Evaluation of Artificial Diets for atlas (: ) in Yogyakarta Special Region, Indonesia Author(s): Sukirno Sukirno , J. Situmorang , S. Sumarmi , R. C. Hidayat Soesilohadi , and R. Pratiwi Source: Journal of Economic Entomology, 106(6):2364-2370. 2013. Published By: Entomological Society of America URL: http://www.bioone.org/doi/full/10.1603/EC13144

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.

BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. ECOLOGY AND BEHAVIOR Evaluation of Artificial Diets for Attacus atlas (Lepidoptera: Saturniidae) in Yogyakarta Special Region, Indonesia

SUKIRNO SUKIRNO,1,2,3 J. SITUMORANG,1 S. SUMARMI,1 R. C. HIDAYAT SOESILOHADI,1 4 AND R. PRATIWI

J. Econ. Entomol. 106(6): 2364Ð2370 (2013); DOI: http://dx.doi.org/10.1603/EC13144 ABSTRACT The objective of this research was to evaluate artiÞcial diets that can be used to successfully culture the atlas , Attacus atlas L. (Lepidoptera: Saturniidae) indoors. Four plant were evaluated as the basic component of each diet, barringtonia (Barringtonia asiatica), cheesewood (Nauclea orientalis), soursop (Annona muricata), and mahogany (Swietenia mahagoni). Evaluation of the nutritional value of each diet was determined by an analysis of the hemolymph proteins of sixth using the FolinÐCiocalteu assay. Survivorship, cocoon quality, and hemolymph protein content of larvae fed the barringtonia diet were higher than those of larvae fed mahogany-, cheesewood-, and soursop-based artiÞcial diets. The average adult emergence of those fed the barringtonia-based diet was 74.5%. The weights of the cocoon in this treatment with the and the empty cocoons were 7.0 and 1.1 g, respectively. Hemolymph of the larvae fed the barringtonia-based artiÞcial diet had the highest concentration of protein with an average of 28.06 mg/ml. The atlas moth reared on the barringtonia-based artiÞcial diet was comparable with those reared only on barringtonia leaves. However, the weight of empty cocoons, adult wingspan, and amount of hemolymph protein were lower than in those reared on barringtonia leaves only. This may suggest that the artiÞcial barringtonia-based diet requires additional protein for maximum efÞciency.

KEY WORDS Attacus atlas, artiÞcial diet, hemolymph protein

Larvae of the atlas moth, Attacus atlas (L.) and known plants for the atlas moth (Peigler 1989). A survey in in Indonesia as “ulat jedung,” form large conspicuous Yogyakarta Special Region, Java, Indonesia in 2009 silken cocoons (Peigler 1989). The atlas moth is found revealed that there were 29 species of plants that were throughout the AsiaÐPaciÞc Region, including Indo- primary or secondary hosts for this moth (Soesilohadi nesia (Peigler 1989). The larvae of this species can be et al. 2009). The culturing of many depends on an important defoliator of cinchona, tea, avocado, and the success of development of an artiÞcial diet mango (Kalshoven 1981). Although this species is (Vanderzant 1974). In Yogyakarta Special Region, sometimes considered a pest of certain cultivated there are four plant species, barringtonia (Barringto- plants, the silk produced when the larvae form co- nia asiatica (L.) Kurz), cheesewood or Leichhardt coons is of higher quality than that produced by the tree (Nauclea orientalis L.), soursop (Annona muricata well-known silk moth, L. (Akai 2000). L.), and mahogany (Swietenia mahagoni (L.) Jacq), Moreover, the atlas moth has the potential to be cul- which may have a high potential when used as basic tured like the tasar silkworm, Antheraea mylitta components for the artiÞcial diets. Previous research (Drury), and the eri silkworm, Samia cynthia ricini using the above mentioned plants to rear the atlas Boisduval. Since 1992, the atlas silk moth has been moth has shown that the overall survivorship of larvae maintained in culture under screen house conditions in the Entomology Laboratory, Faculty of Biology, fed barringtonia leaves was the highest (Situmorang Gadjah Mada University, Indonesia. 1996, Tjiptoro 1997, Subagyo 2000, Widyarto 2001, There are at least 110 species representing 49 plant Situmorang and Wuliandari 2002). However, the mor- families that have been recorded as possible host tality rate of larvae using barringtonia reared outdoors was high, reaching 57%, because of predators, parasi- 1 Entomology Laboratory, Faculty of Biology, Gadjah Mada Uni- toids, and diseases (Purwanto and Sukirno 2006). In versity. Jalan Teknika Selatan, Sekip Utara, Depok Sleman 55281 silk such as Antheraea, Attacus, Callosamia, Plat- Indonesia. 2 Economic Entomology Research Unit, Plant Protection Depart- ysamia cecropia L., Saturnia pyri L., B. mori, and Erio- ment, College of Food and Agriculture Sciences, King Saud Univer- gaster lanestris L., the photoperiod, light intensity, sity, Riyadh, Kingdom of Saudi Arabia. temperature, and humidity have been found to affect 3 Corresponding author, e-mail: [email protected]. cocoon quality and duration of diapause (Kato and 4 Biochemistry Laboratory, Faculty of Biology, Gadjah Mada Uni- versity. Jalan Teknika Selatan, Sekip Utara Depok Sleman 55281 In- Sakate 1980, Peigler 1989, Kato and Miyata 1994, Hari- donesia. zanis 2004).

