Utilization of Fly larvae, Earthworms and Duckweed for Frog production in an Integrated Farming System
 

Latsamy Phounvisouk

LivingAquatic Resources Research Center (LARReC)
NaongThang Villged, Vientiane City, Laos PDR
Meetouna@yahoo.com

1. Introduction

Livestock excreta are a major source of environmental pollution in intensive, specialized animal agriculture (Steinfeld et al., 2006). According to Kirschenmann (2007) "Agro-ecologists increasingly are convinced that the most viable alternative technology will spring from the biological synergies inherent in multispecies systems and that additionalresearch might make such systems the next new technology". Integration of crops and livestock, with recycling of the manure, is considered to be the basis of new management systems, "with the potential to increase the quantity and quality of production and the economic return to the farmers while at the same time placing less degrading pressure on soil and water resources." (Franzluebbers, 2007). One way of improving animal production without negative effects on the environment and increasing the efficiency of resource utilization is by integrating all the processes in the system, in the context of the sustainable use of those resources (Sophin and Preston, 2001).

To solve the problem of pollution, and to make better use of animal excreta, there are several techniques that can be applied.

Biodigesters

Anaerobic biodigestion is one of the most efficient technologies for converting animal manure into useful products: gas for cooking and effluent for fertilizer (Preston and Rodriguez 1998). For small-scale farms the plastic biodigester, because of its low cost and simple construction and management, may be the preferred technology (Bui Xuan An et al., 1997). The effluent is a valuable product from biodigesters and has been shown to be superior to the original manure for fertilization of crops (Le Ha Chau 1998) and fish ponds (Pich Sophin and Preston 2001). The effluents from biodigesters, when suitably diluted, are very effective media for growing duckweed (Le Ha Chau 1998).

Earthworms

The cultivation of earthworms can be another component of manure recycling. They have a comparative advantage over other forms of recycling when the manure is from goats or rabbits (Preston and Rodriguez 1998).

Fly larvae

Manure is the principal food of many insects in nature, house fly (Muscadomestica Linnaeus). The larvae convert residual manure proteins and other nutrients into their biomass, which is rich in high quality animal protein. Also, while occupying the manure they aerate and dry it, reducing odors.

Aquatic plants

Aquatic plants are being promoted as important components in management of livestock wastes, especially in large scale intensive units (Steinfeld et al., 2006). According to Leng (1995).farmers throughout Asia have harvested naturally produced aquatic plants for a number of purposes including animal feed, green manure and for their family feed resources. "The best known of these is "duckweed", which has become prominent, because of its ability to concentrate minerals in heavily polluted water such as that arising from sewage treatment facilities"(Leng 1995). It has also high potential as a feed resource for fish (Leng et al., 1994), ducks (Bui Xuan Men et al., 1997) and pigs (Du Thnh Hang 1998). Duckweed grows on water with relatively high levels of N, P and K and concentrates the minerals and synthesises protein.

Potential of frog cultivation in waste recycling

Live stock play two roles in waste recycling. On the one hand they maybe the cause of pollution if their manure is discharged directly to the environment. However, certain species may be able to benefit directly from products that arise in an efficient system of waste recycling. Aquatic animals are most suited for the utilization of products of recycling since their normal habitat is either wholly or partially in natural or artificial water bodies.

The common lowland frog (Rana rugulosa) appears to be specially adapted for cultivation in integrated farming systems, since its natural feed source includes insects and earth worms. In the Northern region of Laos, there is a high demand for consumption in family households and local markets. The frog is fast growing. From hatching to market size, it takes 3 to 4 months which is similar to other commercial species such as catfish and tilapia. Frogs can be raised in all locations in Laos, as they require small area and lower quantity of water than other aquaculture species (Bounsong, 2001).

2. Objectives

The aims of the present study were to investigate components in a waste recycling system which could be used for the cultivation of frogs, to reduce the present dependency on imported concentrate feeds, and to make the system more attractive for small-scale farmers in Laos.

Two experiments have been carried out. The first experiment was a study of the effect of different sources of household waste, irrigated with biodigester effluent, on the production of fly larvae. The second experiment compared the use of fly larvae and earth worms, with or without duck weed, as the basal diet for growing frogs raised in ponds.

