Mass rearing of Cheilomenessexmaculata(Coleoptera :Coccinellidae) on different diets under laboratory conditions

The ladybird beetle,Cheilomenes sexmaculata is a potential biocontrol agent for use in augmentative release programmes. The survival (%) of coccinellid larvae varied significantly among the seven different selected diets (egg yolk, chicken liver, chicken liver with aphids, chicken liver with legume juice, aphids, house fly maggots, fish meal powder) (F=1478 df =98; 6 p<0.05). The highest survival was recorded when the larvae were fed on aphids (95.0 ± 3.0%). Larvae did not survive when fed on egg yolk and fish meal powder. The survival of L2 larvae was 72% when fed on chicken liver. The total larval duration significantly varied with different diets (F=767 df =63; 6 p<0.05). The shortest total larval duration was recorded when all larval instars were fed exclusively on aphids (7.60 ± 0.1days). When L2 larvae were fed on chicken liver, 10.86 ± 0.13 days were taken to develop into an adult. The highest growth rate was recorded when all larval instars were fed exclusively on aphids. The longest duration was recorded for L1 larvae fed on aphids and other three instars fed on chicken liver. To test the effect of substrates on the number of egg laid eggs laid during the lifespan, five substrates (plastic boxes, paper pieces, fresh bean leaves, bean cotyledons, sand layer) were also tested. It varied significantly among the five different substrates (F=2502.07 df =70: 4 p< 0.05). The highest number of eggs was laid on the sides of plastic boxes (n=823.7 ± 2.2) while the lowest number of eggs was laid on the sand layer (n= 80.1 ± 10.4). The findings indicate that the replacement of aphid feed by chicken liver for larval instars L2, L3 and L4 is possible with a compromised survival (%) of larvae.


INTRODUCTION
Ladybird beetles (Coleoptera: Coccinellidae) are predatory insects with diverse food habits and live in a variety of habitats. Both adults and larvae feed on small soft bodied insects. From Sri Lanka, fifteen coccinellid species belonging to 12 genera in four tribes and three subfamilies have been recorded from the Mid-country (Mayadunnage et al., 2007). Adults and larvae of C. sexmaculata voraciously feed on a wide range of prey including aphids, coccids, diaspids and aleyrodids etc. (Agarwala and Yasuda, 2000).Nymphs and adults of Aphis craccivora(Koch) attack beans and other leguminous crops causing significant damage by feeding on sap of flowers, buds, pods, tender shoots thereby reducing the market value (Srivastava and Singh, 1986). At present, Sri Lankan farmers rely mainly on insecticides to control A. craccivora. Insecticidal control is not only expensive but also has adverse effects on the environment and also leads to many human health problems. Indiscriminate use of pesticides causes phytotoxicity and destruction of beneficial organisms such as predators, parasitoids, microorganisms and pollinators (Luckman and Metcalf, 1978). Subsequently, Integrated Pest Management has been opted in which biological control is one of the major aspects (Debach and Rosen, 1991). Larvae and adults of C. sexmaculatais an effective biocontrol agent and require mass rearing for commercial use. Rearing on natural prey requires the maintenance of host plant and aphid cultures; hence, it is necessary to explore the possibility of mass rearing of C. sexmaculata on artificial diet or on alternative hosts.
Food that sustains larval development and oviposition is considered as essential food while the food that serve only as a source of energy which prolong development period leading to poor survival is considered as alternative food (Hodek, 1996). Cheilomenes sexmaculata are also known to feed on pollen, nectar and fungal spores (Hemptinneet al. 1988). Various formulations of artificial diets have been tested on H. convergens adults (Hagen, 1964). The present study examines the possibility of replacing the natural diet: aphids by an alternative diet source thereafter and develop a mass rearing protocol suitable for mass production of the predatory beetles in the laboratory.

MATERIALS AND METHODS
The study was conducted in the Entomology Research Laboratory, Department of Agricultural Biology, Faculty of Agriculture, University of Peradeniya from January to June, 2015. Experiments were conducted under laboratory conditions of 27ºC and 80 % RH.