0022-0493/13/2364Ð2370$04.00/0 ᭧ 2013 Entomological Society of America December 2013 SUKIRNO ET AL.: EVALUATION OF ARTIFICIAL DIETS FOR Attacus atlas 2365

Research using host plant foliage and artiÞcial diets Table 1. The ingredients of the artificial diets for the rearing for rearing the atlas moth has been conducted. Rela- of atlas moth, A. atlas tive cocoon quality of laboratory-reared moths was Ingredients Barringtonia Cheesewood Mahogany Soursop lower than that of those reared in natural settings (Situmorang 1996, 1997). The atlas moth has been Leaf powder (g) 35 35 35 35 Cellulose powder (g) 5 5 5 5 reared on an artiÞcial diet with modiÞcation of diet Corn bee pollen (g) 30 30 30 30 ingredients originally developed for An. mylitta by Low fat soya bean 5555 powder (g) Situmorang (1997). The diet was modiÞed by replac- Vitamin C (g) 1.5 1.5 1.5 1.5 2Ϫ ing arjun leaf powder (Terminalia arjuna (Roxb.) K2HPO4 (g) 0.8 0.8 0.8 0.8 Wight & Arn.) with barringtonia and Chlorella with CaCO3 (g) 0.96 0.96 0.96 0.96 Fe2PO3 (g) 0.16 0.16 0.16 0.16 bee pollen. Chloramphenicol (g) 0.08 0.08 0.08 0.08 The atlas silk processing industry in Yogyakarta has Acrylic acid (ml) 0.8 0.8 0.8 0.8 relied on harvesting wild cocoons. Recently, it has Vitamin B complex (g) 0.06 0.06 0.06 0.06 Sorbic acid (g) 0.08 0.08 0.08 0.08 become difÞcult to Þnd wild atlas cocoons in numbers Agar (g) 8.43 8.43 8.43 8.43 sufÞcient to support this silk industry. To fulÞll the Distilled water (ml) 400 400 400 400 demand for cocoons, an attempt to successfully cul- ture the atlas moth indoors has become a priority. Moth propagation in the laboratory may also provide Ten petri dishes (90 by 15 mm) with 15 neonate a source of disease-free and neonate larvae to be larvae were reared on each diet with 10 replicates for cultured outdoors. Possible domestication of this each treatment. Portions of the diet (10 by 10 mm) moth, especially with the availability of an artiÞcial were arranged with 5-mm gaps between them and diet, could support both industry demands and reduce were supported from below using small bamboo sticks excessive exploitation of the moth in the wild. (4 by 80 mm) to facilitate larval access. Larvae on the Nutritional value of the artiÞcial diet is the major artiÞcial diet were incubated at room conditions of factor for the survivorship of silk moth larvae, includ- 24Ð30ЊC and 85Ð100% relative humidity (RH). Two ing the atlas moth. The quality of the diet must be days after the larvae underwent second molting, evaluated continuously to improve and maintain op- they were transferred into a bamboo box and fed with timal rearing (Vanderzant 1974). A successful artiÞcial the artiÞcial diet until the fourth instar. Fourth instar diet must have complete nutrition; be easily digested, larvae were maintained individually until pupation. absorbed, and processed through metabolic processes; Each transferred was labeled to keep track of and support respiration and excretion (Trager 1953, which dish the larva came from. Frass was removed Coudron et al. 2004). The objective of this research daily, and the diets were changed at least weekly. was to develop and to evaluate the nutritional value of Seven days after pupation, the cocoons were col- artiÞcial diets used to culture the atlas silk moth in- lected and weighed. The cocoons were then incu- doors. bated in cages (500 by 500 by 750 mm) with mesh walls until emergence. The cages were covered with a wet cloth to maintain humidity and stimulate emergence. Materials and Methods The parameters recorded were larval development This research was undertaken from November 2008 time, cocooning percentage, cocoon weight, adult to April 2009 at the Entomology Laboratory, Faculty emergence, adult wingspan, and percentage of of Biology, Gadjah Mada University, Indonesia. Larvae hatchability. The number of male and female moths used were the progeny fed fresh barringtonia leaves emerged from cocoons for each diet was recorded. for three successive generations in the laboratory. As controls, 50 neonate larvae were reared outdoors The artiÞcial diet ingredients used in this research on barringtonia foliage, and 50 neonates were also were modiÞed from an original diet developed by maintained in the laboratory using only barringtonia Situmorang (1997). The host plant components used leaves. in this research were dry green leaf powders from Selection of the cocoons for commercial use is based barringtonia, mahogany, cheesewood, and soursop. on the parameters of cocoon cleanliness, color, and Fresh young green leaves of each plant were washed the availability of spun Þber (Nunuh 2013). As in using tap water, cut into 3-mm-wide strips, and dried commercial operations, cocoons were graded as in an incubator at 50ЊC for 24 h. The dried cut leaves “good,”“medium,” and “poor” quality. Based on these were ground into powder using a grinder. The ingre- criteria, the cocoon yield for each artiÞcial diet treat- dients (Table 1) of the diets consisted of leaf powder, ment was selected. Good quality cocoons had a com- cellulose powder, bee corn pollen, low fat soybean pact texture, even color, and even thickness; cocoons powder, ascorbic acid, chloramphenicol, potassium with uneven color were graded as medium quality. hydrophosphate, iron phosphate, calcium carbonate, Good and medium quality cocoons are considered acrylic acid, and ground vitamin B complex tablets acceptable commercial grades. If the cocoon was thin, (Wyeth Pharmaceuticals, Madison, NJ). These were not compact, with even or uneven color, and could not homogenized using a blender. The sorbic acid, agar, be spun, it was categorized as poor quality (Nunuh and distilled water were boiled. After the agar solution 2013). reached 50ЊC, all other ingredients were added and Nutritional Value Evaluation of the Diets. Hemo- mixed using a standard laboratory blender. lymph protein was quantiÞed using the FolinÐCiocal- 2366 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 6

