REARING OF THE DIAMONDBACK MOTH, PLUTELLA XYLOSTELLA (L.) (LEPIDOPTERA:YPONOMEUTIDAE) ON ARTIFICIAL DIET IN THE LABORATORY

C Dunhawoor and D Abeeluck

Agricultural Research and Extension Unit

ABSTRACT

A technique to rear Plutella xylostella (L) on artificial diet was developed in the laboratory. The suitability of four types of diets (Biever’s, G⁺, G⁺⁺ and soya diets) was tested. Soya diet was found to be the most suitable one. The life cycle from egg to adult lasts 18 days at 26± 2ºC and 60 – 65% RH. Caged moths lived for about 11 days. A mated female (reared on soya diet) laid an average of 207 eggs during her lifetime. The majority of eggs were collected during the first 5 days. Grooved aluminium foil dipped in fresh cabbage juice was found to be a suitable oviposition substrate for egg collection. Eggs seeded on diet hatched within 3 days. Larval development (1^st to 4^th instar) was completed in 10-11 days and the pupal period was 3-4 days.

Keywords: Plutella xylostella, soya diet, egg, adult and aluminium foil

INTRODUCTION

The diamondback moth (DBM), Plutella xylostella, is a major pest of cabbage and cauliflower in Mauritius. The pest was controlled easily with insecticides until 1980 when severe failures of pesticides began to occur and progressively growers reported resistance problems (Dunhawoor et al. 1997). As an alternative to pesticides, P. xylostella was then considered for control by F1 sterility technique in conjunction with biological and cultural methods. F1 sterility technique has been considered as an advantageous genetic method for suppression of lepidopteran populations by introducing lethal mutations in reared moths and releasing them into wild populations which ultimately decreases fertility in F1 generations (Knipling and Klassen 1976, La Chance 1984, Mastro and Schwalbe 1988, Omar et al. 1993). To implement this technique, mass rearing of P. xylostella is required for releases of large numbers of irradiated moths. The technique for rearing DBM on artificial diet has been developed by Biever et al. (1971), Koshihara (1976) and Hsiao et al. (1978).

Plutella xylostella was first reared on cabbage plants. This method was abandoned because it required large quantities of cabbage plants and many cages. Rearing of P. xylostella on artificial diet was thus initiated in 1998. A colony was established from field collected larvae and pupae but was destroyed after 4 months because of infection with a microsporidian pathogen ( Nosema spp ). Microbial contamination is one of the major problems affecting the rearing of insects (Sikorowski et al. 1994).

The paper reports on procedures for:

1. establishment of a disease free colony in greenhouse and laboratory

2. selection of a suitable artificial diet

3. fecundity and longevity of moths raised on diets and

4. development time and method to maximise egg collection in laboratory.

MATERIALS AND METHODS

Establishment of a disease free colony

An effective sanitation programme was set-up and included frequent cleaning of laboratory and disinfecting of walls, shelves, floor and insectary equipment with a detergent sanitiser (ANTEC DSC-1000) and was maintained to keep a clean environment for the rearing programme.

DBM larvae and pupae were collected from isolated sites (Plaine Sophie, Henrietta, Palma, Midlands, La Chartreuse, Providence, L’Escalier, Belle-Vue and Terre Rouge) and reared on cabbage leaves in separate cages in the greenhouse. Moths were dissected and those from Henrietta, Palma, Midlands and Belle-Vue were found free from disease. Males and females from 10 cages from the above mentioned sites were allowed to mate and single pair colonies were set. They were reared for 10 generations. At each generation, 25 adults were randomly selected and were dissected to check the presence of microporidian spores. The adults were found free from spores.

The individual colonies from the 4 sites were then mixed to form a single colony. This greenhouse colony generated eggs for the establishment of DBM colony on artificial diet.

Eggs were first surface sterilised (dipped in 12 % of sodium hypochlorite solution), washed with distilled water and dried under a Laminar flow with HEPA filters.

Selection of a suitable diet for rearing DBM larvae

13 types of diets were initially tested in the laboratory. Four promising diets, Biever’, G⁺, G⁺⁺ and soya diets, were retained for further studies in the selection of a suitable one for DBM rearing.

The 4 diets (Biever’s, G⁺, G⁺⁺ and soya) were prepared using ingredients given in Table 1. The diets were cut into cubes (1x 1 x 1cm) and 2 cubes of each diet were placed in a solo plastic cup under a Laminar flow to avoid mould contamination. Four sets of cups with diets were prepared.