Development of mass rearing protocol for C. sexmaculataunder laboratory conditions
Adults of C. sexmaculata were collected from bean fields in Dodangolla, Mahakanda and Doluwa areas. The field collected beetles were sexed in the laboratory and pairs were selected for the oviposition.

Preparation of egg laying cages
Polymate Plastic boxes were used as egg laying cages. A 2.5 cm sand layer was placed at the bottom of the box. Water mixed with (100:1) hydroponic solution was added to the sand layer and mixed properly. Bean seeds were placed on the sand bed for germination. Ventilation pores were made on the lid of plastic box. Two days after seeding, aphids were introduced into the box. After 4 days of placing the bean seeds, field collected mated pairs of C. sexmaculata were introduced into one box. Three paper pieces were placed in the box to facilitate egg laying by C. sexmaculata introduced into the container. The numbers of eggs laid were counted daily.

Preparation of rearing cages
Paper on which eggs were laid was collected for separation of each egg. Egg masses glued on to the papers were removed and put into water. Eggs in an egg mass were separated using a small paint brush. A layer of wetted coir dust was placed in small plastic cups and a few holes were made for aeration. Subsequently, bean seeds were then placed on the coir dust layer. After 2 days of germination of bean seeds, aphids were placed on the germinated cotyledons. Eggs were placed individually in the plastic cup and covered using a polythene sheet. The durations of the larval stage were recorded.

Rearing of Pupae
Soon after pupation, each pupae was placed in a Petri dish that had ventilation holes on the lid. A wet cotton plug was placed on the Petri dish to avoid desiccation. Adults were sexed. Durations of pupal stage were recorded. Newly emerged pairs were transferred for separate rearing.

Survival and Development of C. sexmaculataLarvae on Natural and Formulated Diets
Adults of C. sexmaculatathat were obtained from laboratory cultures were sexed and confined in pairs into Petri dishes (15 cm diameter) with aphids for oviposition. Egg masses were separated and incubated. After the eggs hatched, the young larvae that emerged individually were transferred into the Petri dishes (15 cm diameter). L1 larvae were fed only with Aphis craccivora. L2 larvae were used for the experimentation. The survival rate and development duration of larvae were recorded. Each treatment was replicated 15 times.
Larvae were provided with seven different diets. Diet 1 (D1) consisted of chicken egg yolk kept in Petri dishes as small droplets. Fresh feed was supplied to the larvae daily. Diet 2 (D2) was prepared by boiling 10 g of chicken liver at 60 0 C for 10 minutes in 100 ml of water. Boiled chicken liver was ground to a paste using a motar and pestle. The diet was given to larvae in the same manner as in D1. Diet 3 (D3) consisted of boiled chicken liver as in D2 ground to a paste while adding 60 adult aphids. Diet 4 (D4) was boiled chicken liver as in D2 and ground to a paste by adding legume juice (5 ml). Bean leaves were used to extract 5 ml of legume juice. Diet 5 (D5) was the natural diet: aphids. Diet 6 (D6) was prepared by grinding house fly maggots using a motor and a pestle. As the diet 7 (D7), fish meal powder was used in dried form. The survival rates of C. sexmaculata larvae and total development durations of larvae were recorded for each diet. Each treatment was replicated 15 times. Data were analyzed using one-way ANOVA, followed by LSD mean separation using Minitab Statistical software (Version 14).

Development durations of C. sexmaculatalarvae and Survival on different feeding plan on a formulated diets
Adults of C. sexmaculatathat obtained from laboratory cultures were sexed and placed in pairs in Petri dishes (15 cm diameter) with aphids for oviposition. The collected egg masses were separated and incubated. After the eggs hatch, immature larvae were transferred individually into Petri dishes (15 cm diameter). Three diets were used to feed the larvae. Diet 1 (D1) consisted only of live aphids. Diet 2 (D2) was prepared by boiling 10 g of chicken liver (10g) in 60 0 C hot water for 10 minutes and ground by using the motor and pestle. Diet 3 (D3) was prepared by boiling 10 g of chicken liver in water heated to 60 0 C for 10 minutes and ground using a motor and pestle until a paste was formed while adding 60 adult aphids. The three different diets were administered as nine treatments (Table 1).
The survival rate C. sexmaculatalarvae and the total development durations of larvae were recorded. Each treatment was replicated 15 times. Data were analyzed using one-way ANOVA, followed by LSD mean separation using Minitab statistical software.