Table 2. Quality of cocoon shell weights (mean ؎ SEM) from atlas moth larvae fed on different diets

ArtiÞcial diets Barringtonia leaf Cocoon Quality N Barringtonia N Mahogany N Cheesewood N Soursop N Outdoor N Indoor Weight (g) Good 50 1.1 (0.03)a 11 1.03 (0.05)a 10 0.91 (0.04)a 3 0.98 (0.08)a 15 1.69 (0.0)b 17 1.89 (0.0)b Medium 27 1.0 (0.02)ab 3 1.13 (0.13)b 7 0.91 (0.08)ab 2 0.78 (0.03)a ÑÑÑÑ Poor 48 0.79 (0.03)a 3 0.7 (0.24)a 9 0.76 (0.03)a 2 0.78 (0.03)a ÑÑÑÑ Proportion (%) Good 45.19 64.71 38.46 50.0 68.18 43.59 Medium 20.0 17.65 26.92 25.0 22.73 38.46 Poor 34.81 17.65 34.62 25.0 9.09 17.95

Numbers in the same rows followed by a different letter show signiÞcant differences at ␣ 0.05. teu assays (Plummer 1971) to evaluate the nutritional ing Curve Expert ver. 1.3 (Curve Expert Software, value of each artiÞcial diet. Two days after the Þfth Chattanooga, TN). molt, hemolymph was obtained by severing two dorsal abdominal tubercles. The hemolymph was collected Results in a 5-ml test tube and added to a small amount of 1-phenyl-2-thiourea crystals (Magro et al. 2006). The Study of the Artificial Diet’s Effect on Atlas Moth isolated hemolymph was centrifuged at 10,000 rpm for Survivorship. The weights of good quality cocoons 5 min. The protein pellets were added to 1 ml of produced by larvae fed different diets were signiÞ- demineralized water. Next, 25-␮l samples were added cantly different (F ϭ 86.4; df ϭ 5,100; P Ͻ 0.001). to 1 ml of alkaline solution (2% Na2CO3 in 0.1 N NaOH Larvae fed the barringtonia leaves produced heavier and 0.5% CuSO4 in 1% NaK; SigmaÐAldrich, St. Louis, cocoons than those fed the artiÞcial diets (Table 2). In MO), homogenized, and then incubated at room tem- the medium quality category, larvae fed the mahog- perature for 10 min. Then, 3 ml of FolinÐCiocalteu any-based diet were signiÞcantly heavier (F ϭ 1.97; reagents (SigmaÐAldrich) was added, stirred, and in- df ϭ 3, 35; P Ͻ 0.05) than those fed the soursop diet. cubated for 30 min at room temperature. Protein In contrast, poor quality cocoons produced by the quantiÞcation of the solution was measured using a different diets were not signiÞcant (F ϭ 1.9; df ϭ 3, 58; spectrophotometer (Spectronic 21DV; Milton Roy P Ͼ 0.05). Company Analytical Products Division, Rochester, The proportion of good quality cocoons on the NY) at a wavelength of 595 nm and bovine serum artiÞcial diet treatments ranged between 38 and 65%. albumin (BSA) as a standard. Two-day-old sixth in- Larvae reared on barringtonia leaves indoors yielded stars reared indoors on barringtonia leaves were used good quality cocoons 43% of the time. These propor- as controls. Seven larvae that were reared separately tions were lower than those of larvae reared on bar- in identical conditions for each diet were tested. ringtonia leaves in outdoor conditions. Cocoons of Data Analysis. The research design used in this poor quality produced by larvae on artiÞcial diets were experiment was completely randomized. An analysis 18Ð35%, and this percentage was higher than that of of variance (␣ ϭ 0.05) was conducted to compare the cocoons produced by larvae fed barringtonia leaves diets and was followed by DuncanÕs multiple range outdoors. test to separate means. To estimate the potential in- The diet formulae affected larval survival. There teraction between hemolymph protein content and were signiÞcant differences (F ϭ 179.2; df ϭ 5, 36; P Ͻ atlas moth survivorship, regression analysis was per- 0.001) in the cocoon yield percentage of larvae fed the formed using SPSS 13 (SPSS Inc. 2005). By considering different diets (Table 3). Cocoon yields of larvae fed the correlation values and its P value, the curve equa- barringtonia leaves and barringtonia artiÞcial diets tion estimation procedures were then performed us- were higher than those of larvae on other treatments.