Table of contents

3. General discussion. 9

3.1. Biological characteristics and feeding behaviour of frog species. 9

3.1.1. The common lowland frog, (Rana Rugulosa)9

3.1.2. .Natural habitat10

3.1.3. Food and feeding. 10

3.1.4. Stocking density. 10

3.1.5. Nutrient Requirements. 10

3.2. Frog production in Aquaculture systems. 10

3.3. Earthworm as a feed source for livestock and frog production. 11

3.3.1. Significance of Earthworm in animal production. 11

3.3.2. Feed and feeding of Earthworm.. 11

3.3.3. Nutritive value of earthworms. 11

3.4. Nutritive value of fly larvae. 12

3.4.1. Larvae as a feed source for livestock production. 13

3.4.2. Research on fly larvae in the past13

3.4.3. Production of fly larvae. 13

3.4.4 Fly larvae and chitin. 14

3.5. Biodigesters in integrated farming systems. 17

3.6. Duckweed. 17

3.7. Water quality parameters for Frogs. 18

4. Conclusions and recommendations. 19

5. References. 21

3. General discussion

3.1. Biological characteristics and feeding behaviourof frog species

3.1.1. The common lowland frog, (RanaRugulosa)

The local lowland frog, Rana rugulosa Wiegmann, locally called "Kob Thong" or "Kob Jarn" is one of 38 frog species found in Laos. At present, four native and one introduced species, RanaRugulosa, Rana tigerina, Rana marcrodon, Rana blythii and Rana catesbeiana (bullfrog), respectively, are most preferred in both local and foreign markets. Because of a high demand, frog farms continue to expand throughout the SE Asia region (Thongyount, 2004).

3.1.2. .Natural habitat

The lowland frog is generally found in various water bodies and wet or damp areas. They can be found in a considerable distance from their aquatic habitat because of their wandering behavior.

3.1.3. Food and feeding

The lowland frog (Rana rugulosa)is a carnivorous amphibian, feeding on earthworms, insects, spiders, worms, small fish and small frogs (Akasay, 1994). In their natural habitat, frogs have a variety of foods to select except at times of unfavorable environment or seasonal conditions. In Thailand, frog culture is usually conducted in concrete tanks, floating net cages, or in ponds with commercial feed (Kamonphone, 1977).

3.1.4. Stocking density

After they are taken from tadpole ponds, young frogs can be stocked in culture pens or concrete tank at up 30 frogs per square meter (Grey Lutz and Jimmy, 1999).

3.1.5. Nutrient Requirements

The lowland frog require similar nutrients as other aquatic animal species, especially those that are carnivorous, to maintain normal growth and metabolic function. The major nutrients such as protein, lipids, essential fatty acids, carbohydrates, vitamins and minerals are required for normal growth (LARReC, 2001).

The dietary protein requirements for frogs have been determined in various studies. According to Thongyount (2004), protein levels start at 38% in DM for tadpoles (1-30 days), reducing to 32% for small frogs (30-60 days) and 26% for fattening frog (60-90 days). Artificial feed of 35 % crude protein supported faster growth in Rana pipen tadpoles than young tilapia fish of 23 % protein (Ling et al., 2003). Feeding of ensiled fish, over a period of 4 months, enabled frogs to reach a live weight of 200 to 400g (Marttinez, 1993). According to Martinez et al., (1993) commercial feeds with 39% protein feed are suitable for frogs in intensive culture.

3.2. Frog production in Aquaculture systems

In general, there are three forms of frog culture: in ponds, cages and tanks. Each system has its own important characteristics and the decision as to which system to use will depend on the purpose of the operation. Cage and tank culture are the preferred systems for commercial production. However, for the experiment described in Paper II, earthen ponds lined with plastic sheets were used as this facilitated establishing the required degree of replication of the treatments.

3.3. Earthworm as a feed source for livestock and frog production

Contribution of earthworm to our ecosystem is often underestimated. Not only improving degradation rate of the waste, they could be used as animal feed as containing high protein content and many important amino acids (Parlevliet 1997).