Most suitable egg laying substrate for C. sexamculata under the laboratory conditions
Cheilomenes sexmaculata, females laid eggs on the walls of the plastic petri dishes. Five different substrates, plastic boxes, paper pieces, fresh bean leaves, bean cotyledons and sand layer were tested to see an optimum substrate for egg laying. Three pieces of papers and bean leaves were inserted into each experimental setting. The number of eggs laid / day / female beetle and the total egg number / substrate were recorded. The experiment was replicated 15 times.

Development of mass rearing protocol for C. sexmaculataunder the laboratory condition
Polymate plastic boxes containing bean cotyledons were found to be suited for rearing of coccinellid adults. Larval rearing cups were also suitable for rearing.

Survival and Development of C. sexmaculataLarvae on natural and formulated Diets
The survival percentage of larvae was significantly different among the treatments (F=264 df=72; 8 p<0.05). The highest survival percentage was recorded when larvae were fed only on aphids (93.33 ± 2.35 %). The lowest survival percentage was recorded when larvae reared only on chicken liver diet (10.00 ± 2.35 %). Survival percentage of larvae increased with the addition of aphids into the diets. Diet D 7 appears to be a suitable replacement for aphids with chicken liver while maintaining a 78.88% survival of larvae. The total larval duration varied significantly with different treatments (F=56 df =72; 8p<0.05). The highest development rate was recorded when all larval instars were fed exclusively on aphids and when L1, L2, and L3 instars were fed exclusively on aphids and L4 stage on D3. The highest larval duration was when L1 larvae were fed on aphids and other three instars on chicken liver.

Table 3. The total larval duration and survival percentages of C. sexmaculatawhen reared on aphids (D1) chicken liver (D2), and their combination (D3)
Mean Values within a column followed by the same letter are not significantly different at 5% level

Survival and Development of C.sexmaculataLarvae on Natural and Formulated Diets
The percentage of survival was 72% when L2 larvae were fed on chicken liver. Replacement of aphids by chicken liver for L2, L3 and L4 was possible with a compromise of survival percentage of larvae. The total larval duration significantly varied with different treatments (F=767 df =63; p<0.05). The fastest growth rate was recorded when all larval instars were fed exclusively on aphids. Total larval development duration was nearly one week. When the L2 larvae were provided with chicken liver, 10.86 ± 0.13 days were taken to become a pupa.  and Coccinella transversoguttataFaldermann increased in the body weight but failed to produce eggs when fed on weevil larvae (Richards and Evans 1998). Pollen is an unsuitable source of food because pollen grains become clumpy and adheres to larval cuticle. Subsequently, larvae become desiccated and die. This has been observed in Coleomegilla maculate (Michaud and Grant 2005). Allen (1985) reported that ox liver is a nutritionally adequate diet for predator development but no eggs were laid. It is evident that the artificial diet containing chicken liver, yeast and sucrose support the growth of C. sexmaculata (Hussein et al., 1986). According to the present findings, the most suitable diet of the predatory larvae is the natural diet of aphids but chicken liver can be used to replace the natural diet to some extent for L3 and L4 instars of larvae.

Egg laying substrate for C.sexamculata
The highest number of eggs per female was laid on the walls of plastic box (823.7 ± 2.2 / entire female lifespan) and the lowest on the sand layer (80.1 ± 10.4 / entire female life span). Thus best egg laying substrate for C. semaculata, is the wall of the rearing cages or boxes.

CONCLUSIONS
Replacement of aphids by chicken liver for L2, L3 and L4 was possible with a compromise of survival percentage. A wall of the rearing cages was the preferred substrate for oviposition by C. sexmaculata under the laboratory conditions.