Table 3. Atlas moth stage survivals, developmental times, cocoon weights, wingspans, and fecundity (mean ؎ SEM) on four different artificial diets containing powdered host leaves

ArtiÞcial diet Barringtonia leaf Parameters N Barringtonia N Cheesewood N Mahogany N Soursop N Outdoor N Indoor & larval duration (d) 46 37.26 (0.50)ab 9 40.22 (0.70)b 7 40.00 (0.87)ab 3 37.00 (2.89)a Ñ Ñ Ñ Ñ ( larval duration (d) 42 38.35 (1.42)a 12 40.35 (1.17)a 8 40.33 (2.60)a 3 35.50 (0.58)a Ñ Ñ Ñ Ñ Cocoon yields (%) 10 86.00 (2.89)d 10 20.00 (2.43)b 10 14.7 (1.66)ab 10 6.7 (1.41)a 1 44 (0.0)c 1 82 (0.0)d Cocoon wt (g) 129 7.81 (0.16)c 30 6.34 (0.28)a 22 7.12 (0.21)bc 10 6.97 (0.97)ab 22 9.12 (2.04)c 41 8.85 (1.84)c & pupal duration (d) 46 24.13 (1.08)a 9 24.67 (0.65)a 7 25.00 (0.58)a 3 25.50 (1.00)a Ñ Ñ Ñ Ñ ( pupal duration (d) 42 23.29 (1.42)a 12 23.45 (0.58)a 8 22.67 (1.20)a 3 26.50 (2.73)a Ñ Ñ Ñ Ñ Adults emergence (%) 10 74.53 (2.78)c 10 16.57 (1.81)a 10 13.2 (1.37)a 10 6.10 (1.53)a 2 38.18 (1.3)b 2 76.18 (1.3)c & wing span (cm) 46 18.98 (0.37)bc 9 15.94 (0.89)a 8 16.36 (1.00)ab 3 18.25 (0.73)abc 10 20.89 (0.18)c 18 20.83 (0.26)c ( wing span (cm) 42 17.21 (0.29)abc 12 16.83 (0.79)ab 3 15.48 (2.46)a 3 17.00 (0.84)abc 12 20.23 (0.32)c 23 18.86 (0.79)bc Fecundity 17 273.5 (8.99)b 5 174.4 (21.00)a 4 149.3 (53.03)a Ñ Ñ Ñ Ñ Ñ Ñ Eggs hatch (%) 17 87.39 (2.56)a 5 76.84 (7.52)a 4 75.82 (4.49)a Ñ Ñ Ñ Ñ Ñ Ñ

Numbers in the same rows followed by a different letter show signiÞcant differences at ␣ 0.05. December 2013 SUKIRNO ET AL.: EVALUATION OF ARTIFICIAL DIETS FOR Attacus atlas 2367