Greenhouse eggs were surface sterilized and kept at 22 ± 2ºC in an incubator for 48 hours. 250 newly hatched larvae (<24 hr) were selected. 10 individuals were placed in a cup with each type of diet. As control, 10 larvae were placed in a cup with cabbage leaves. The experiment was conducted in 5 replicates.

All cups were placed in the larval rearing room at 26 ± 2ºC and 60 – 65% RH. A diet cube was added at 8 day-intervals and cabbage leaves in the control cup every 2 days.

The number of pupae from each type of diet was recorded and kept separately in cages. Emerging moths were weighed and number of emergences was also recorded.

Longevity and fecundity of Plutella xylostella

DBM larvae reared on Biever’s, G⁺, G⁺⁺ and soya diets and cabbage leaves were kept separately until pupation. Collected pupae were kept in individual gelatin capsules. Newly emerged males and females (1day old) were allowed to mate.

Five pairs of moths (mated female and male) from each diet and cabbage were placed in 5 solo plastic cups (4 x 4 cm in size) and fed with 20 % honey solution. A grooved aluminium foil (2 x 1.5 cm in size) dipped in boiled cabbage juice was placed in each cage to collect eggs.

Aluminium eggsheets were replaced daily and eggs on sheets and on the interior of cages were counted. Male and female longevity was also recorded.

Development Time

5 sets of eggs (300/set) from mated females (reared on soya diet) were surface sterilized and seeded in plastic cups with 100 mL of soya diet. The cups were examined every 24 hr to determine the time when eggs hatched and the development time from 1^st instar larva to pupal stage.

Oviposition Substrate

Six types of substrates used as eggsheets (kitchen towel, plain aluminium foil, crinkled aluminium foil, grooved aluminium foil, grooved aluminium foil soaked in fresh cabbage juice and grooved aluminium foil soaked in boiled cabbage juice) were tested as eggsheets for egg deposition from cages. Each substrate measured 8 x 7.5 cm in size.

100 males & 100 females were placed in a cage (6x 6 cm) and fed with 10 % honey solution. 6 such cages were set up and one type of substrate (2 foils) was placed in each of them. The experiment was replicated three times.

Eggsheets from each cage were collected every 24 hours for two consecutive days and eggs counted.

RESULTS

Selection of a suitable diet for rearing DBM larvae

An average of 75 % of the greenhouse larvae reared on cabbage attained pupal stage. Among the diets, pupal yield from soya diet was highest (52 %). The percentage of pupal harvest on Biever’s, G⁺ and G⁺⁺ diets were 44 %, 34 % and 46 % respectively (Table 2).

Moth emergence from larvae on cabbage and soya diet was 54 % and 42 % respectively. The percentage of moth emergence from Biever’s, G⁺ , G⁺⁺ diets ranged from 20 % to 28 %.

The number of males emerging from Biever’s, G⁺ and G⁺⁺ diets was higher whereas the number of females from soya diet was higher.

The weight of females was significantly higher than that of males irrespective of diets. However, the mean weight of female reared on soya diet was significantly higher (0.0056 g) than those raised on other diets.

Table 1 List of ingredients required to prepare 1 Litre of diet

Ingredients		Biever’s diet	G^+	G^++	Soya
Agar	g	24.0	22.5	22.5	22.5
Alphacel	g	6.0	5.0	5.0	5.0
Ascorbic acid	g	4.0	4.0	4.0	4.0
Aureomycin	g	1.0	1.0	1.0	1.0
Brewer’s yeast	g			16.2	16.2
Cabbage powder	g		30.0	30.0	30.0
Cabbage powder	g		30.0	30.0	30.0
Casein	g	32.0	35.0	35.0	35.0
Cholesterol	g			2.5	2.5
Choline chloride	g		1.0	1.0	1.0
L. Inositol	g		0.2	0.2	0.2
Methyl P **	g	1.4	2.0	2.0	2.0
Potassium sorbate *	g	1.0
Sorbic acid	g		2.0	2.0	2.0
Soya flour	g		30.0	30.0	30.0
Sucrose	g	34.0	35.0	35.0	35.0
USDA vitamin premix	g	10.0	10.0	10.0	10.0
Wesson salts	g	9.0	10.0	10.0	10.0
Wheat germ raw	g	44.0	46.2
KOH solution	ml	5.0	5.0	5.0	5.0
Linseed oil (raw)	ml	6.5
Soya oil	ml		7.0	7.0	7.0
Triton – x solution	ml			10.0	10.0
Water	ml	750.0	840.0	840.0	840.0

