (Fig.2.8) and this is the period associated with onset of exogenous feeding in these species (Çelik et al. 2014; Gomes et al. 2010).

Figure 2.8. Larval ontogeny of the freshwater giant trahira Hoplias lacerdae, from 1 to 7 DPH. (a) 1 DPH: otic vesicle (ov), encephalic vesicle (ev), yolk sac (ys) and embryonic fin (ef). (b) 2 DPH: pectoral fin (pf), miomeres (mm) and future mouth place (mo). (c) 3 DPH: heart (he) and opened mouth (mo). (d) 4 DPH: notochord (nt). (e) 5 DPH: eye (e) and gut (g). (f) DPH: operculum (op) and chromatophores (cr). (g) 7 DPH: mesenchymal rays (mr). All drawings are at the same magnification and the scale bar represents 1 mm (Gomes et al. 2010).

Goblet cells develop in the buccal cavity around 6 DPH and by the onset of exogenous feeding, they are reported to be covering the whole buccal pharynx epithelia with teeth differentiation from areolar connective tissue underlying buccal pharyngeal epithelia around the same time (Govoni et al. 1986; Asgari et al. 2014; Bilal, 2012). Reports suggest that in butter catfish, Ompok bimaculatus, by 9 DPH the pharyngeal papillae close to the connection of pharynx and oesophagus are already differentiated and increase in size during larval development with no histological growth (Pradhan et al. 2012). However, in the oesophagus, the elongated and epithelial lining becomes columnar with thin epithelial tissue, which disappears on the transition to the stomach and no further changes are observed after 9 DPH except for an increase in size and number of mucous cells (Pradhan et al. 2012).

2.7.3 Stomach At hatching, catfishes and other freshwater fish species have advanced digestive systems with a functional stomach, unlike larvae of many marine fish species with no functional stomach at hatching that thus undergo significant changes in the stomach during the first larval days (Carter, 2015). The stomach develops from the undifferentiated anterior ventral region of the yolk sac made up of multi-layered squamous epithelia, and starts to differentiate into the glandular (cardiac) stomach and non-glandular (pyloric) stomach by 2 DPH (Govoni, 1986; Verreth et al. 1992; Pradhan et al. 2012; Nyang’ate Onura et al. 2018). The glandular stomach starts to differentiate at 3 DPH with epithelium changing from squamous to columnar epithelium, which surrounds the gastric lumen still having yolk remnants and this time. This is followed by differentiation of gastric glands in this glandular region as clusters of cubic cells between 9 and 11 DPH (Govoni et al. 1986; Pradhan et al. 2012). The non-glandular epithelia start to differentiate between 6 and 7 DPH close to the anterior intestine, developing columnar cells with basal nuclei, apical vacuoles and PAS (positive acidophilic) microvilli (Govoni et al. 1986; Pradhan et al. 2012).

Between 8 and 14 DPH, the stomach develops to occupy most of the abdominal cavity and the longitudinal tubular glands having secretory cells with microvilli develop on the apical border. The cubic epithelial base and the pyloric sphincter separates the stomach from anterior intestines (Govoni et al. 1986; Verreth et al. 1992; Pradhan et al. 2012). The gastric

glands increase up to 14 DPH and reduced muscle thickness of the glandular stomach and gastric glands at the junction between the glandular and non-glandular stomach is observed. Stomach histological ontogeny generally stops at 14 DPH and further development involves an increase in size and complexity in relation to mucosal folds in the glandular and non- glandular region, the development of the gastric glands in the glandular region and tunica muscularis in the non-glandular region (Govoni et al. 1986). However, some freshwater fishes, mainly the Cyprinids like common carp (Cyprinus carpio), remain with no stomach but develop a functional digestive tract (Carter, 2015).

2.7.4 Intestines At hatching the incipient intestine appears as a straight translucent tubule segment dorsal to the yolk sac lined with columnar epithelium, microvilli at the apical surface and absorptive cells (enterocytes) arranged in a single layer with median nuclei (Pradhan et al. 2012; Mitra et al. 2015). The posterior region of the intestine is bent at 90° in butter catfish O. bimaculatus larvae by 2 DPH and the intestinal valve divided into posterior and anterior intestines with the rectum distinguishable (Pradhan et al. 2012). In giat trahira Hoplias larcerdae, the intestines are fully developed by 7 DPH with the rectum identifiable (Gomes et al. 2010). However, in featherback Chitala chitala the rectum is not fully distinguishable up to 8 DPH where it appears as a short flattened intestinal segment with no folds ( Mitra et al. 2015).

The first goblet cells are observed scattered in the intestinal mucosa at 2 DPH and they increase between 3 and 4 DPH from the proximal to the distal intestine, but decrease from 6 DPH (Pradhan et al. 2012; Mitra et al. 2015; Nyang’ate Onura et al. 2018). During the same time lipid droplets are observed in the enterocytes, followed by lipid accumulation due to inter- and intracellular fat deposits in the posterior region characterized by large vacuoles (Pradhan et al. 2012; Mitra et al. 2015). By 12 DPH the intestines appear to be properly coiled with lipid inclusion levels lowered to only the intestinal mucosa, eosinophilic supra-nucleus inclusion vesicles appear in the posterior gut and the intestinal mucosa is surrounded by circular internal and external musculature. The musculature is separated by a thin connective tissue and the pyloric caeca is pronounced and differentiated (Pradhan et al. 2012; Mitra et al. 2015). No further relevant modification is observed thereafter, except for an increase in size and length of intestines and number of folds (Pradhan et al. 2012; Mitra et al. 2015; Nyang’ate Onura et al. 2018).

2.7.5 Liver and pancreas The liver and pancreas are not differentiated at hatching and the primordial liver appears in the ventral yolk sac region as a mass of polyhedral hepatocyte cells with yolk pigment granules and large lipid vacuoles (Bilal et al. 2015; Mitra et al. 2015). Reports have however pointed out the pancreas to be partially differentiated by 1 DPH with the exocrine pancreas having polyhedral basophilic cells bearing round-shaped zymogen granules (Verreth et al. 1992; Pradhan et al. 2012). By 2 DPH, the pancreatic duct is surrounded by rosette pattern cells around a central canal, having squamous epithelia opened to the central part of the anterior intestines before the pyloric sphincter (Pradhan et al. 2012). At the same time, the hepatocytes of the liver exhibit basophilic homogenous cytoplasm and a nucleus having a prominent nucleolus. However, the cytoplasm is characterized by granulation and vascularization as proteins, lipids and glucagon are synthesized between 3 and 4 DPH ( Pradhan et al. 2012).

Various histological changes appear in the liver between 8 and 12 DPH, including lipid vacuole increase followed by a decrease between 8 and 10 DPH, increase in eosinophilic granules, hepatocyte differentiation and the yolk sac becoming completely dissolved, the gallbladder and bile duct becoming visible and increased hepatic lipid deposits (Bilal et al. 2015; Pradhan et al. 2012; Mitra et al. 2015) (Fig. 2.9). The pancreas as well shows numerous changes before 10 DPH from endocrine pancreas differentiation to the formation of islets of Langerhans by endocrine cell arrangement around numerous capillaries ( Pradhan et al. 2012). Reports have shown no major histological developments in both pancreas and liver after 12 DPH, except for an increase in size and number of pancreatic acini and islets of Langerhans (for the pancreas), and an increase in liver size occupying most anterior abdominal cavity ( Verreth et al. 1992; Pradhan et al. 2012; Bilal et al. 2015; Mitra et al. 2015) (Fig. 2.9).

Figure 2.9. Main histological ontogenetical landmarks during larval development of Snakehead murrel, Channa striatus (Bilal et al. 2015).

CHAPTER 3: MATERIALS AND METHODS

3.1 Sampling and histological procedures 3.1.1Tissue processing The development of the gastrointestinal tract was analysed using histological sections randomly obtained from selecting larvae samples from 17, 20, 22, 24 and 26 DPH with two samples selected to represent each DPH. The larvae were fixed by placing in Bouin’s solution to preserve normal morphology and facilitate further processing (Genten et al. 2009; Mokhtar, 2017). Dehydration was done with a series of graded alcohol using STP 120 Microm Tissue Processor and dehydrated larvae were placed head down into embedding moulds of fresh paraffin wax using a Thermo scientific Microm EC 350 modular tissue embedding centre according to Humason (1979). Embedded larvae could harden at room temperature hence the hot melted paraffin replaced the clearing agent in the process.

3.1.2 Tissue sectioning Paraffin-embedded blocks were placed on a rotary microtome (HM 360 Microtome), oriented properly in relation to the knife and trimmed according to Humason (1979). Serial transverse sections of 8 µm thickness were cut along the whole length of the specimen. Tissue sections along with wax were transferred using forceps to an alcohol bath at 10°C to soften the paraffin and expand the sections (Drury & Wallington, 1980). The sections were then transferred using a glass slide to a gelatine bath for 5 min at 37° C to help them adhere to the slide, later mounted on to on a glass slide and air dried overnight (Drury & Wallington, 1980).

3.1.3 Staining and cover slipping Staining with Hematoxylin and Eosin (H&E) was used for general observations where hematoxylin stained cell nucleus and other acidic structures blue, whereas eosin stained cytoplasmic proteins and extracellular structures from pink to red. This was done using a Thermo scientific Gemini AS automated slide stainer.

Periodic Acid Schiff (PAS) staining was used to detect neutral and acid muco-substances in mucous cells and glycogen deposits in the liver staining PAS positive sites magenta/red while Alcian Blue (AB) positive sites remained blue (Genten et al. 2009; Gurcan et al. 2009). This was done manually involving deparaffinisation and dehydration in a series of ethanol followed

by rinsing in flowing tap water for 15 min, the slides were then immersed in periodic acid for 10 min and flushed with tap water for 10 min. A 20 min immersion in Schiff reagent was done followed by 20 min washing in running water to rinse the slides before a 5 min hematoxylin immersion. This was followed by a 5 min rinsing of the slides in running tap water. The stained sections were covered with glass to protect the tissue from being scratched, to produce better optical properties for viewing under the microscope and preservation of the tissue sections (Genten et al. 2009; Mokhtar, 2017). This was done by taking stained slides through a series of alcohol solutions (70, 90 and 100%) to remove the water and clearing agents before placing a permanent resinous medium (Thermo scientific DPX mounting media) beneath the coverslip over the section.

3.1.4 Histological analysis Histological analysis of the tissue samples was done using an Olympus BX61 light microscope and an Olympus DP73 camera. CellSens Dimension software by Olympus life science solutions was used to take random measurements in the spiral valve which were averaged to determine diameter at a point.