Table 4. Hemolymph protein contents of the larvae fed on Evaluation of Artificial Diet Quality. There were different diets signiÞcant differences (F ϭ 14.88; df ϭ 4, 30; P Ͻ 0.05) in the hemolymph protein content among larvae fed Treatments N Protein content (mg/ml) different diets (Table 4). The hemolymph protein Barringtonia-based diet 7 28.06 (8.52)b content of larvae fed barringtonia leaves was signiÞ- Cheesewood-based diet 7 17.92 (5.90)ab Mahogany-based diet 7 16.05 (2.86)ab cantly higher than that of those fed the four artiÞcial Soursop-based diet 7 3.09 (2.02)a diets. In larvae fed the barringtonia-based artiÞcial Barringtonia leaf (indoor) 7 56.91 (7.42)c diet, hemolymph protein content was signiÞcantly higher than in larvae fed the soursop diet. However, Numbers followed by a different letter show signiÞcant differences at ␣ 0.05. an analysis of the hemolymph protein content of lar- vae fed the barringtonia diet was higher than those fed the cheesewood and mahogany diets but not signiÞ- The weight of the cocoon with an enclosed pupa from cantly different. the barringtonia leaves treatment was signiÞcantly Correlation of Hemolymph Protein and Atlas Moth higher than those on cheesewood and soursop diets 2 Ն ϭ ϭ Ͻ Survivorships. There was a positive correlation (R (F 13.34; df 5, 248; P 0.001). Different diets 0.95; r Ն0.95; P Յ 0.05) between hemolymph protein affected the developmental period of female larvae content and male larval duration, pupal duration, adult (F ϭ 2.13; df ϭ 3, 61; P Ͻ 0.10), but developmental emergence, fecundity, egg hatchability, and weight of periods of male larvae were not signiÞcantly different good quality cocoons (Table 5). The relationship be- (F ϭ 0.87; df ϭ 3, 61; P Ͼ 0.05). There were no tween protein content and female pupal duration was signiÞcant differences between female (F ϭ 0.22; df ϭ 3, 61; P Ͼ 0.05) and male pupal durations (F ϭ 0.28; linear, whereas other relationships were nonlinear df ϭ 3, 61; P Ͼ 0.05). Emergence rates of moths fed on with an optimum point. barringtonia leaves indoors and on the barringtonia artiÞcial diet were higher than those of moths in other treatments (F ϭ 263.9; df ϭ 5, 46; P Ͻ 0.05). Few adults Discussion emerged from cocoons of larvae that were fed cheese- wood and soursop. The weight of cocoons without enclosed pupae pro- The diets affected adult wingspan, fecundity, and duced by larvae fed a barringtonia-based artiÞcial diet egg viability (Table 3). The wingspan of females on was lower than those of larvae fed only barringtonia the barringtonia leaves treatment was signiÞcantly leaves. Average cocoon shell weight for larvae fed wider than that of those on cheesewood- and mahog- barringtonia leaves was 1.89 g or 42% higher than that any-based diets (F ϭ 8.99; df ϭ 5, 88; P Ͻ 0.001). In for those on the diet. This may be the result of nutri- contrast, for males, the wingspan of those reared on ents in the natural diet being higher and apparently barringtonia leaves were signiÞcantly wider than that more complete than those available in artiÞcial diets. of moths reared on the mahogany diet (F ϭ 10.2; df ϭ Fresh leaves of barringtonia contain triterpene com- 5, 89; P Ͻ 0.001). Females produced by larvae fed the pounds such as germanicol and cameliogenone (Ra- barringtonia artiÞcial diet laid signiÞcantly more eggs gasa et al. 2011). They also contain spinasterol, sitos- (F ϭ 5.96; df ϭ 2, 23; P Ͻ 0.01). The number of viable terol, squalene, lutein, and trinolein (Ragasa et al. eggs produced by moths fed the barringtonia-based 2011). The terpenes may play important roles in atlas artiÞcial diet was not signiÞcantly different (F ϭ 2.60; moth larval food preferences (Nuringtyas et al. 2007). df ϭ 2, 23; P Ͼ 0.05) than that produced by moths In the artiÞcial diet containing leaf powders, these reared on mahogany- and cheesewood-based diets. compounds are lost during the drying process.

Table 5. Regression and correlation of hemolymph protein content and atlas moth survivorship

Interaction R2 Pattern rPvalue Equation Female larval duration (d) 0.99 Quadratic 0.99 0.07 y ϭ 35.28 ϩ 0.61x Ϫ 0.02x2 Male larval duration (d) 1.0 Quadratic 0.99 0.01 y ϭ 33.3 ϩ 0.78x Ϫ 0.02x2 Cocooning % 0.75 Quadratic 0.86 0.25 y ϭϪ15.5 ϩ 3.5x Ϫ 0.03x2 Cocoon wt (g) 0.80 Quadratic 0.89 0.20 y ϭ 6.79 ϩ 0.002x ϩ 0.0006x2 Female pupal duration (d) 0.96 Linear 0.98 0.02 y ϭ 25.72 Ϫ 0.05x 22.61x Male pupal duration (d) 0.93 S 0.97 0.04 y ϭ Ϫ0.45 ϩ x Adult emergence (%) 0.99 Quadratic 0.99 0.04 y ϭ 14.3 Ϫ 3.2x ϩ 0.19x2 Female wing span (cm) 0.98 Quadratic 0.99 0.16 y ϭ 19.59 Ϫ 0.48x ϩ 0.02x2 Male wing span (cm) 0.54 Quadratic 0.74 0.68 y ϭ 17.48 Ϫ 019x Ϫ 0.006x2 Ϫ4,368x Fecundity 1.0 S 0.99 0.01 y ϭ Ϫ475.8 ϩ x Eggs hatchability (%) 0.99 Exponential 0.99 0.04 y ϭ 62.1e0.01x Good cocoon wt (g) 0.99 Quadratic 0.99 0.01 y ϭ 0.99 Ϫ 0.01x ϩ 0.0004x2 1,407 ϩ 1.04x3 Medium cocoon wt (g) 0.69 S 1 0.17 y ϭ 1,828 ϩ x3 Poor cocoon wt (g) 0.60 Quadratic 0.77 0.63 y ϭ 0.81 Ϫ 0.01x ϩ 0.0004x2 2368 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 6