3.3.1. Significance of Earthworm in animal production

Earthworm can be used as feed ingredient for fish, poultry, amphibians and pigs. They can be mixed with other feed ingredients such as rice brume, broken rice and others (Samphone et al., 1999). Such mixed feed ensure rapid growth of animals which can tolerate hash environmental conditions and diseases Parlevliet (1997).

3.3.2. Feed and feeding of Earthworm

Earthworms live in holes they made in the soil. Earthworm intake food through the mouth and discharge through the other end the holes in the soil are very small in size and folds themselves in the holes. They normally do not appear on the surface during day time except when there is a heavy rain. Usually Earthworm can be found on soil surface at night especially after the rain. When the weather is dry and cold digs their holes as deep as 2.5 meters, and under such environmental condition, Earthworm will twist themselves around one another to form a crump and release sticky liquid substance. At night Earthworm will feed on plant materials on the surface and return to their holes after discharging waste materials close to their holes (Vineset et al., 1997).

3.3.3. Nutritive value of earthworms

Sun-dried earthworms, Perionyx excavatus, has 93.62% dry matter, 59.90% crude protein, 402.09 Kcal/100 g gross energy, 7.43% fatty acid, 7.43% crude fibre, 1.73% Ca, and 0.118% P (Bay, 2002). Earthworm meal is very high in protein, and with variable oil content (Table 1). The dry matter content of earthworms ranges between about 15 and 20%. The fatty acid composition of the lipid extracted from worms is quite similar to the lipid composition of some fish oils, being high in φ-3 polyunsaturated lipids. In our study natural earthworms had average protein (70% in dry matter (DM)) and fat (13 % in DM) contents. However, they have a comparative advantage over other forms of recycling when the faeces are from goats or rabbits (Nguyen Quang Suc et al., 2000). Recently, the use of the earthworm (Eisenia foetida) as an agent for recycling live stock manure has received increasing attention (Preston and Rodríguez, 2002).

3.4. Nutritivevalue of fly larvae

House fly (Muscadomestica) larvae has 30% DM and, in the DM, 63% protein and 15% fat according to Newton et al., (1977). By contrast, in Paper 11 it was found that the protein was lower (49% in DM) and the fat much higher (31% in DM). Due to the protein content, the fly larval meal is considered to be a suitable replacement for conventional protein and fat sources. Comparative amino acid profiles of the proteins of fishmeal and house fly larvae are in Table 3.

3.4.1. Larvae as a feed source for livestock production

Fly larvae (Mosca spp) have been fed experimentally to several animals, with larvae or prepupae used to replace soybean or fish meal in a formulated diet (Sheppard. No. date). Fly larvae feeding tests have utilized cockerels (Hale, 1973), pigs (Newton et al., 1977) and catfish (Bondarie et al, 1987). The larvae have been shown to be generally equal to soybean meal (and other conventional ingredients) in feed value when fed to chicks (Calvert et al., 1970).

3.4.2. Research on fly larvae in the past

Complete replacement of the protein supplemented by fresh termites or fly larvae produced on farm from waste bagasse, wood or fresh pigs manure, and managed and harvested by household labour, decreased the chicken feed costs by 40% and 51% respectively, compared to the control diet (Men, 2005).

3.4.3. Production of fly larvae

Viet Chuong (2001) investigated a production of fly larvae from a rectangular holes, 50 x 50 cm and deep which dug and filled with soft, damp rice straw and fresh cattle manure in alternate 15 cm thick layers. A thin layer of rice gruel was placed on the top straw layer. Around one kg of spoilt fish was added to attract flies to lay their eggs, and small shelters were constructed over the holes to protect them from rain and direct sunlight, and to allow the flies to enter the hole easily. To maintain high humidity 2-3 litres of water were poured over the holes when the eggs had been laid. Growing larvae were harvested 4-5 days after putting the bait into the holes, the yields of larvae harvested after the 5 days period were very low. It was found that this method is not suitable for producing fly larvae under these particular experimental conditions.