The epithelial tissue was described and classified based on the number of cell layers from the basement, the shape of cells at the free surface and surface modifications of the cells according to Jennings & Premanandan (2017).

3.2 3D Reconstruction 3.2.1 Selection of slides for reconstruction The quality of the slides from 17 and 26 DPH larvae was evaluated basing to completeness, clarity and general appearance of structures using an Olympus BX53 light microscope. The best set of slides was selected to be used for reconstruction while the remaining slides for one larva were kept as a reference to be used later in verifying and validating data obtained from 3D reconstructed images according to Cornillie et al. (2008).

3.2.2 2D image acquisition protocol 2D digital images for the selected slides were captured using an Olympus DP73 digital camera mounted on an Olympus BX61 light microscope with a 4X and 2X magnification objective lens for 17 and 26 DPH larvae, respectively. A total of 1593 and 1457 images were obtained from

a single 17 DPH and 26 DPH larva, respectively. Image stacks of optical sections obtained in a TIF were converted and stored in jpeg format with a high XY resolution (pixel size of 4.34 µm and 2.17 µm, respectively) to enable proper recognition and differentiation of organs (Rudskiy & Khodorova, 2012).

3.2.3 Reconstruction procedure Image import and alignment of image slices A separate file was created for 17 and 26 DPH images having relative distance of each slice from the position of the first slice with an increment of 8 µm according to Cornillie et al. (2008). The images were loaded separately into Amira 6.7 software in stacked slice file format and were automatically aligned using automated least square alignment followed by manual alignment for images with physical cross section shifted during image acquisition.

Image segmentation Aligned image slices were resampled in high-quality mode into Amira mesh file format to produce a data set with a coarser resolution. Deformed and damaged image slices were recomputed from adjacent slides using the interpolate command in the Amira 6.7 application. Image segmentation was done according to Cornillie et al. (2008). This involved assigning every pixel of each picture a value between 0-255 visually represented by the grey tone value (0 = black and 255= white using the standard setting in the Amira 6.7 application). The number attributed to the pixel formed the basis for the semi-automated labelling of the sections using the original red- green- blue colour field value.

The segmentation editor of the Amira software was used to label contours of the buccal cavity, oesophagus, spiral valve and other important structures. Semi-automatic labelling was done where possible involving manual contour drawing every 10 to 20 selections followed by labelling selections in-between through the interpolating command of the segmentation editor.

Surface generation, editing and visualisation The organs labelled were checked for continuity on previous and next slices and correct labelling was confirmed by comparing original histological slices with labelled digital slices. Small patches in organs that had been left out were corrected using the “remove islands” command in the segmentation editor section of the software.

The voxel size of all bricks was down-sampled to 3X3X2 in the X, Y and Z direction, respectively, using the resampling module to create a 3D image from the bricks of the labelled sections. All surfaces were built from triangles with an edge length of 0.4 times down-sampled to voxel sizes as the last post-processing compact step. The generation of 3D surfaces was done using the Generate surface command and were visualised through the Surface view module of the application. Surfaces were smoothed by selecting the Vertex normal module in more options under the surface view module. The resulting surfaces were later closed after confirmation of boundaries of the lungfish larvae with the exterior. The obtained images were compared with histological slices of the reference lungfish larvae of the same age according to Cornillie et al (2008).

CHAPTER 4: RESULTS

4.1 Summary of histological and morphological landmarks between 17 and 26 DPH Histological and morphological developments between 17 and 26 DPH in various structures of organs including the buccal cavity, pharynx, oesophagus, spiral valve, liver and pancreas were highlighted by distinct landmark features (Table 4.1). These features included change in shape of the teeth and appearance of the pharynx in case of the buccal-pharyngeal cavity. Progressive changes in connective tissue composition was used as landmark feature for the oesophagus whereas lumen size, presence of lymphocytes and supranuclear vacuoles and the number of coils in the posterior part of the valve were used for the spiral valve. Shape of hepatocytes and number of lobes were used as landmarks for the liver and pancreas, respectively.

Table 4.1. Histological and morphological landmarks in lungfish larvae as observed between 17 and 26 DPH. LANDMARK AT RESPECTIVE DAY POST HATCHING (DPH)

ORGAN Structure 17 20 22 24 26

BUCCAL CAVITY Teeth Canine-like Canine-like Canine-like Tooth Tooth teeth with teeth with teeth plates plates no enamel an enamel forming present present cap cap teeth plates

PHARYNX Appearance Folded and Folded and Straight and Straight Straight wide wide narrow and narrow and narrow

OESOPHAGUS Connective Loose Loose and Loose and Loose and Loose and tissue connective dense dense dense dense tissue connective connective connective connective tissue tissue tissue tissue

SPIRAL VALVE Anterior part Partially Fully open Fully open Fully open Fully open closed

Lymphocytes Absent Absent Absent Present Present

Supranuclear Absent Absent Absent Present Present vacuoles

Complete One One One Two Three spiral coils

Yolk sac Present Present Present Only in Absent residues throughout throughout throughout posterior

CLOACA Invagination Long and Long and Short and Short and Short and and length slightly slightly slightly highly highly invaginated invaginated invaginated invaginated invaginated

LIVER Hepatocytes Irregular in Polyhedral Polyhedral Polyhedral Polyhedral shape with large with large vacuoles vacuoles

PANCREAS Lobes One lobe One lobe One lobe Two lobes Two lobes

4.2 3D Reconstruction The internal and external morphology of both the 17 and 26 DPH lungfish larvae were all conserved thus making it possible for proper 3D reconstruction of the selected samples. In both 17 and 26 DPH (Fig. 4.1(a & b)), a short buccal cavity linked the long and elongated pharynx to the spiral valve via a laterally constricting narrow oesophagus. The spiral valve connected to the cloaca which had an anal opening. The pancreas appeared to occupy a narrow area at the beginning of the spiral valve and dorsal to the liver. The reconstruction revealed lateral elongation of both the larvae and internal body organs between 17 and 26 DPH (Fig. 4.1(a & b)).

Pronounced morphological changes occurred in the spiral valve and the cloaca as shown in Fig. 4.2 (a & b). The exterior of the spiral valve at 17 DPH appeared as a laterally compressed tube that coiled once at the end of the anterior part (Fig. 4.2a). By 26 DPH, the coiling structure of the spiral valve appeared to form 3 complete coiling spiral turns from the middle of the valve forming laterally enlarging coils that extended slightly after compressing to form the cloaca (Fig.4.2b). The cloaca appeared to turn alternately to the right and left laterally towards the anal opening by 17 DPH but it only appeared as a constricted, smaller and shorter invaginated tube by 26 DPH (Fig. 4.2 (a & b)).

Figure 4.2. Three-dimensional images showing a lungfish spiral valve (A) and cloaca (B) at 17DPH (a) and 26 DPH (b). C= Spiral turns/coils. (1 square = 1µm2).

4.3 Histological observations between 17 and 26 DPH 4.3.1 Buccal cavity By 17 DPH the buccal cavity had a lot of taste buds, more so on the upper side. The buccal cavity had an evolving stratified squamous epithelium with cells having basal nuclei and goblet cells protruding into the lumen. The epithelium appeared to be actively growing as several cells were observed to be undergoing mitosis. These secretory cells stained purple with PAS staining indicating the presence of neutral goblet cells. Tooth cusps were observed piercing through the epithelia on each side of the membrane (Fig. 4.3a).

Between 20 DPH and 22 DPH, the buccal cavity was lined with bi-stratified squamous epithelia that became multi-stratified in fields close to the tooth cusps. More external and internal taste buds appear along buccal cavity epithelia. There was an increase in the number of goblet cells in the buccal cavity. The tooth cusps appeared to be emerging further out of the epithelial tissue of the buccal cavity into the lumen as evidenced by 3 tooth ridges on each side of the cavity, with teeth on the upper side appearing to be originating from a single tooth plate (Fig. 4.3a). The squamous epithelial tissue covering the teeth by 20 DPH started to peel off, revealing the enamel cap of the canine-like teeth (Fig. 4.3a).

By 24 DPH the outer layer of the buccal cavity was lined with a simple squamous epithelium which had apical nuclei and a thick basement membrane. This epithelium got stratified inside the buccal cavity to form a stratified squamous epithelia with numerous mucous cells and taste buds protruding into the lumen of the buccal cavity (Fig. 4.3b, Fig. 4.3c). There was an enormous increase in the number of mucous cells as compared to 20 DPH larvae. The teeth at this stage had already protruded into the lumen of the buccal cavity and lost the initial canine-like edges to merge into a single tooth plate on the upper cavity and two tooth plates on the lower cavity. The tooth plates had pointed edges from the precursor tooth ridges that had been seen protruding out of the buccal cavity at 20 DPH (Fig. 4.3c). The teeth forming the tooth plates started to resemble fully developed teeth having an enamel cap, dentine layer and pulp core made up of cartilage. The tooth plates appeared to be immovable as they lacked the basal ligament. New immature teeth cusps could be seen under the epithelia of the buccal

cavity towards the pharynx by 26 DPH and there was increased calcification of the tooth plates (Fig. 4.3b).

4.3.2 Pharynx At the posterior end of the buccal cavity after teeth, there was a reduction in diameter to form the pharynx lined by a thin bi-stratified squamous epithelium on the upper part and a simple squamous epithelium on the lower part by 17 DPH. The pharynx had an enormous number of taste buds compared to the anterior part of the buccal cavity as observed on 17 DPH. A thin basement membrane separated epithelial tissue from loose connective tissue and a thick muscularis layer made of smooth muscles covered the pharynx which led to the oesophagus. There was a constriction and separation of the pharynx distally to form a narrow tube which was the oesophagus and the gill arches on opposite sides which were lined with a simple squamous epithelium. The pharynx had a tongue with a tip lined with a simple squamous epithelium which got stratified with mucous cells and taste buds towards the end of the tongue where it formed the lower cavity of the pharynx. Between 17 and 26 DPH, there was an increase in both the goblet cells and taste buds in the pharynx (Fig. 4.3d).

Figure 4.3 PAS staining light microscopy cross sections of the buccal cavity (B) and pharynx (C) of the African lungfish larvae at 20 DPH (a) and 24 DPH (b, c and d). This reveals the eyes (A), emerged tooth plates (thin arrows), the stratified epithelia of the buccal cavity (H) and the simple epithelia along the lower cavity of the pharynx. (a) shows the buccal cavity (B) with immature canine-like teeth (big arrows) protruding out of the epithelia. (d) reveals the taste buds (E), mucous cells (F) and loose connective tissue (G) surrounding the epithelia of the pharynx lumen (C) at 24 DPH. Scale bar (a, b and c) = 200µm while d=100 µm. 4.3.3 Oesophagus By 17 DPH, the anterior part of the oesophagus appeared to have the bistratified squamous epithelium lined with a thin basement membrane separating it from the surrounding connective tissue. Both dense and loose connective tissue surrounded the epithelia with the outermost muscularis composed of bundles of longitudinal smooth muscles that surrounded the whole oesophagus with a small adipose layer. The anterior part had single secretory cells that appeared to be scattered in the epithelia protruding into the lumen (Fig. 4.4). Towards

the posterior, the whole epithelia appeared to be covered by secretory cells surrounding the lumen after the constriction of the oesophagus to form the respiratory organ.