In outdoor rearing, an average cocoon weight of eggs laid by these adults was comparable with those larvae fed mahogany was 1.35 g (Subagyo 2000) and laid by adults reared on barringtonia leaves (Situmo- that of larvae fed soursop and cheesewood was 1.26 rang 1996), ranging from 108 to 326 eggs. This fact is and 0.8 g, respectively (Wahyudi 2000). Larvae rear- important for producing eggs for commercial-scale ing of the atlas moth using the barringtonia-based atlas moth production. artiÞcial diet produced a better grade cocoon than QuantiÞcation of hemolymph proteins has been re- those fed other diets, but cocoon grades were still ported by Cremonez et al. (1998) for the honey bee lower than those of larvae fed solely fresh leaves of the and by Magro et al. (2006) for the braconid wasp, same plant. Bracon hebetor Say. QuantiÞcation of hemolymph pro- The percentage of larvae successfully forming a tein is possible to evaluate diet quality. Total hemo- cocoon after being fed the barringtonia diet was lymph protein levels of last instar atlas moth larvae higher than that of larvae fed SitumorangÕs artiÞcial appear to be an accurate method to evaluate the diet diet (63%) (Situmorang 1997) and barringtonia leaves quality. Our analyses showed variability in protein (Table 3). This suggests that the artiÞcial diet used in titers in the hemolymph of atlas moths fed four arti- our experiment is better than previous diets available Þcial diets reared under identical conditions (Table because larvae yield better cocoons, and this artiÞcial 4). The FolinÐCiocalteu assay indicated high reactiv- diet has the potential to be used for mass indoor ity with amino acids, tyrosine, cysteine, and trypto- rearing. phan. It also showed high reactivity with phenols, Some research is available regarding rearing of the thiols, vitamin derivatives, inorganic ions, nucleotide atlas moth indoors using only fresh leaves. Only 30% guanine, trioses glyceraldehydes, and dihydroxyac- of the larvae reared on cheesewood leaves produced etone (Everette et al. 2010). Protein measurements a cocoon with larval duration and cocoon yield of are relatively inexpensive and rapid. Measurement 22Ð31 d and 4.79Ð12.92 g, respectively (Tjiptoro 1997). requires only small amounts of relatively inexpensive While on the Erythrina (Zebua 1997), soursop (San- reagents and causes no larval mortality. The extraction tika 1997), and barringtonia leaves (Situmorang 1996), of amino acids in the last instar of A. atlas larvae the larval duration was 31, 35, and 28 d, respectively. showed that methionine was dominant ( 2001). The average cocoon weight and larval duration in the The use of hemolymph proteins to evaluate diet qual- previous artiÞcial diet were 7.23 g and 32.7 d, respec- ity is an important tool in mass rearing of the atlas tively (Situmorang 1997). Although the larval dura- moth. tion of the reared on the barringtonia-based diet Protein variability affects male larval duration, pu- was Ϸ5 d longer than the previous diet, the cocoon pal duration, adult emergence, fecundity, egg hatch- weight was almost 10% higher and comparable with ability, and the weight of good quality cocoons. The those fed leaves only. Other studies have indicated protein content of the diet affects larval growth and that barringtonia is the most preferred host plant (Pur- wanto and Garjito 2002, Purwanto et al. 2002). relative free amino acid composition of the hemo- The proportions of good quality cocoons in indoor lymph (Horie and Watanabe 1983). In B. mori, the rearing were less than those in outdoor rearing (Table presence of carotenoid-binding proteins in hemo- 2). Besides the nutritional value of the diet, both the lymph plays an important role in cocoon pigmentation dietÕs water content and environmental conditions (Tabunoki et al. 2004). Embryonic development and under which the larvae are reared are important in egg hatching of this species may require the presence growth and development. A satisfactory diet for an of proteins (Zhong et al. 2005). In Cecropia silk worm, insect usually has similar water content as in natural proteins control the pupal duration and termination of food. The water content is important for metabolism diapauses (Telfer and Williams 1960). (Kikuchi et al. 1980, Mathews and Stephen 1999). Based on the results of our experiment, a barringto- Temperature and relative humidity are important to nia-based artiÞcial diet was the most successful diet for maintain suitable metabolism processes. Relative hu- laboratory rearing of the atlas moth. The rearing of the midity of Ͼ80% increases survivorship because of the atlas moth on the barringtonia-based artiÞcial diet was higher nutritional value of the diet (Mathews and comparable with rearing moths on preferred host Stephen 1999). It has been shown that light intensity plants, although further reÞnement is still needed to enhances proper cocooning in B. mori (Krishnaswami improve the empty cocoon weight and adult size. et al. 1972). It has been suggested in the atlas moth Further studies on the optimization of rearing condi- cocoon that quality is better in outdoor rearing be- tions such as humidity, temperature, and light inten- cause of natural light conditions (Situmorang 1996). In sity for maximum larval survival are also needed. indoor rearing of the atlas moth, optimal light regime In conclusion, barringtonia leaves produced the levels are required for ideal growth and development. best artiÞcial diet for rearing the atlas moth under Adult emergence of the atlas moth larvae fed the indoor conditions. Continuous evaluation of the nu- barringtonia-based diet was comparable with that of tritional value of diets using hemolymph protein con- those fed barringtonia leaves. Emergence rates were tent is a simple, fast, and inexpensive approach to higher under indoor conditions. Although the adults evaluate the development of a satisfactory diet. How- had relatively smaller wingspans than those fed leaves, ever, hemolymph protein content analysis indicates fecundity was satisfactory. In this treatment, adults that the artiÞcial diet formula as used in this study laid 125Ð389 eggs with 87% viability. The number of requires some additional reÞnement. December 2013 SUKIRNO ET AL.: EVALUATION OF ARTIFICIAL DIETS FOR Attacus atlas 2369