* Dissolved in 15 mL of water ** Dissolved in 15 mL of alcohol

Procedure for preparing diet:

1. Weigh ingredients separately.

2. Mix dry ingredients, soya oil, KOH and Triton-x solutions in 220 mL of hot water (70ºC) in a blender.

3. Dissolve agar in 620 mL of water in a cooking pot at 80ºC

4. Cool dissolved agar to 70ºC and pour it in the blender containing the mixed ingredients.

5. Ground thoroughly the mixture for 1-3 minutes

6. Dispense the hot diet in cups

7. Scarify’ the top of the diet (when cooled) with a scarification tool.

8. Store the cups with diet in plastic bags in refrigerator

Table 2 Percentage pupal harvest and moth emergence and mean weight of moths from greenhouse larvae reared on different types of diet

Diet	Pupal Harvest (%)	Moth Emergence (%)	Sex Ratio (M:F)	Mean Weight of Moth (g)
				male	female
Cabbage	75	54	1:1	0.0027	0.0032
Biever's diet	44	26	2:1	0.0026	0.0032
G^+	34	20	3:2	0.0024	0.0032
G^++	46	28	2:1	0.0028	0.0030
Soya	52	42	2:3	0.0044	0.0056

Longevity and fecundity of Plutella xylostella

Adult longevity of DBM males and females reared from the 5 types of diets are shown in Figure 2. Males reared from all diets lived longer (11 – 13 days) compared to females (8 – 11 days). On soya diet, females lived for about 9 days.

All mated females produced eggs for 9 days. Egg laying started 24 hours after mating. Data on the cumulative number of eggs laid by females reared on the 5 diets are represented in Figure 1. The average number of eggs per female reared on cabbage leaves during her life time was 225. A female (from soya diet) laid 207 eggs whereas those reared from Biever’s, G⁺, and G⁺⁺ diets laid 103, 163 and 135 eggs respectively. About 78 % of the eggs were collected during the first 5 days.

The average number of eggs per female raised on soya diet or cabbage was similar and was significantly higher than the number per female raised on the other 3 diets (P < 0.05).

Development Time

Hatching of eggs started after 48 hours of incubation. Larval development (1^st to 4^th instar) was completed in 10-11 days and the pupal period was 3-4 days.

Oviposition substrate

Among the 6 types of substrates, the number of eggs recorded on aluminium foil dipped in boiled cabbage juice was significantly higher (Table 3). Egg numbers on aluminium foil dipped in boiled cabbage juice was 12 times higher than aluminium foil dipped in fresh cabbage juice.

Figure 1 Cumulative numbers of eggs laid by females reared on different diets

Figure 2 Male and Female longevity of DBM moths reared on artificial diet.

Table 3 Number of eggs collected on different substrates

Oviposition Surface	Egg count	Average
Towel Paper	1 806	602 b
Aluminuim foil plain	4 437	1 479 b
Aluminuim foil crinkled	2 075	692 b
Aluminuim foil grooved	2 985	995 b
Aluminuim foil grooved + fresh cabbage juice	938	312.7 c
Aluminuim foil grooved + boiled cabbage juice	11 154	3 718 a

a numbers within a column with the same letter are not significantly different (P < 0.05)

DISCUSSION

Soya diet was the most suitable one for DBM rearing. In addition to the basic ingredients in Biever’s diet, Brewer’s yeast, Sorbic acid, Cholesterol, L. Inositol, Choline Chloride and Triton - x solution were incorporated in soya diet (Table 1). The poor larval development on Biever’s diet could be related to the absence of Cholesterol that is essential for successful insect molting and wing formation (Chapman, 1991 and Inositol, a phagostimulant (Hsiao & Hou, 1978). Sorbic acid controls microbial contamination (Carpenter et al. 20002). DBM larval development period on soya diet was almost similar to that on cabbage (10-11 days).

DBM can be successfully reared on soya diet with strict sanitary measures in the rearing facility. Occasional contamination (microbial and mould) is almost inevitable and can cause high mortality in DBM larvae and adults. This can be suppressed by maintaining a clean environment and sterilising laboratory equipment, eggs and diets.

It is now desirable to determine whether there is any change in the biological performance of these laboratory reared moths.

ACKNOWLEDGEMENTS

We are grateful to the staff of the Entomology Division and Mr R.K. Ramnauth (Biometrician) for their assistance.

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