The posterior oesophagus was made up of columnar epithelia lined with a thin layer of loose connective tissue and the smooth muscle forming the muscularis was greatly reduced (Fig. 4.4). The posterior of the oesophagus appeared to be folded on the inside and folding increased greatly towards the start of the spiral valve. No changes were observed in the structure of the oesophagus between 17 and 26 DPH apart from an increase in dense connective tissue at the point of constriction to form the respiratory surface and the secretory cells covering the lumen.

Figure 4.4. PAS stain cross section of a 24 DPH larvae oesophagus under a light microscope. The oesophagus lumen (B) surrounded by mucous cells (arrows), loose (D) and thick (F) connective tissue and a thick layer of smooth muscles (E). A= vertebral column, C = blood vessel. Scale bar= 50 µm.

4.3.4 Spiral valve At the end of the oesophagus, a simple columnar epithelium appeared to transform into bistratified columnar epithelia as seen by 17 DPH. The diameter of the gut rapidly increased as the epithelial tissue later changed from bi-stratified columnar epithelia to a simple columnar epithelia made up of enterocytes marking the anterior part of the spiral valve (Fig. 4.5(a, b & c)). The enterocytes in the anterior part of the valve had a length of about 24 µm. In the spiral valve, a thin basement membrane separated the epithelium from a thin loose connective tissue which was surrounded by a simple layer of mesenchymal tissue lining the whole spiral valve (Fig. 4.5c). Particles that stained purple in PAS appeared in the epithelium indicating the presence of goblet cells in the spiral valve at this stage and yolk sac residues were evident in the anterior part of the spiral valve by 17 DPH (Fig. 4.5(a, b & c)). The lumen of the anterior part of the valve was narrow with a diameter of less than 1 µm but open in the posterior with goblet cells protruding into the lumen of the spiral valve increasing towards the posterior (Fig. 4.5(a, b & c)).

Between 20 and 22 DPH, there were less yolk sac residues in the posterior part of the spiral valve as compared to the anterior, with the lumen in the anterior having a diameter of about 5 µm and epithelial tissue with a thickness of about 10 µm (Fig. 4.5d). PAS staining positive particles identified as food were observed starting from 20 DPH in the posterior part of the spiral valve. Enterocytes in the posterior parts of the valve with these particles appeared compressed thus acquiring a cuboidal structure (Fig. 4.5d).

Figure 4.5. Light microscopy cross-sectional view of the spiral valve of the lungfish larvae at 17 DPH (a, b and c) and 20 DPH (d); a and d are PAS stains while b and c are H&E. (a) reveals the 17 DPH anterior spiral valve closed with numerous goblet cells in the spiral valve (blue to magenta) together with high yolk sac residues (red droplets in spiral valve). The mucous cells in the oesophagus (D) are also shown (magenta colour). (b) and (c) show the mid spiral valve at 17 DPH with (c) having a magnified view to the yellow box in (b). The enterocytes (black arrow) of the spiral valve (E) form the simple columnar epithelium while the oesophagus (D) has a stratified columnar epithelium (white arrow). (d) shows the posterior spiral valve at 20 DPH with food particles (H) and mucous cells protruding into the lumen (a blue patch in between cells (E). A= Liver, B= Pancreas, C= spleen, F= lungs, G= Kidney. Scale bar (a, b and d) = 100 µm while c = 50 µm.

By 24 DPH, the spiral valve had an increased number of mucous cells as compared to 22 DPH larvae, an indistinct fringe at the surface of the epithelium towards the lumen and thin basement membrane throughout the whole mucosal tissue (Fig 4.6a). The anterior spiral valve also appeared to become more coiled to the inside forming a complete second fold with a thickness of around 13 µm and a lumen diameter of around 8 µm (Fig. 4.6a). The nuclei appeared to take different positions within the enterocytes of the spiral valve (basal, central

and apical) but cells shared the same thin basement membrane (Fig. 4.6a). Supranuclear vacuoles having a diameter of around 3 µm were observed in the basal part of epithelial cells spaced throughout the anterior part (Fig. 4.6a). Goblet cells were also observed to be actively secreting into the spiral valve lumen in the superficial layer of the epithelium but still had their bases deep in the epithelium (Fig. 4.6a). At this stage lymphocytes could be seen scattered in the mesenchymal tissue of the spiral valve and the residues of the yolk sac were almost depleted in the spiral valve (Fig. 4.6a).

On 26 DPH the spiral valve had depleted yolk sac deposits and there was an increase in the number of supranuclear vacuoles in the anterior part, although they still had around the same size as on 24 DPH. There was increased vasculature in the mesenchymal tissue surrounding the spiral valve epithelium. The diameter of the lumen had decreased to around 5 µm and the thickness increased to around 17 µm in the anterior part of the valve. The spiral valve appeared to divide in the posterior end to form three longitudinal tubes that later combine to form one valve (Fig. 6(b & c)). It can also be observed that in the rear end of the valve the spiral coils compressed laterally forming a highly invaginated tube marking the start of the cloaca.

Figure 4.6. Cross section PAS staining light microscopy of the African lungfish larvae posterior spiral valve at 24 (a) and 26 (b & c) DPH. a shows the supercoiling of the spiral valve forming two super folds (G) having numerous mucous cells (*) protruding into the lumen of the valve with supranuclear vacuoles (white arrows) close to the thin basement membrane (H) separating epithelial cells (D) from mesenchymal tissue (B). The spiral valve had blood vessels (A) and lymphocytes (C). b & c reveal the spiral valve forming partitions (PT) starting at the end of the posterior side. FD =Food, E =Kidney, and F=Lungs. Scale bar (a, b and c) = 100 µm.

4.3.5 Cloaca and anal opening As observed from 17 DPH till 26 DPH, the gut appeared to constrict laterally towards the end of the spiral valve forming the cloaca which appeared as a hollow tube in the anterior, while the posterior end was heavily invaginated as it led to the anal opening. The cloaca was made up of stratified columnar epithelia that appeared cuboidal when extended (Fig. 4.7a). A thick basement membrane separated epithelia from loose connective tissue which connected the

kidney and the cloaca (Fig. 4.7a). The lumen of the cloaca had a few secretory cells scattered throughout and these increased between 17 and 26 DPH. The muscularis covering the cloaca epithelia was made up of a thick layer of smooth muscles (Fig. 4.7a). Lateral constriction of the cloaca formed a highly invaginated structure which led to the anal opening forming the last part of the gastro-intestinal tract, as shown in (Fig. 4.7b). The anal opening was surrounded by a thick layer of smooth muscles and opened to the outside (Fig. 4.7b).

Figure 4.7. Light microscopy PAS staining of the cross (1) and longitudinal (2) section of a lungfish larva cloaca at 20 and 26 DPH, respectively. The columnar epithelia of the cloaca (thick black arrows) appear multi-stratified at 20 DPH, yet bi-stratified at 26 DPH with a thick basement membrane (white arrows) surrounded by a loose connective tissue (*) surrounded by a thick layer of smooth muscles (F). At 26 DPH (2) the cloaca (E) is short and highly invaginated connecting the spiral valve (D) to the anal opening (G). 4.3.6 Liver and pancreas The liver appeared as a large organ close to both the oesophagus and spiral valve with irregularly shaped hepatocytes with peripheral nuclei by 17 DPH that transformed into clear vacuolar polyhedral hepatocytes that appear enlarged with a central nucleus by 26 DPH (Fig. 4.8a,b and c). The liver was lined with a simple squamous epithelium and thin connective tissue. Intrahepatic ducts were observed scattered throughout the hepatocyte cells forming the bile canaliculi fully visible from 24 DPH (Fig. 4.8c). PAS staining by 24 DPH revealed small amounts of collagen between hepatocyte cells in the liver. Lipid granules were scattered in the hepatocytes by 17 DPH but not present from 22 DPH onwards (Fig. 4.8a & b). By 24 DPH some hepatocytes appeared stained magenta by PAS staining, representing the presence of polysaccharides hence glycogen in the liver (Fig. 4.8c). There was formation of the bile duct

as a round tube made of a simple cuboidal epithelium surrounded by a thin connective tissue connecting to the gall bladder (Fig. 4.8d and e).

The exocrine pancreas appeared as a discrete organ lying in between the spiral valve and the liver. It appeared to be composed of a single lobe by 17 DPH, which divided to form two lobes of different sizes and irregular in shape by 24 DPH (Fig. 4.8f). The lobes were made up of masses of exocrine acini and acinar units separated from one another by connective tissue. The acinar units were fully developed secretory eosinophilic granules (Fig. 4.8f). The pancreatic duct was fully developed by 24 DPH with endocrine islets appearing to be dispersed among exocrine cells as shown in (Fig. 4.8f).

Figure 4.8. Cross sections of lungfish larvae liver and pancreas in PAS (a, b, c & f) and H&E (d & e). The liver (B) of a 17 DPH larvae (a) and magnified in (b) reveals lipid droplets (Lp) and

irregularly shaped hepatocytes (Hp) while that of a 24 DPH larva (c) has glycogen deposits (magenta colouring) and regular polyhedral hepatocytes with numerous intrahepatic ducts (Hd). The liver at 24 DPH has a fully formed gall bladder (C) as revealed (d) with a simple columnar epithelium (white arrows) connected to it via the bile duct (black arrows) (the portion in the yellow box in d is magnified in e). The pancreas by 24 DPH (f) appears well developed with two lobes (H) having endocrine secretory acinar units (Ac) and dispersed exocrine islets (G). A=Lungs, D= spleen, E=Oesophagus lumen, Ms= oesophagus secretory cells, Sp= spiral valve lumen, F=Kidney and Pv= Portal vein.

CHAPTER 5: DISCUSSION

5.1 Larval digestive ontogeny Larval digestive ontogeny has been described in various species using methods that target gross morphology, histology, ultrastructure and this maybe in combination with biochemical, immunological or molecular techniques (Rønnestad et al. 2013). There are differences in ontogeny, feeding physiology and nutrient requirements even among fish species of the same family, thus a holistic understanding of the nutrient supply line for each species is important for larval diet formulation and rearing conditions adaptations if optimum larval growth is to be realised (Hamre et al. 2013). Most research on larval feeding physiology has focused on altricial fish larvae (larvae that develop a functional stomach during metamorphosis) and few studies on agastric (stomach-less) larvae, probably due to their recent unpopularity in aquaculture (Rønnestad et al. 2013).