Acknowledgments Nunuh, A. 2013. Serikultur: budidaya sutera alam (Bombyx mori Linn.). A Guide Manual for Silk Worm Cultivation. The rector of Gadjah Mada University and the Dean of Bandung, Jawa Barat, Indonesia. Biology Faculty provided Þnancial support. We thank the Nuringtyas, T. R., H. Purwanto, and A. Rochayati. 2007. head of Entomology Laboratory for facilitating research in Senyawa metabolit sekunder pada daun muda dan tua the laboratory and Sawit Sari Research Station. empat jenis tanaman pakan alami ulat atakas (Attacus Boris C. Kondratieff, Colorado State University USA assisted atlas L.). Berkala Ilmiah Biologi 6: 63Ð70. in a review of the manuscript. We thank Suparmin and Peigler, R. S. 1989. A revision of The Indo-Australian genus Paimin for their technical support. We gratefully acknowl- Attacus. The Lepidopteran Research Foundation Inc., edge anonymous reviewers who gave valuable suggestions Beverly Hill, CA. for improving this manuscript. Plummer, D. T. 1971. An introduction to practical bio- chemistry. Tata McGrawÐHill Publishing Company Ltd., New Delhi, . References Cited Purwanto, H., and T. A. Garjito. 2002. Eggs deposition pref- erence of Attacus atlas (L.) (Lepidoptera: Saturniidae) Akai, H. 2000. Review of cocoon Þlament characters and on Several of Host Plants. The 4th International Confer- post cocoon technology. Int. J. Wild Silkmoth and Silk 5: ence on Wild Silkmoth, Yogyakarta. 71Ð84. Purwanto, H., and Sukirno. 2006. Food availability of keben Coudron, T. A., C. L. Goodman, W. A. Jones, and R. Leopold. (Barringtonia asiatica Kurzt.) to the development of the 2004. Development of an artiÞcial diet and evaluation of late instar of Attacus atlas (L.) reared outdoor without artiÞcial ovipositional substrates for the in vitro rearing of screen. In The 3rd International Life Sciences Postgrad- Gonatocerus spp. parasitoids of the eggs of the glassy- uate Conference, 2006, Penang, Malaysia. winged sharshooter, pp. 304Ð305. U.S. Department of Purwanto, H., R. Widyarto, and A. Wahyudi. 2002. Does Agriculture Technical Bulletin Agricultural Research species of parentÕs host plant of Attacus atlas (L.) (Lep- Service, Columbia. idoptera: Saturniidae) has effect on the survivorship of Cremonez, T. M., D. D. Jong, and M. C. Bitondi. 1998. progeny when reared on other plant species? The 4th QuantiÞcation of hemolymph protein as a fast method for International Conference on Wild Silkmoth, Yogyakarta, testing protein diets for honey bees (Hymenoptera : Api- Indonesia. dae). J. Econ. Entomol. 91: 1284Ð1289. Everette, J.D.Q., M. Bryant, A. M. Green, Y. A. Abbey, Ragasa, C. Y., D. L. Espineli, and C. C. Shen. 2011. New G. W. Wangila, and R. B. Walker. 2010. Thorough triterpenes from Barringtonia asiatica. Chem. Pharm. study of reactivity of various compound classes toward Bull. 59: 778Ð782. the FolinÐCiocalteu reagent. J. Agric. Food Chem. 58: Santika, I.G.K. 1997. Daur hidup Attacus atlas Linn. dengan 8139Ð8144. pemberian pakan sirsat (Annona muricata Linn.) di labo- Harizanis, P. C. 2004. Manual of : silkworm rear- ratorium. Undergraduate, Atmajaya University Yogya- ing and mulberry cultivation. Agricultural University of karta, Yogyakarta, Indonesia. Athens Laboratory of Sericulture and Apiculture, Athens, Sari, N. I. 2001. Komposisi asam amino hemolimfe dan Greece. sutera cair kelenjar sutera larva Attacus atlas (L.) (Lep- Horie, Y., and K. Watanabe. 1983. Effect of various kinds of idoptera: Saturniidae) yang diberi pakan daun keben. dietary protein and supplementation with limiting amino Undergraduate thesis, Faculty of Biology, Gadjah Mada acids on growth, haemolymph components and uric acid Uniersity, Yogyakarta, Indonesia. excretion in the silk worm, Bombyx mori. J. Insect Physiol. Situmorang, J. 1996. An attemp to produce Attacus atlas L., 29: 187Ð199. using baringtonia leaves as plant fooder. Int. J. Wild Silk- Kalshoven, L.G.E. 1981. Pest of crop in Indonesia. PT moth Silk: 55Ð57. Ichtiar Baru- Van Hoeve, Jakarta, Indonesia. Situmorang, J. 1997. Optimasi susunan pakan buatan untuk Kato, Y., and S. Sakate. 1980. Photoperiodic and tempera- pertumbuhan dan kualitas kokon ulat sutera atakas, At- ture conditions affecting summer diapause of Antheraea tacus atlas (Linn.) (Lepidoptera: Saturniidae). Research yamamai pupa. In Symposium in XVI International Con- Report Faculty of Biology, Gadjah Mada University, In- gress of Entomology 1980, 1981, Kyoto, Japan. donesia. Kato, Y., and T. Miyata. 1994. Cocoon coloration and its Situmorang, J., and J. R. Wuliandari. 2002. The effects of determination factor in Rhodinia fugax. Int. J. Wild Silk- different of food on growth of Attacus atlas Linn. reared moth Silk 1: 53Ð55. indoor and outdoor. In The 4th International Conference Kikuchi, Y., S. Hotta, and Y. Higuchi. 1980. Studies on the on Wild Silkmoth 23Ð27 April 2002, Yogyakarta. artiÞcial dry food for non-mulberry silkworm, p. 3338. In Soesilohadi, R.C.H., S. Sumarmi, Sukirno, and R. Susanda- Symposium in XVI International Congress of Entomology rini. 2009. Konservasi ulat sutera liar atakas, Attacus spp., 1980, 1980, Kyoto, Japan. Research Report National Priority Competitive Research Krishnaswami, S., N.R.M. Rao, S.S.K. Suryanarayan, and Grant. Indonesian Ministry of Higher Education. Gadjah S.T.S. Sundaramurthi. 1972. Sericulture manual 3-silk Mada University, Yogyakarta, Indonesia. reeling. FAO Agricultural Services Bulletin. p. 15. SPSS Inc. 2005. SPSS for windows userÕs guide, version 13.0. Magro, S. R., A. B. Dias, W. R. Terra, and J.R.P. Parra. SPPS Inc., Chicago, IL. 2006. Biological, nutritional, and histochemical basis Subagyo, A. 2000. Daur hidup Attacus atlas (L.) (Lepidop- for improving an artiÞcial diet for Bracon hebetor Say. tera: Saturniidae) dengan pakan alami daun mahoni (Swi- (Hymenoptera: Braconidae). Neotrop. Entomol. 35: etenia mahagoni Jacq.) di lapangan. Undergraduate, Gad- 215Ð222. jah Mada University Yogyakarta. Mathews, P. L., and F. M. Stephen. 1999. Effects of an Tabunoki, H., S. Higurashi, O. Ninagi, H. Fujii, Y. Banno, M. artiÞcial diet and varied environmental conditions on Nozaki, M. Kitajima, N. Miura. S. Atsumi, K. Tsuchida, et the longevity of Coeloides pissodis (Hymenoptera: Bra- al. 2004. A carotenoid-binding protein (CBP) plays a conidae), a parasitoid of Dendroctonus frontalis (Co- crucial role in cocoon pigmentation of silkworm (Bmbyx leoptera: Scolytidae). Environ. Entomol. 28: 729Ð734. mori) larvae. FEBS Lett. 567: 175Ð178. 2370 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 106, no. 6