3.4.4 Fly larvae and chitin

Chitin is the most widespread amino polysaccharide in nature and is estimated annually to be produced almost as much as cellulose. It is mainly found in arthropod exoskeletons, fungal cell walls or nematode egg shells. However, derivatives of chitin oligomershave also been implicated as morphogenic factors in the communication between leguminous plants and Rhizobium and even in vertebrates, where they may be important during early stages of embryogenesis. Chitin is one of the most important biopolymers in nature. It is mainly produced by fungi, arthropods and nematodes. In insects, it functions as scaffold material, supporting the cuticles of the epidermis and trachea as well as the peritrophic matrices lining the gut epithelium. Insect growth and morphogenesis are strictly dependent on the capability to remodel chitin-containing structures(Bakkers et al., 1999).

Chitin is composed largely of alternating Nacetylglucosamine residues, which are linked by b-(1-4)- glycosidic bonds. Since hydrolysis of chitin by chitinase treatment leads to the release of glucosamine in addition to Nacetylglucosamine, it was concluded that glucosamine might be a significant portion of the polymer. However, solids NMR analysis of tobacco hornworm cuticle preparations suggested that little or no glucosamine is present (Kramer et al., 1995). Chitin polymers tend to form microfibrils (also referred to as rods or crystallites) of ~3·nm in diameter that are stabilized by hydrogen bonds formed between the amine and carbonyl groups. Chitin microfibrils of peritrophic matrices may even exceed 0.5·mm in length and frequently associate in bundles containing parallel groups of 10 or more single microfibrils (Peters et al., 1979; Lehane, 1997). X-ray diffraction analysis suggested that chitin is a polymorphic substance that occurs in three different crystalline modifications, termed a-, b- and g- chitin. They mainly differ in the degree of hydration, in the size of the unit cell and in the number of chitin chains per unit cell (Rudall and Kenchington, 1973; Kramer and Koga, 1986). In the a form, all chains exhibit an anti-parallel orientation; in the b form the chains are arranged in a parallel manner; in the g form sets of two parallel strands alternate with single antiparallel strands. In addition, non-crystalline, transient states have also been reported in a fungal system (Vermeulen and Wessels, 1986). All three crystalline modifications are actually found in chitinous structures of insects. The a form is most prevalent in chitinous cuticles, whereas the b and g forms are frequently found in cocoons (Kenchington, 1976; Peters, 1992). Peritrophic matrices usually consist of a- and b-chitin. Sometimes the presence of b-chitin in cocoons is traced back to the fact that some cocoons are formed from peritrophic matrices; for example, those of Australian spider beetle Ptinus tectus, a specialized beetle (Rudall and Kenchington, 1973). The anti-parallel arrangement of chitin molecules in the a form allows tight packaging into chitin microfibrils, consisting of ~20 single chitin chains that are stabilized by a high number of hydrogen bonds formed within and between the molecules. This arrangement may contribute significantly to the physicochemical properties of the cuticle such as mechanical strength and stability (Giraud-Guille and Bouligand, 1986). By contrast, in the b- and g-chains, packing tightness and numbers of inter-chain hydrogen bonds are reduced, resulting in an increased number of hydrogen bonds with water. The high degree of hydration and reduced packaging tightness result in more flexible and soft chitinous structures, as are found in peritrophic matrices or cocoons. The picture drawn above is certainly oversimplified and does not explain the physicochemical properties of cuticles and peritrophic matrices adequately because it is reduced to only one component of a complex structure. However, differences in the arrangement of chitin microfibrils between cuticles and peritrophic matrices may help to understand their function. The cuticle is secreted in the form of thin layers by the apical microvilli of epidermal cells. The chitin microfibrils are embedded into the protein matrix and stabilize it in a way that resembles constructions of steelreinforced concrete. Since horizontal microfibrils, in parallel with the cuticle plane, rotate either progressively or abruptly from one level to another, complex patterns (e.g. helicoidal) and textures (e.g. plywood-like structures) arise, depending on the degree of rotational displacement (Bouligand, 1972). By contrast, in peritrophic matrices, the microfibrils are normally arranged as a network of randomly organized, felt-like structures embedded in an amorphous matrix, and only in a few cases have higher ordered configurations been reported (Lehane, 1997).

Figure.1. Biosynthesis of chitin in insects. The pathway starts with trehalose, the main hemolymph sugar in most insects, and ends with the chitin polymer. The diagrammatic representation is based on previously published pathways (Kramer and Koga, 1986; Cohen, 2001).