Studies on histological and morphological development of digestive systems in fish shed light on the digestive physiology, thus providing information for synchronisation of domestication procedures with environmental conditions for proper larval rearing (Kumar et al. 2019). As the case in most fish species including gilthead seabream, Sparus aurata (Calzada et al. 1998), stripped murrel, Channa striatus (Bilal, 2012), butter catfish, Ompok bimaculatus (Pradhan et al. 2012), featherback, Chitala chitala (Mitra et al. 2015) and stinging catfish, Heteropneustes fossilis (Kumar et al. 2019), the alimentary canal in African lungfish is undifferentiated at hatching but fully developed and functional by the end of the larval stage (Govoni et al. 1986).

Following a histological study of African lungfish larvae between 6 and 17 DPH (Sserwadda et al. under review), the current study utilised morphological and histological methods to analyse developmental ontogeny of African lungfish gastro-intestinal tract between 17 and 26 DPH. Morphological analysis of the gastro-intestinal tract with the help of 3D reconstruction provided an architectural visualisation of the gastro-intestinal tract (Gunasekara et al. 2011), and this enabled the evaluation of development by observing structural differences between 17 and 26 DPH larvae.

5.2 Morphological development Morphological follow-up in larval development not only helps in assessment of maturation in the gastro-intestinal tract but is as well very essential in evaluating its changes in ability to utilise food during larval development (Santos et al. 2016). This helps in development of

proper larval feeding regimes in new aquaculture species (Zaiss et al. 2006). Larval intestinal morphological development has been analysed utilising different techniques other than 3D simulation in various fish species, including change in mucosa morphometric parameters in African catfish (Nyang’ate Onura et al. 2018) and digestive enzymatic activity changes in common carp (Farhoudi et al. 2013). The evolution of 3D models in biological research has enabled proper observations of organisms anatomical features providing insights into morphology and functional enhancements for even smaller animals. This has been made possible by using digital image acquisition techniques like microscopy and scanner-based non- invasive virtual sectioning techniques like X-ray computed tomography (CT), magnetic resonance imaging (MRI) and micro CT (Laforsch et al. 2012). Morphology and spatial organisation of the digestive system is challenging using 2D histological sections, more so when the larval gut gets coiled and elongated during ontogeny (Kamisaka & Rønnestad, 2011) as is the case with African lungfish. However, depending on the resolution of the imaging technique used, 3D reconstruction may miss relative details of some biological structures and pitfalls in correcting and viewing of the image may also attribute to reconstruction artefacts (Cornillie et al. 2008; Laforsch et al. 2012). In the current study, the disadvantages of using histological sections for 3D reconstruction did not hinder the study due to use of high- resolution images and manual correction of artefacts.

Various studies have been done on morphology of organs using 3D simulation in both plants and animals (Cornillie et al. 2008; Gunasekara et al. 2011; Rudskiy & Khodorova 2012). The first study of fish larval digestive ontogeny was done on Atlantic cod (Gadus morhua), providing a deeper understanding of the feeding mechanism and digestive physiology during ontogeny in addition to digestive organ morphology (Kamisaka & Rønnestad, 2011). Recent studies using 3D models have as well proved to be a strong breakthrough in larval nutritional research as observed in Atlantic halibut Hippoglossus hippoglossus (Gomes et al. 2014), African lungfish (Sserwadda et al. under review) and long snouted seahorse Hippocampus guttulatus (Ofelio et al. 2019).

In the current study, 3D images revealed a lateral elongation of both larvae and its internal organs between 17 and 26 DPH; similar results have been reported using 3D models in studying ontogeny in Atlantic halibut (Hippoglossus hippoglossus) by Gomes et al (2014). The elongation is more pronounced in the spiral valve reflecting the fusiform shape described in

juvenile Australian lungfish (Neoceratodus fosteri) intestines by Hassanpour & Joss (2009) and in West African lungfish (Protopterus annectens) by Icardo et al. (2010). These finds are as well in line with findings on Atlantic cod where a 3D model revealed a large volume of the anterior midgut forming a bulk like appearance (Kamisaka & Rønnestad, 2011). This provides for an increase in residence time of food enabling more time for food mixing with secretions from the pancreas and bile (Ueberschar et al. 2013). Studies have proved that the oesophagus in larval fish becomes open, narrower and longer after yolk sac absorption, marking the start of exogenous feeding which was proved to depend on both oesophagus diameter and prey width (Busch, 1996). The current study also proves that the spiral valve develops from a single coil (17 DPH) to 3 spiral coils (26 DPH), indicating an increase in the absorptive surface (Zambino-Infante et al. 2008; Ofelio et al. 2019) and a development towards the juvenile stage which is represented by 6 spiral coils in West African lungfish (Icardo et al. 2010) and 9 spiral coils in Australian lungfish (Hassanpour & Joss, 2009). Such features are almost difficult to illustrate in histological sections, thus showing the importance of 3D models. These morphological developments represent a need to increase the feeding ration around 26 DPH due to increased digestion and absorptive surface in the spiral valve together with the functional development of the accessory organs.

5.3 Histological development 5.3.1 Buccal and pharyngeal cavities The timing of histological ontogeny events in fishes differs from species to species, with temperature being a major factor, as higher temperatures have been associated with faster histological ontogeny of the gastro intestinal tract (Kumar et al. 2019). The buccal and pharyngeal cavity had stratified squamous epithelia that was fully developed by 26 DPH with numerous taste buds and goblet cells (Govoni et al. 1986; Moghadam et al. 2014). The stratification and number of goblet cells and taste buds (both internal and external) increased during larval development between 17 and 26 DPH. Similar observations were reported in different other fish species (Govoni et al. 1986; Genten et al. 2009; Moghadam et al. 2014).

The appearance of taste buds and mucous secreting cells (goblet cells) during larval ontogeny varies from species to species in fish, depending on the organ and related function (Moghadam et al. 2014). By 17 DPH they were observed along the epithelia of the buccal and pharyngeal cavities in African lungfish. The presence of goblet cells by 17 DPH indicates that

at this stage the fish had started exogenous feeding using goblet cells for production of mucin in the buccal and pharyngeal cavity (Zambonino-Infante et al. 2008; Ostaszewska et al. 2011). The presence of taste buds by 17 DPH indicates that the African lungfish larvae at this stage fully had the ability to select prey on an organoleptic basis even before the full transition to exogenous feeding (Moghadam et al. 2014; Sserwadda et al. under review). The taste buds became numerous and bigger in size by 20, 22, 24 and 26 DPH to accommodate changes in the diet during the larval stage (Govoni et al. 1986), since the larvae had been provided with a mixed diet during larviculture (Sserwadda et al. under review). African lungfish is predominately carnivorous as an adult with over 50% of its diet comprising of Nile tilapia (Oreochromis niloticus) according to Mlewa et al. (2006). The availability of a tongue by 17 DPH and its development by thickening of the epithelia floor with the stratified epithelia by 26 DPH prepares the fish to this type of feeding involving descaling of prey. This has also been reported in marine fishes to coincide with exogenous feeding (Zambonino-Infante et al. 2008).

The current study reveals that between 17 and 24 DPH, teeth changed from sharp canine like cusps protruding from the oral epithelium to molariform tooth plates surrounded by a stratified columnar gingival epithelium. Similar results had earlier been reported in Australian lungfish by Kemp (2002) and African cichlid fish species Burton’s haplo, Astatotilapia burtoni and rainbow krib, Pelvicachromis pulcher (Genten et al. 2009). This change in teeth structure during larval development in African lungfish is correlated with the change in jaw functions from tooth cusps that just grasp prey, to tooth plates that can be used for crushing and rotational grinding of prey as fish progress from larvae to the highly carnivorous juveniles over time (Kemp, 2002; Zambonino-Infante et al. 2008). Teeth at the start of the pharynx have been reported to act as barriers preventing backflow of material from the oesophagus into the buccal cavity (O’Connell, 1981). The formation of the inceptive tooth plate observed in 26 DPH larvae highlights the possibility of feeding with artificial diets, which may represent an option of weaning in the larval rearing of African lungfish at this stage.

5.3.2 Oesophagus The structure of the oesophagus in African lungfish between 17 and 24 DPH is similar to that described in a lot of teleost fishes (Arellano et al. 2002; Zambonino-Infante et al. 2008) with mucosa having stratified epithelia and muscularis with striated muscle fibres. By 24 DPH, the

oesophagus had both stratified squamous epithelia and columnar epithelia depending on the part of the oesophagus with columnar epithelia towards the start of the spiral valve. Similar results were reported in the oesophagus epithelia of large yellow croaker (Larimichthys crocea) by Mai et al. (2005). As described in Senegal sole Solea senegalensis, Nile tilapia Oreochromis niloticus, African catfish Clarias gariepinus and other fish species, the oesophagus in African lungfish larvae lacked taste buds (Awaad et al. 2014; Genten et al. 2009; Arellano et al. 2002; Martin & Blaber, 1984).

The goblet cells present in the oesophagus produce muco-substances, which not only act as lubricants protecting the mucosa from abrasion, but reports have also proved they contain sialic acid residues which prevent viruses from recognising their receptor determinants and prevent bacterial attack of sialidase (Genten et al. 2009; Scocco et al. 1998; Zimmer et al. 1992). The presence and increase in the number and size of goblet cells between 17 and 26 DPH thus epitomises the possibility of endo-exotrophic feeding (Moghadam et al. 2014), while representing an increased immunity and protection of the oesophagus mucosa on transition to exogenous first feeding. In agastric fish species, it has also been proved that goblet cells in the oesophagus can be a morphological adaptation to replace the functional stomach depending on their specialisation, which was however out of scope for the current study (Ofelio et al. 2019). The oesophagus by 17 DPH is characterised by longitudinal folds and intraepithelial capillaries, which allows maximum distension for larger prey intake and breaking down food particles (Grosell et al. 2011; Genten et al. 2009; Zambonino-Infante et al. 2008).