Telfer, W. H., and C. M. Williams. 1960. The effects of Widyarto, R. 2001. Keberhasilan hidup Attacus atlas (L.) diapause, development, and injury on the incorporation (Lepidoptera: Saturniidae) yang berasal dari tanaman of radioactive glycine into the blood proteins of Cecropia dadap dan gempol, yang dipelihara pada tanaman keben, silk worm. J. Insect Physiol. 5: 61Ð72. sirsat, dadap dan gempol di lapangan. Undergraduate, Tjiptoro. 1997. Daur hidup Attacus atlas L. dengan pembe- Gadjah Mada University, Yogyakarta, Indonesia. rian pakan daun gempol (Nauclea orientalis L.) di labo- Zebua, B.T.U. 1997. Daur hidup Attacus atlas (L.) dengan ratorium. Undergraduate, Atmajaya University Yogya- pemberian pakan daun dadap (Erythrina lithosperma karta, Indonesia. Miq.) di laboratorium. Undergraduate, Atmajaya Univer- Trager, W. 1953. Nutrition. Wiley Inc., New York, NY. sity, Yogyakarta, Indonesia. Vanderzant, E. 1974. Development, signiÞcance, and appli- Zhong, B., J. Li, J. Lin, J. Liang, S. Su, H. Xu, H. Yan, P. Zhang, cation of artiÞcial diets for insects. Annu. Rev. Entomol. and H. Fujii. 2005. Possible effect of 30K proteins in 19: 139Ð160. embryonic development of silkworm Bombyx mori. Acta Wahyudi, A. 2000. Keberhasilan hidup Attacus atlas (Lepi- Biochimica et Biophysica Sinica. 37: 355Ð361. doptera: Saturniidae) pada tanaman dadap dan gempol di lapangan. Gadjah Mada University, Yogyakarta, Indone- sia. Received 26 March 2013; accepted 6 September 2013.