Chitin, an integral part of the invertebrate cuticle (exoskeleton), can be estimated by determining the acid detergent fiber fraction corrected for ash. Since chitin contains about 7% nitrogen, each 1% of ADF (presumed to be chitin) contains the equivalent of 0.4% crude protein (1 x 0.07 x 6.25). It has been reported, that some insectivores have an intestinal chitinase, while others may rely on chitinases produced by gut microorganisms. Chitin digestibility in three species of mammals has been shown to range from 2-20%. However, there is no evidence that the nitrogen released can contribute to the protein available for absorption by the insectivore (Bernard et al., 1997).

Assuming the chitin content of the fly larvae in Paper 2 was 25%, then the protein present in the chitin would be about 6%. This would reduce the available protein from 49% of the DM to 43% which is still above the estimated requirements of 37% in DM.

3.5. Biodigestersin integrated farming systems

Biodigester technology for converting manure into methane for fuel is neither new nor uncommon. In many parts of Asia, Central America and Europe, biodigester use is widespread. Biodigesters are installed in these areas in response to organic waste (manure) disposal problems and/or high-energy costs (Figure 2.)

Figure.2.Biodigester Schematic (Halter, www.csale.usask.ca/PDF Documents/biodigester Develop.pdf)

3.6. Duckweed

Conventional feed such as soya bean meal and fish meal are a source of protein in diets for pigs and poultry. Soya bean meal and fish meal are expensive, which results in high costs of production. Using of local feed resources can be a way to reduce cost of production, and as a result, increasing income. Thus, these improve the standard of living of smallholder farmers. Duckweeds have potential as protein sources when combined with energy-rich feeds which are low in fiber such as broken rice, cassava root meal or sugar cane juice (Becerra et al., 1995). Duckweed grows well on pond surfaces. They are more resistant to pests and diseases than other aquatic plants and have high protein and carotene content. Protein content of duckweed responds quickly to nutrients in a water environment (Leng et al., 1994). The effluents from biodigesters, when suitably diluted, are very effective media for growing duckweed (Le Ha Chau 1998). Duckweed has high crude protein content and a well-balanced amino acid profile and is also a good source of vitamins and minerals for livestock. Even though the moisture content of duckweed can be a limiting factor for fish and also frog production, duckweed can play important role in aquatic systems. The effluent leaving the biodigester retains the minerals and with suitable dilution is a good media for duckweed ponds. Ponds fertilized with effluent can produce up to 100 g fresh duckweed/m²/day which is equivalent to about 6 tonnes protein/ha/year (Rodriguéz et al., no date).

3.7. Water quality parameters for Frogs

An abundant supply of high quality water must be readily available to the frogs throughout the growing season. (Diana et al., 1997) Water temperatures of 18^o-22^oC are generally suitable for rearing larval salamander species found in Laos, and temperatures of 18^o-22^oC are suitable for most species of frog. Uodone (2004), the data from frog training course at the fishery station belong to Living Aquatic Recourse Research Center found that water quality parameters for available to the frogs growing in Laos in (Table. 5) suitable for most species of frog.

4. Conclusions and recommendations

Based on the results in this thesis, it is concluded that:

• Frog cultivation in a waste recycling system is important for sustainable aquaculture, to reduce expenditure on costly feeds.

• Earthworms, Larvae and Duckweed could be the basis of a system to replace the high protein concentrate feed traditionally used in intensive frog culture.

• Pig manure mixed with fermented fish waste was a better substrate for growing larvae than Jackfruit waste and pig manure.

• There appeared to be no advantages from applying biodigester effluent to the substrates.

• Growth rates and feed conversion (for DM and crude protein) were better, and mortality was lower, when frogs were fed earthworms rather than larvae and when they had access to fresh duckweed mixed with the larvae/earthworms.

• The net increase in live weight (252 g in 90 days) on the best diet (earthworms plus duckweed) was better than in one report concerning frogs fed an artificial diet (200 g in 120 days).

• Comparisons with another aquatic species (catfish) indicated broadly similar results for growth rate and feed conversion.

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