5.3.3 Spiral valve The spiral valve of the African lungfish by 17 DPH was connected to the anterior digestive tract by a narrow oesophagus as it appears in many other stomach-less fishes (Hassanpour & Joss, 2009; Wallace et al. 2005). The stratified columnar epithelia in the posterior oesophagus is replaced by a simple columnar epithelium on connecting to the anterior part of the spiral valve. Similar results have been reported in other stomach-less fish like the guppy Poecilia reticulata (Genten et al. 2009; Zambonino-Infante et al. 2008). Absorptive enterocytes and goblet cells are observed increasing more from the anterior to the posterior of the spiral valve by 24 DPH as observed in adult freshwater stingray Himantura signifer (Chatchavalvanich & Marcos, 2006) and a lot of marine fish larvae (Zambonino-Infante et al. 2008).

The presence of food particles in the lumen of the mid and posterior parts of the spiral valve that were PAS positive in the present study proved that by 20 DPH the African lungfish larvae can be weaned to proper exogenous feeding. This represents a properly developed spiral valve supported by an increase in the number of mucous cells protruding through the epithelia forming the proper brush border by 24 DPH for lubrication, nutrient absorption and protection of the intestinal wall from mechanical damage (Zambonino-Infante et al. 2008; Mokhtar, 2017). Due to the ability of the spiral valve to ingest by pinocytosis, muco- substances from the goblet cells of the spiral valve brush border have been reported to regulate protein transfer into the epithelium (Domeneghinil et al. 1998). The current study also shows a decrease in lumen diameter with increasing folding of the spiral valve by 26 DPH, which can be explained by the need to create enough pressure along the valve for movement of food particles as well as absorption into the blood streams, which is a reason for increased vascularisation by 26 DPH. This decrease in lumen diameter also increases the retention time for proper food digestion and absorption.

Lymphocytes were observed scattered in the mesenchymal tissue along the inner border of the spiral valve by 24 DPH. Similar cells have been reported in adult west African lungfish (Icardo et al. 2010, 2014; Tacchi et al. 2015), zebrafish Danio rerio (Wallace et al. 2005) and in Australian lungfish (Hassanpour & Joss, 2009). These node-like cells appear in aggregates in adult Australian lungfish and provide immune surveillance in the intestine and general immune response within the mucosa (Hassanpour & Joss, 2009). These lymphocytes are believed to be produced by the connective tissue surrounding the spiral valve (Icardo et al. 2014). The appearance of lymphocytes in the spiral valve by 24 DPH in this study suggests that the African lungfish larvae develop an immune system around the intestines at the start of exogenous feeding. This information is essential for developing probiotics and immune boosters for African lungfish in the early larval stages.

The current study reveals the presence of supranuclear vacuoles in the spiral valve by 24 DPH and their increase in number by 26 DPH. These results are not in agreement with earlier findings in stinging catfish (Heteropneustes fossilis), neotropical carnivorous freshwater catfish (Hemisorubim platyrhynchos), shi drum (Umbrina arrosa) and sea bream (Sparus aurata), which all proved that supranuclear vacuoles reduced and disappeared after their initial appearance in larval development (Kumar et al. 2019; Kuhn et al. 2016; Zaiss et al. 2006;

Calzada et al. 1998). In gastric fish (fish with true stomach) the reduction in supranuclear vacuoles is always correlated with the development of the gastric glands since these vacuoles help in pinocytotic protein absorption and intracellular ingestion in teleost larvae before the development of a functional stomach during larval development (Govoni et al. 1986; Kumar et al. 2019). However, no reduction in number of supranuclear vacuoles was observed in flat fish brill (Scophthalmus rhombus) larvae, even though this species is gastric, this probably implies combined intra and extracellular protein digestion in such fish larvae (Hachero- cruzado et al. 2009). These vacuoles thus help in optimum protein utilisation, more so in agastric fish like African lungfish (Domeneghinil et al. 1998). The lack of a functional gastric stomach in African lungfish probably explains the increase in supranuclear vacuoles in the current study between 24 and 26 DPH due to the need for protein digestion by the growing larvae. The lack of gastric glands explains the absence of the pyloric caeca observed in the present study since its major role is to neutralise gastric acids from the stomach to the intestines (Zambonino-Infante et al. 2008). For proper development of the feeding regime in African lungfish, more investigations should be done to determine how the number and size of supranuclear vacuoles change both at the end of the larval stage and in the early juvenile stages, to determine the exact onset of digestive enzyme secretion in the spiral valve.

Residues of the yolk sac were still present in the spiral valve, however, reducing from 17 DPH onwards and almost completely absent by 26 DPH. Similar results have been reported in other freshwater carnivores like snook Petenia splendida where the residues were depleted by 24 DPH (Trevino et al. 2011). Reports have proved these to be oil globules that can as well persist for only 32 h in marine reef rabbit fish (Siganus guttatus), 88 h in marine carnivores barramundi (Lates calcarifer) and over 10 days in many other teleost fish (Bagarinao, 1986; Dabrowski, 1989). However, no such oil globules were observed during the larval stage of marine carnivore Atlantic halibut (Hippoglossus hippoglossus) as reported by Jaroszewska & Dabrowski, (2011). The reduction in oil globules in the current study is supported by Iwamatsu et al. (2008) who reported their reduction after yolk sac absorption, with their exhaustion correlating with catabolism of neutral lipids in Japanese rice fish Oryzias latipes. The resorption of the oil globules has been reported to represent a time of endo-exotrophic nutrition (mixed feeding) (Jaroszewska & Dabrowski, 2011). The neutral lipids in these oil globules have been reported to be a great source of energy between hatching and first

feeding after depletion of the fish amino acids during embryogenesis and their resorption was reported to increase with feed intake as observed in yellowtail kingfish Seriola lalandi (Hilton et al. 2008). This keeps lipid provision an important factor to investigate when developing a feeding regime for African lungfish larvae, with more research still needed to determine the role of the oil droplets in larval growth and survival.

5.3.4 Cloaca The cloaca appeared to have highly stratified columnar epithelia with a thick layer of smooth muscles to enable transfer of faeces from the body. The secretory goblet cells of the cloaca increased in number between 17 and 26 DPH. The muco-substances produced by the cloaca goblet cells have been reported to help in lubrication of faeces (Domeneghinil et al. 1998; Zambonino-Infante et al. 2008), thus their increase represents increased transport ability along the cloaca in response to exogenous feeding onset.

5.3.5 Liver and pancreas The current study reveals that between 17 DPH to 26 DPH liver hepatocytes changed from an irregular shape with a peripheral nucleus to regular shaped hepatocytes with a large central nucleus by 26 DPH with an increase in number and size. Similar results have also been reported in Hamun mani (Schizothorax zarudnyi) between 2 and 5 DPH by Moghadam et al. (2014). The position and shape of hepatocyte nuclei indicates the availability of lipids, with the peripheral nuclei indicating a high level of lipid deposition by 17 DPH while the large central nucleus indicates low lipid deposition by 26 DPH (Gisbert et al. 2005). With the hepatocytes increase in size during larval ontogeny visualising the onset of exogenous feeding (Zambonino-Infante et al. 2008), the reduction in lipid levels in the liver highlights the need of a lipid inclusion into larval diets for first feeding African lungfish larvae, since the liver synthesises and stores the lipids. These lipids could have been reduced due to their use in energy production in a protein sparing way, common in carnivorous fish, or the initiation of hormone production and increased use for normal cell function (Sargent et al. 1999). However, Trevino et al. (2011) observed an increased lipid accumulation in the liver after the onset of exogenous feeding which is not in agreement with the current study. This was attributed to the compound trout diet whose lipids were digested and absorbed by the larvae but did not fit the nutritional requirements of snook Petenia splendida. This highlights the need for a species-specific diet for optimum larviculture of a given fish species.

Morphological development followed by onset of bile synthesis and secretion determines the functionality of the liver in larval fish (Zambonino-Infante et al. 2008). In the current study, fully developed bile canaliculi were observed by 24 DPH, representing the functional liver (Genten et al. 2009). The liver also had glycogen deposits (PAS positive) by 24 DPH with high vacuolisation. This can be due to the reconstitution of glycogen that was accumulated during lecithotrophic feeding and reabsorbed on transition to exogenous feeding (Zambonino- Infante et al. 2008). This takes place after yolk sac absorption, thus the liver at this stage can utilise exogenous feeds for carbohydrate synthesis with the storage reserves increasing the vacuolisation of the liver hepatocytes. The presence of glycogen in the liver by 24 DPH coupled with the fully developed pancreatic ducts represents a fully functional endocrine pancreas that has the ability to deliver hormones for regulation of carbohydrate metabolism by the larvae at this stage (Zambonino-Infante et al. 2008; Moghadam et al. 2014). The importance of pancreatic islets in larval metabolism and development has been described by various authors (Hachero-cruzado et al. 2009; Zambonino-Infante et al. 2008).

5.4 Conclusions and recommendations By 26 DPH, African lungfish larvae proved to have an almost well-developed gastro-intestinal tract with tooth plates developing in the buccal cavity, a narrow oesophagus and half of the intestinal spiral coils already present. The spiral valve had evolved from a long narrow tube to a three-coiled valve. The immunity of the spiral valve was boosted by the presence of lymphocytes after the start of exogenous feeding (20 to 24 DPH). Liver glycogen accumulation and formation of supranuclear vacuoles in enterocytes proved initiation of carbohydrate and protein synthesis activity respectively, while a reduction in liver lipid content and resorption of spiral valve oil globules did highlight the importance of dietary lipids during first feeding in African lungfish.

Mixed feeding with various starter feeds was done due to lack of knowledge on the best starter feed for the species at the onset of exogenous feeding. Food particles were found in the spiral valve from 17 DPH onwards after a mixed feeding treatment. Future research should hence focus on understanding the impact of different starter feeds on African lungfish larval intestinal development by understanding how each feed type does influence intestinal development aspects like goblet cell evolution and intestinal mucosa and associated

microbiota development. This will provide information for the development of a proper feeding regime for both larvae and juveniles of African lungfish.

Provision of micro- and macro-nutrients specific to African lungfish larvae should also be an area to investigate. This will help maximise larval nutrient provision for the species, with more focus on the evolution of supranuclear vacuoles during further developmental stages to understand protein digestion and utilization in this agastric fish species, and the exact onset of digestive enzyme secretion in the spiral valve.

Research into establishment of proper breeding lines for African lungfish should also be investigated in order to fully close the cycle and produce good quality seed, as this determines the growth and survival of fish larvae and the overall success of an aquaculture fish species. This may involve the use of genetic techniques and cross-breeding of strains originating from different water bodies, in order to produce brood stock having desirable aquaculture traits like fast growth, disease resistance, high fecundity and water quality tolerance.

CHAPTER 6: REFERENCES

Ababouch, L., Alder, J., Anganuzzi, A., Barg, U., Bartley, D., Bernal, M. & Vannuccini, S. (2016). The state of world fisheries and aquaculture.

Abobi, S. M., Alhassan, E. H. & Asare, I. O. (2015). Analysis of the fecundity and gonadal stages of African carp, Labeo senegalensis (Valenciennes, 1842) from the White Volta, Ghana. Elixir International Journal, 79.

Absalom, K. V., Anpe, J. A., Igoche, L. E. & Okunsebor, S. A. (2017). Fecundity and egg size of Clarias Gariepinus in Pandam lake, Quan-Pan L.G.A. plateau state, Nigeria. IOSR Journal of Agriculture and Veterinary Science, 10(04), 19–22.

Aditya, K., Charan, R., Thongam, I., Sudhanshu, M., Uday, K. & Pratap, C. (2017). Effect of different weaning strategies on survival and growth of endangered fish Pengba, Osteobrama belangeri (Valenciennes, 1844 ) larvae. International Journal of Pure & Applied Biosciences, 5(5).

Ando, Y., Shigeno, I., Maruyama, I., Hirayama, K. & Nakao, T. (2013). Application of unicellular algae Chlorella vulgaris for the mass-culture of marine rotifer Brachionus. Live Food in Aquaculture, 133–138.

Arellano, J. M., Storch, V. & Sarasquete, C. (2002). The muntifunctional gut of fish. In Brauner, J.C. & Farrell P.A (Eds.), Fish Physiology, 45, 279–294.

Asgari, R., Rafiee, G., Eagderi, S., Shahrooz, R., Poorbagher, H., Agh, N. & Gisbert, E. (2014). Ontogeny of the digestive system in hatchery produced Beluga Huso huso (Linnaeus, 1758); A comparative study between Beluga and genus Acipenser. Aquaculture Nutrition, 20(6), 595–608.

Awaad, A. S., Moawad, U. K. & Tawfiek, M. G. (2014). Comparative histomorphological and histochemical studies on the oesophagus of Nile tilapia Oreochromis niloticus and African Catfish Clarias gariepinus. Journal of Histology. http://dx.doi.org/10.1155/2014/987041.

Bagarinao, T. (1986). Yolk resorption, onset of feeding and survival potential of larvae of three tropical marine fish species reared in the hatchery. Marine Biology, 91, 449–459.

Bemis, W. E. & Lauder, G. V. (1986). Morphology and function of the feeding apparatus of the lungfish, Lepidosiren paradoxa (Dipnoi). Journal of Morphology, 187(1), 81–108.

Berra. (2001). Freshwater fish distribution. Berra, T. M. (Ed), Academic Press (1st ed.). Academic Press, Cambridge, UK.

Bilal, A. P. (2012). Histological studies on ontogenic development and larval rearing of striped Murrel, Channa strialus. PhD Thesis, Manonmaniam Sundaranar University, Tirunelveli, India.

Bouillon, J. (1961). The lungfish of Africa. Natural History, 70(3), 62–71.

Boulenger G.A. (1870). Catalogue of the fishes in the British museum., 1, 549.

Brein, P. (1959). Ethologie du Protopterus dolloi (Boulenger) et de ses larves. Signification des sacs pulmonaires des Dipneustes. Annales de la Société royale zoologique de Belgique,

89, 9-48.

Brinkmann, H., Venkatesh, B., Brenner, S. & Meyer, A. (2004). Nuclear protein-coding genes support lungfish and not the coelacanth as the closest living relatives of land vertebrates. Proceedings of the National Academy of Sciences, 101(14), 4900–4905.

Busch, K.E.T., Peruzzi, F. & Falk-Petersen, I.B. (2011). Effect of prey type and size on the growth, survival and pigmentation of cod (Gadus morhua L.) larvae. Aquaculture Nutrition, 17, 595–603.

Busch, A. (1996). Transition from endogenous to exogenous nutrition : larval size parameters determining the start of external feeding and size of prey ingested by Ruegen spring herring Clupea harengus. Marine Ecology Progress Series, 130, 39–46.

Calzada, A., Medina, A. & González De Canales, M. L. (1998). Fine structure of the intestine development in cultured sea bream larvae. Journal of Fish Biology, 53(2), 340–365.

Carter, D. G. (2015). Feeding in hatcheries. In Davis, D.A. (Ed.), Feeding and feeding practices in aquaculture (Series in). Woodhead publishing Limited, Cambridge, United Kingdom.

Chapman, J. L., Witte, Goudswaard, K. (2002). Decline of the African lungfish ( Protopterus aethiopicus ) in Lake Victoria ( East Africa ). African Journal of ecology, 40, 42–52.

Chatchavalvanich, K. & Marcos, R. (2006). Histology of the digestive tract of the freshwater stingray Himantura signifer Compagno and Roberts, 1982 (Elasmobranchii, Dasyatidae ). Anatomy and Embryology, 211, 507–518.

Cornillie, P., Simoens, P. & Van den Broeck, W. (2008). Three-Dimensional reconstruction of the remodeling of the systemic vasculature in early pig embryos. Microscopy Research and Technique, 75, 105–111.

Coutteau, P. (1996). Micro-algae. In Sorgeloos, P & Lavens, P. (Eds.), Manual on the production and use of live feeds for aquaculture. Rome: FAO fisheries technical paper 361.

Dabrowski, K. (1989). Fomulation of a bioenergenic modules for coregoninae early life history. In Transactions of the American Fisheries Society, 38–50.

Davis, D A., Ii, T. J. D. & Head, M. E. (2018). Culture of zmall Zooplankton for the feeding of larval fish. SRAC Publication, (0701), 1–6.

Defaux, V. & Hjort, A. (2012). Regional market assesment (supply and demand).

Dhert, P., Rombaut, G., Suantika, G. & Sorgeloos, P. (2001). Advancement of rotifer culture and manipulation techniques in Europe. Aquaculture, 200,129-146.

Dhont, J., Dierckens, K., Stottrup, J., Van Stappen, G., Wille, M. & Sorgeloos, P. (2013). Rotifers, Artemia and copepods as live feeds for fish larvae in aquaculture. In Burnell A. & Gavin G. (Eds.), Advances in aquaculture hatchery technology (1st ed.). Woodhead publishing Limited, Cambridge, United Kingdom & NY., USA.

Diamond, J. (2002). Diamond (2002) - Evolution, consequences and future of plant and animal domestication, Nature, 418.

Domeneghinil, C., Strainil, R. P. & Veggetti, A. (1998). Gut glycoconjugates in Sparus aurata L . (Pisces, Teleostei ). A comparative histochemical study in larval and adult ages. Histology and Histopathology, 13, 359–372.

Drury, R. A. & Wallington, E. (1980). Carleton’s histological techniqued (5th ed.). Oxford University Press, Oxford, United Kingdom.

FAO, IFAD, UNICEF, WFP. and WHO. (2018). The State of Food Security and Nutrition in the World 2018. Building climate resilience for food security and nutrition. Rome.

FAO. (2012). The State of World Fisheries and Aquaculture. Sofia. Rome.

FAO. (2018). The State of World Fisheries and Aquaculture 2018 - Meeting the sustainable development goals. Rome.

Farhoudi, A., Kenari, A. A. M., Nazari, R. M. & Makhadoomi, C. (2013). Changes of digestive enzymes activity in common carp ( Cyprinus carpio ) during larval ontogeny. Iranian Journal of Fisheries Sciences, 12(2), 320–334.

Fernández, R. G. (2001). Artemia bioencapsulation. Effect of particle sizes on the filtering behavior of Artemia franciscana. Journal of crustacean biology, 21(2), 435-442.

Gagnat, M. R., Wold, P.A., Bardal, T., Øie, G. & Kjørsvik, E. (2016). Allometric growth and development of organs in ballan wrasse Labrus bergylta (Ascanius, 1767) larvae in relation to different live prey diets and growth rates. Biology Open, 5(9), 1241–1251.

Genten, F., Terwinghe, E. & Danguy, A. (2009). Atlas of fish histology. Science Publishers, Enfield, NH., USA.

Giacometti, M., Célia, M., Roberto, J., Teresa, M. & Conceição, L. E. C. (2009). Co-feeding of live feed and inert diet from first-feeding affects Artemia lipid digestibility and retention in Senegalese sole ( Solea senegalensis ) larvae. Aquaculture, 296, 284–291.

Gisbert, E, & Sarasquete, C. (2008). Review; Nutritional cellular biomarkers in early life stages of fish. Histology & Histopathology, 23, 1525–1539.

Gisbert, E., Villeneuve, L., Cahu, C. L., & Zambonino-infante, J. L. (2005). Dietary phospholipids are more efficient than neutral lipids for long chain polyunsaturated fatty acid supply in European sea bass Dicentrarchus labrax Larval development. The American Oil Chemists’society, 40(6), 609–618.

Gjedrem, T. (2012). Genetic improvement for the development of efficient global aquaculture: A personal opinion review. Aquaculture, 344–349(0044), 12–22.

Gogoi, B., Safi, V. & Das, D. N. (2016). The Cladoceran as live Feed in Fish Culture : A Brief Review The Cladoceran as live Feed in Fish Culture : A Brief Review. Research journal of animal veterinary and fisheries sciences,4(3), 7-12.

Gomes, A. S., Kamisaka, Y., Harboe, T., Power, D. M. & Rønnestad, I. (2014). Functional modifications associated with gastrointestinal tract organogenesis during metamorphosis in Atlantic halibut ( Hippoglossus hippoglossus ). Developmental Biology, 14(11), 1–16.

Gomes, M. L. M., Matta, S. L., Aruajos, V. A., Silva, G. M. F. & Zacaro, A. A. (2010). Larval ontogeny and morphology of giant trahira Hoplias lacerdae. Journal of Fish Biology, 76, 852–861. Govoni, J J., Boehlert, G. W. & Watanabe, J. (1986). The physiology of digestion in fish larvae. Environinental Biology of Fishes, 16(7), 59–77.

Graham B. J. (1997). Air-Breathing Fishes: evolution, diversity and adaptation (1st ed.). Academic press, San Diego, CA., USA.

Greenwood, P. H. (1958). Reproductiion in the East African lungfish Protopterus aethiopicus Hechel. In Proceedings of the Zoological Society of London (pp. 547–567). British Museum of National History, London, UK.

Greenwood, P. H. (1986). Natural history of African lungfishes. Journal of Morphology, 190(S1), 163–179.

Grosell, M., Farrell, P. A. & Brauner, J., C. (2011). The multifunctional gut of fish. In Brauner, C. J. & Farrell, P.A. (Eds.), Fish Physiology (1st ed.), 2–4). Academic press, London, UK.

Gunasekara, R. A. Y. S. A., Rekecki, A., Cornillie, P., Cornelissen, M., Sorgeloos, P., Simoens, P., Bossier, P. & Van den Broeck, W. (2011). Morphological characteristics of the digestive tract of gnotobiotic Artemia franciscana nauplii. Aquaculture, 321(1–2), 1–7.

Gurcan, M. N., Member, S., Boucheron, L. E. & Can, A. (2009). Histopathological image analysis : A review. IEE Reviews in Biomedical Engineering, 2, 147–171.

Hachero-cruzado, I., Ortiz-delgado, J. B., Borrega, B., Herrera, M., Navas, J. I. & Sarasquete, C. (2009). Larval organogenesis of flat fish brill Scophthalmus rhombus L : Histological and histochemical aspects. Aquaculture, 286(1–2), 138–149.

Hagiwara, A., Gallardo, W. G., Assavaaree, M., Kotani, T. & Araujo, A. B. (2001). Live food production in Japan: Recent progress and future aspects. Aquaculture, 200, 111–127.

Hamlin, H. J., Hunt Von Herbing, I., & Kling, L. J. (2000). Histological and morphological evaluations of the digestive tract and associated organs of haddock throughout post- hatching ontogeny. Journal of Fish Biology, 57(3), 716–732.

Hanke, W., Kloas, W., Reinecke, M., Storch, V., & Segner, H. (1994). The development of functional digestive and metabolic organs in turbot, Scophthalmus maximus. Marine Biology, 119, 471–472.

Harrod, C. (2016). Climate change and freshwater fisheries. John, C. K. (Ed.), Freshwater Fisheries Ecology (1st ed). John Wiley and Sons, NJ., USA.

Harvey, B., Soto, D., Carolsfeld, J., Beveridge, M. & Bartley, D. (2017). FAO technical workshop, 23–25 June 2016, FAO Rome. In Harvey D. M., Soto, D., Carolsfeld, J., Beveridge, M. & Bartley (Ed.), Planning for aquaculture diversification: the importance of climate change and other drivers (p. 166). Rome.

Hassanpour, M. & Joss, J. (2009). Anatomy and histology of the spiral valve intestine in juvenile Australian lungfish, Neoceratodus forsteri. The Open Zoology Journal, 2, 62–85.

Helfman, G. S., Collette, B. B., Facey, D. E. & Bowen, B. W. (Eds.). (2009). The diversity of fishes; Biology Evolution and Ecology (2nd Edit). Wiley-Blackwell, Oxford, UK.

Hilton, Z., Poortenaar, C. W. & Sewell, M. A. (2008). Lipid and protein utilisation during early development of yellowtail kingfish ( Seriola lalandi ). Marine Biology., 154, 855–865.

Hossan, S., Ulka, S. B. & Motin, A. (2013). Egg and fry production performance of female tilapia related to fluctuating temperature and size variation Egg and Fry Production Performance of Female Tilapia Related to Fluctuating Temperature and Size Variation. 4th International Conference on Environmental Aspects of Bangladesh, At Fukuoka, Japan. 105-108

Humason, G. (1979). Histological techniques. In Animal tissue techniques (4th Edit.). W H Freeman & Co., San Francisco, CA., USA.

Icardo, J. M., Wong, W. P. & Colvee, E. (2014). Lympho-granulocytic tissue associated with the wall of the spiral valve in the African lungfish Protopterus annectens. Cell Tissue Research, 355, 397–407.

Icardo, J. M., Wong, W. P., Colvee, E., Loong, A. M., & Yuen, K. (2010). The Anatomy of the gastrointestinal tract of the African Lungfish, Protopterus annectens. The Anatomical Record, 293(1), 1146–1154.

Intergovernmental Panel on Climate Change (IPCC). (2014). Climate Change 2014 Synthesis Report Summary for Policymakers.Cambridge University Press, Cambridge, UK. https://doi.org/10.1017/CBO9781107415324

Ishimatsu, A. (2012). Evolution of the cardiorespiratory system in air-breathing fishes. Aqua- BioScience Monographs, 5(1), 1–28.

Iwamatsu, T., Muramatsu, T. & Kabohashi, S. (2008). Oil droplets and yolk spheres during development of Medaka embryos. Ichthological Research, 55, 344–348.

Jamu D.M & Ayinla O.A. (2003). Potential for the development of aquaculture in Africa. NAGA, WorldFish Center Quarterly, 26(3), 9-13.

Jaroszewska, M. & Dabrowski, K. (2011). Utilisation of yolk: Transition from endogenous to exogenous nutrition in fish. In Holt, J.G. (Ed.), Larval fish nutrition (1st ed.), 12, 43–51. John Wiley & Sons, N. J., USA.

Jennings, R. & Premanandan, C. (2017). Veterinary histology. Ohio State University Library.

Johanson, A. P. (2010). Pylogeny of Lungfishes. In Jorgensen, J.M & Joss, J. (Ed.), The Biology of Lungfishes (1st ed.), 43–60. Science Publishers, Enfield, N.H., USA.

Johnels, A.G. & Svensson, G. S. O. (1954). On the biology of Protopterus annectens (Owen). 131–164. In Arkiv fo¨r Zoologi, Stockholm, Sweden.

Kamisaka, Y., & Rønnestad, I. (2011). Reconstructed 3D models of digestive organs of developing Atlantic cod ( Gadus morhua ) larvae. Marine Biology, 158, 233–243.

Kemp, A. (1986). The biology of the Australian lungfish, Neoceratodus forsteri (Krefft 1870).

Journal of Morphology, 190(1S), 181–198.

Kemp, A. (2002). Growth and hard tissue remodelling in the dentition of the Australian lungfish, Neoceratodus forsteri(Osteichthyes: Dipnoi). Journal of Zoology, 257, 219–235.

Kemp, A. (1981). Rearing of embryos and larvae of the Australian lungfish, Neoceratodus forsteri, under laboratory conditions. Copeia, 4, 776–784.

Kjørsvik, E., Pittman, K. & Pavlov, D. (2004). From fertilisation to the end of metamorphosis- functional development. In Moksness, E., Kjorsvik, E. & Olsen, Y. (Eds) Culture of cold- water marine fish, 204–278. Blackwell Publishing, Hoboken, N.J., USA.

Kuhn, C., Alari, R., Hideo, R., Carlos, A., Alberto, R., Belmont, F., Vicentini, B.F. & Aberto, C. (2016). Organogenesis of the digestive system in neotropical carnivorous freshwater catfish Hemisorubim platyrhynchos (Siluriformes : Pimelodidae ). Aquaculture, 451, 205– 212.

Kumar, A., Pradhan, P. K., & Chadha, N. K. (2019). Ontogeny of the digestive tract in stinging catfish, Heteropneustes fossilis ( Bloch ) larvae. Fish Physiology and Biochemistry, 45(2), 667–679.

Kumar, P., Jena, J., Mitra, G., Sood, N., & Gisbert, E. (2014). Effects of different weaning strategies on survival, growth and digestive system development in butter cat fish Ompok bimaculatus ( Bloch ) larvae. Aquaculture, 424–425, 120–130.

Laforsch, C., Imhof, H., Sigl, R., Settles, M., Heß, M., & Wanninger, A. (2012). Applications of computational 3D – modeling in organismal biology. In Gocovski, Z. (eds) Modeling and simulation in engineering, 117–142. Arcler Press, Oakville, Canada.

Liao, I. C. & Huang, Y. S. (2000). Methodological approach used for the domestication of potential candidates for aquaculture. Recent Advances in Mediterranean Aquaculture Finfish Species Diversification, 47, 97–107.

Machena, C. & Moehl, J. (2000). Sub-Saharan African aquaculture: Regional summary. In Subasinghe, R.P., Bueno, P., Phillips, M.J., Hough, C., McGladdery S.E. & Arthur, J.R. (eds). Aquaculture in the Third Millennium. Technical Proceedings of the Conference on Aquaculture in the Third Millennium, Bangkok, Thailand, 20-25 February 2000. pp. 341- 355. NACA, Bangkok and FAO, Rome, Italy.

Mai, K., Yu, H., Ma, H., Duan, Q., & Gisbert, E. (2005). A histological study on the development of the digestive system of Pseudosciaena crocea larvae and juveniles. Journal of Fish Biology, 67(4), 1094–1106.

Marimuthu, K. & Haniffa, M. A. (2007). Embryonic and larval development of the striped snakehead Channa striatus. Taiwania, 52(1), 84–92.

Martin, J. & Blaber, S. (1984). Morphology and histology of the alimentary tracts of Ambassidae (Cuvier) (Teleostei) in relation to feeding. Journal of Morphology, 182, 295– 305.

Meehl, G. A., Stocker, T. F., Collins, W. D., Friedlingstein, P., Gaye, A. T., Gregory, J. M., … Zhao,

Z.-C. (2007). In: Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change [Solomon, S., D. Qin, M. Manning, Z. Chen, M. Marquis, K.B. Averyt, M. Tignor and H.L. Cambridge, UK.

Mitra, A., Mukhopadhyay, P. K., & Homechaudhuri, S. (2015). Histomorphological study of the gut developmental pattern in early life history stages of featherback, Chitala chitala (Hamilton). Archives of Polish Fisheries, 23(1), 25–35.

Mlewa C., Green J M. & Dunbrack, R. L. (2006). Marbled lungfish growth rates in Lake Baringo, Kenya, estimated by mark-recapture. Journal of Fish Biology, 68(2), 443–449.

Mlewa C., Green J M. & Dunbrack, R. L. (2010). The General natural history of the African lungfishes. In Jorgensen J M. & Joss, J. The Biology of Lungfishes. (eds.) (1st ed.). Science Publishers, Enfield, New Hampshire, USA.

Mokhtar, D. M. (2017). Fish histology : from Cells to Organs. Apple academic press, Oakville, Canada.

Naoko Takezaki & Nishihara, H. (2017). Support for lungfish as the closest relative of tetrapods by using slowly evolving ray-finned fish as the Outgroup. Genome Biol. Evol., 155(1–4), 93–101.

Nelson S. J., Wilson V.H. & Grande, C. T. (2016). Fishes of the World (5th ed.). John Wiley & Sons Hoboken, N.J, USA.

Nyang’ate Onura, C., Van den Broeck, W., Nevejan, N., Muendo, P. & Van Stappen, G. (2018). Growth performance and intestinal morphology of African catfish (Clarias gariepinus, Burchell, 1822) larvae fed on live and dry feeds. Aquaculture, 489, 70–79.

O’Connell, C. P. (1981). Development of organ systems in the northern anchovy, Engraulis mordax, and other teleosts. Integrative and Comparative Biology, 21(2), 429–446.

Ofelio, C., Cohen, S., Adriaens, D., Radaelli, G. & Díaz, A. O. (2019). Histochemistry of goblet cells and micro-computed tomography to study the digestive system in the long-snouted seahorse Hippocampus guttulatus. Aquaculture, 502, 400–409.

Ostaszewska, T., Kolman, R., Kamaszewski, M. & Wiszniewski, G. (2011). Morphological changes in digestive tract of Atlantic sturgeon Acipenser oxyrinchus during organogenesis. International Aquatic Research, 3, 101–105.

Otero, O. (2011). Current knowledge and new assumptions on the evolutionary history of the African lungfish, Protopterus, based on a review of its fossil record. Fish and Fisheries, 12(3), 235–255.

Otuogbai, T. M., Ikhenoba, A. & Elakhame, I. (2004). Food and feeding habits of the African lungfish, Protopterus annectens (Owens)(Pisces: Sarcopterygii) in the flood plains of River Niger in Etasako east of Edo State, Nigeria. African Journal of Tropical Hydrobiology and Fisheries, 10(1), 14–26.

Paray, B. A., Haniffa, M. A., War, M. U. D., Al-Sadoon, M. K., Park, Y.H. & Rather, I. A. (2015).

Histological changes in the digestive tract of striped murrel larvae during ontogeny. Indian Journal of Geo-Marine Sciences, 44(7).

Peña-Aguado, F., Nandini, S. & Sarma, S. S. (2005). Differences in population growth of rotifers and cladocerans raised on algal diets supplemented with yeast. Limnologica - Ecology and Management of Inland Waters, 35(4), 298–303.

Pradhan, P. K., Jena, J. K., Mitra, G., Sood, N. & Gisbert, E. (2012). Ontogeny of the digestive tract in butter catfish Ompok bimaculatus (Bloch) larvae. Fish Physiology and Biochemistry, 38(6), 1601–1617.

Pradhan, P. K., Jena, J., Mitra, G., Sood, N. & Gisbert, E. (2014). Effects of different weaning strategies on survival, growth and digestive system development in butter catfish Ompok bimaculatus (Bloch) larvae. Aquaculture, 424–425, 120–130.

Rønnestad, I., Boglione, C., Conceic, L. E. C., Hamre, K. & Yu, M. (2013). Fish larval nutrition and feed formulation : knowledge gaps and bottlenecks for advances in larval rearing. Reviews in Aquaculture, 5(1), 26–58.

Rudskiy, I. V. & Khodorova, N. V. (2012). Reconstruction of the 3D structure and developmental history of plant cells and tissues, 1–15. Retrieved from http://arxiv.org/abs/1205.0225

Ruyet, J. P. (1989). Early weaning of marine fish larvae onto microdiets : constraints and perspectives. Advances in Tropical Aquaculture, 625–642.

Sales, J. (2011). First feeding of freshwater fish larvae with live feed versus compound diets: A meta-analysis. Aquaculture International, 19(6), 1217–1228.

Santos, A. E., Pedreira, M. M., Santos, T. G. & Souza, G. De. (2016). Development of the digestive system in larvae of the Neotropical fish Prochilodus argenteus (Characiformes, Prochilodontidae ). Animal Sciences, 36(1), 9–16.

Sargent, J., Bell, G., Mcevoy, L., Tocher, D. & Estevez, A. (1999). Recent developments in the essential fatty acid nutrition of fish. Aquaculture, 1777, 191–199.

Sayer, C. A., Máiz-Tomé, L. & Darwall, W. R. T. (2018). Freshwater biodiversity in the Lake Victoria Basin: Guidance for species conservation, site protection, climate resilience and sustainable livelihoods. Cambidge, UK and Gland, Switzerland.

Scocco, P., Acilli, D., Menghi, G. & Ceccarelli, P. (1998). Unusual gylcoconjugates in the oesophagus of a tilapine poly hybrid. Journal of Fish Biology, 53(1), 39-48.

Shahriari Moghadam, M., Abtahi, B., Rezaei, S. & Rahdari, A. (2014). Early ontogenetic development of digestive system in Schizothorax zarudnyi Nikolskii, 1897 (Actinopterygii: Cyprinidae) larvae. Italian Journal of Zoology, 81(2), 194–203.

Soltan, M. (2015). Effect of early weaning on growth and survival rate of gilthead seabream ( Sparus aurata ) larvae. Proceeding of the second international Conference on Biotechnology Applications in Agriculture, Moshtohor and Hurghada, 8-11 April 2014. Faculty of Agriculture, Benha University. Animal Biotechnology (poultry and fish), 49-60.

Southgate, P. C. (2012). Foods and feeding. In Lucas, J. S. & Southgate, P. A. (Eds.), Aquaculture; Farming of aquatic animals and plants. (2nd Ed, 188–212). John Wiley & Sons, Inc, Sussex, UK.

Sserwadda, M., Nevejan, N., Cornillie, P., Van den Broeck, W. & Van Stappen, G. (under review). Ontogenetic development of the gastrointestinal tract of African lungfish larvae Protepterus aethiopicus (Heckel 1851): a light microscopy study.

Stoss, J., Hamre, K. & Ottera, H. (2004). Weaning and Nursery. In Moksness, E. Kjorsvik, E. & Olsen, Y. (Eds.), Culture of Cold-water Marine Fish. Fishing News Books, Oxford, UK.

Tacchi, L., Larragoite, E. T., Amemiya, C. T. & Salinas, I. (2015). African lungfish reveal the evolutionary origins of organized mucosal lymphoid tissue in vertebrates. Current Biology, 25, 2417–2424.

Teletchea, F. (2015). Domestication of marine fish species: Update and perspectives. Journal of Marine Science and Engineering, 3(4), 1227–1243.

Tokita, M., Okamoto, T. and Hikida, T. (2005). Evolutionary history of African lungfish: a hypothesis from molecular phylogeny. Molecular Phylogenetics and Evolution, 35,(1), 281–286.

Tredici, M. R., Biondi, N., Ponis, E., Rodolfi, L. & Zittelli, G. C. (2009). Advances in microalgal culture for aquaculture feed and other uses. In Burnell, G. & Allan, G. (Eds.), New Technologies in Aquaculture: Improving Production Efficiency, Quality and Environmental Management. (610–676). Woodhead Publishing, Boca Raton, FL., USA.

Trenberth, K. E. & Josey, S. A. (2007). Global Climate Projections. In: Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change [Solomon, S., D. Qin, M. Manning, Z. Chen, M. Marquis, K.B. A (Vol. 164). Cambridge, United Kingdom and New York, NY, USA.: Cambridge University Press, Cambridge, UK.

Trevino, L., Perales-garcı, N., Alvarez-Gonzalez, C. ., Uscanga-Martınez, A., Gisbert, E., Fernandez, I., … Arevalo-Galan, L. (2011). A histological study of the organogenesis of the digestive system in bay snook Petenia splendida Gunther , 1862 from hatching to the juvenile stage. Journal of Applied Ichthyology, 27, 73–82. Troell, M., Naylor, R. L., Metian, M., Beveridge, M., Tyedmers, P. H., Folke, C., … de Zeeuw, A. (2014). Does aquaculture add resilience to the global food system? Proceedings of the National Academy of Sciences, 111(37), 13257–13263.

Ueberscha, B., Ribeiro, L., Sæle, Ø. & Boglione, C. (2013). Feeding behaviour and digestive physiology in larval fish : current knowledge, and gaps and bottlenecks in research. Reviews in Aquaculture, 5(1), 59–98.

United Nations. (2002). Protein and amino acid requirements in Human nutrition. WHO Technical Report Series (Vol. 935).

United Nations. (2017). World population prospects; The 2017 revision key findings and advance tables. World Population Prospects The 2017, 1–46.

Van Can, N., Dierckens, K., Nguyen, H. T., Minh, T., Hoang, T., Luu, T., … Sorgeloos, P. (2010). Effect of early co-feeding and different weaning diets on the performance of cobia ( Rachycentron canadum ) larvae and juveniles. Aquaculture, 305(1–4), 52–58.

Van Stappen, G., Bossier, P., Boeckx, P., Sorgeloos, P. & Toi, H. T. (2013). Bacteria contribute to Artemia nutrition in algae-limited conditions: A laboratory study. Aquaculture, 388– 391, 1–7.

Verreth, J. A. J., Torreele, E., Spazier, E., Van der Sluiszen, A., Rombout, J. H. W. M., Booms, R. & Segner, H. (1992). The development of a functional digestive system in the African catfish Clarias gariepinus (Burchell). Journal of the World Aquaculture Society, 23(4), 286- 298.

Walakira, J. K., Atukunda, G., Molnar, J. J. & Veverica, K. L. (2012). Prospects and potential for aquaculture of African lungfish in Uganda. World Aquaculture, 43(3), 38–42.

Walakira, J. K., Molnar, J. J., Phelps, R. & Terhune, J. (2014). Culturing the African lungfish in Uganda: Effects of exogenous fish feed on growth performance in tanks. Uganda Journal of Agricultural Sciences, 15(2), 137–155.

Wallace, K. N., Akhter, S., Smith, E. M., Lorent, K. & Pack, M. (2005). Intestinal growth and differentiation in zebrafish. Mechanisms of Development, 122(122), 157–173.

Wang, Y., Hu, M., Wang, W. & Cao, L. (2009). Effects on growth and survival of loach ( Misgurnus anguillicaudatus ) larvae when co-fed on live and microparticle diets. Aquaculture Research, 385–394.

World Bank. (2013). Fish to 2030: Prospects for fisheries and aquaculture. Agriculture and Environmental Services Discussion Paper, 3(83177), 102.

Zaiss, M. M., Papadakis, I. E., Maingot, E., Divanach, P. & Mylonas, C. C. (2006). Ontogeny of the digestive tract in shi drum (Umbrina cirrosa L .) reared using the mesocosm larval rearing system. Aquaculture, 260, 357–368.

Zambonino-Infante, J., Sarasquete, C., Cahu, C., Navarro, I., Gisbert, E. & Gutiérrez, J. (2014). Ontogeny and physiology of the digestive system of marine fish larvae. In Cryrino, J.E.P., Bureau, D.P. & Kapoor, B.G.(Eds) Feeding and Digestive Functions in Fishes (1st ed), 281– 348. Science Publishers, Enfield, NH., USA.

Zimmer, G., Reuter, G. & Schauer, R. (1992). Use of influenza C virus for detection of 9-0- acetylated sialic acids on immobilized glycoconjugates by esterase activity. European Journal of Biochemistry, 204, 209–215.

Zitzow, R. & Millard, J. (1988). Survival and growth of lake whitefish (Coregonus clupeaformis) larvae fed only on formulated dry diets. Aquaculture, 69, 105–113.