Int. J. Indust. Entomol. 30(2) 45-49 (2015) IJIE ISSN 1598-3579, http://dx.doi.org/10.7852/ijie.2015.30.2.45 Developmental characteristics of Zophobas atratus (Coleoptera: Tenebrionidae) larvae in different instars Sun Young Kim1, Hong Geun Kim1, Sung Ho Song2 and Nam Jung Kim1*

1Applied Entomology Division, National Academy of Agricultural Science, RDA, Wanju-gun, Jeollabuk-do, 565-851, Republic of Korea 2Gyeonggi Do Agricultural Research & Extension Services, Hwaseong-city Gyeonggi-do, 445-784, Republic of Korea

Abstract

The giant , Zophobas atratus (Coleoptera: Tenebrionidae), is reared for commercial purposes as a live feed for . In 2011, it was officially introduced in Korea, and since then it has been considered commercially important. This beetle is a good feed resource because of its high protein content with balanced nutrients. However, its life stage characteristics have not been clearly described, especially of the larval stage that can be used as commercial products. To this end, in our study, we determined the number of Z. atratus larval instars, and described their characteristics at each stage, providing basic information about this beetle. Z. atratus larvae required eight to nine d of incubation period before hatching. The first instar period comprised three to four d. There were relatively large Received : 8 May 2015 variations in each instar period, except for the first instar. Before the adults emerged, most Revised : 15 Jun 2015 of the individuals passed through15 to 18 instars. The highest pupation rate, 25.71%, was Accepted :16 Jun 2015 observed in both 16th and 17th instars. Body length gradually increased with each successive Keywords: instar and it reached its maximum at the 18th instar. The color of larvae was white at the first Zophobas atratus, instar, and gradually turned brown after the second instar. , © 2015 The Korean Society of Sericultural Sciences body length, Int. J. Indust. Entomol. 30(2), 45-49 (2015) instar

Introduction included in the list of excluded quarantine pests in 2011 , and has been allowed to be imported live as animal feed (Animal and Zophobas atratus Fabricius (Coleoptera: Tenebrionidae) is a Plant Quarantine Agency, 2013). large neotropical beetle. It is found on fruit bat guano and organic Z. atratus is larger than T. molitor, and is also known as super litter in its natural environment (Tschinkel and Willson, 1971; worm or king worm (Park et al., 2013). It has been used as a Quennedey et al., 1995). The shape of this beetle is similar to protein source for small pets such as birds, reptiles, and small that of mealworm, Tenebrio molitor, a well-known food resource mammals (Jabir et al., 2012; Park et al., 2013). Moreover, for small pets, but it is several times larger than a mealworm. It because of the rising prices of fish meal, which is widely used is not a domestic species of Korea, but originated from South a protein source in animal feed, alternative protein sources are and Central America (Park et al., 2013). In the Animal and Plant sought to reduce the production cost of various animal feeds. Quarantine Agency notification 2013-118, this beetle has been Recently, -based diet has become a strong candidate as

*Corresponding author. Nam Jung Kim Applied Entomology Division, National Academy of Agricultural Science (NAAS), RDA, 166, Nongsaengmyeong-ro, Iseo- myeon, Wanju-gun, Jeollabuk-do, 565-851, Republic of Korea Tel: +82-63-238-2810 / FAX: +82-63-238-3833 E-mail: [email protected] © 2015 The Korean Society of Sericultural Sciences

PB 45 Sun Young Kim et al. Developmental characteristics of Zophobas atratus (Coleoptera: Tenebrionidae) larvae in different instars an alternative to fish meal because fish raised on insect-based diameter × 1 cm height) containing bran. Eggs with hardened diet showed similar growth performance to those raised on shells were obtained after three d. We collected 40 eggs to count conventional fish meal-based diet (Jabiret al., 2012). the number of larval instar and to measure the body size. Each Much attention has been focused on Z. atratus because of its egg was transferred to a petri dish (5 cm diameter × 1.5 cm economic benefits. Previous studies have reported that Z. atratus height) containing wheat bran and Chinese cabbage (1 g). This is highly dependent on isolation for the onset of metamorphosis experiment was repeated 3 times. (Quennedey et al., 1995; Aribi et al., 1997). In crowded conditions, Z. atratus larvae showed increasing size and weight, Counting the number of larval instar and forming supernumerary larval instars that did not pupate until measuring body length of each instar death (Tschinkel and Willson, 1971). This inhibition of pupation induced by crowding conditions has also been described in other Each egg was reared in a petri dish (5 cm diameter × 1.5 cm tenebrionid species, for example, Tribolium freeman (Nakakita, height) with wheat bran at 25±3°C, 50–70% RH, and 14L:10D 1982; Kotake et al., 1993; Quennedey et al., 1995). photoperiod. The condition of the larvae was checked every day The number of post-embryonic molts in T. molitor is known to determine the number of instars. When the larval exuvium to range from eight to more than 20 larval molts, depending was observed, the larvae were removed from the petri dishes. on various intrinsic and extrinsic factors, such as food quality After pupation, instar counting was stopped. and individual density (Connat et al., 1991). Previous studies To measure the body length of each instar, the body lengths of have focused on various abiotic factors, including food quality, ten larvae were measured using a Vernier caliper after each larval humidity (Murray, 1968; Urs and Hopkins, 1973), temperature molting. Generally, the width of the head capsule was measured (Ludwig, 1956), and photoperiod (Tyschchenko and Sheyk because it exhibits distinct variation between the larval stages Ba, 1986) that affected the number of larval instars. However, (Hsia and Kao, 1987). In this experiment, however, the body the characteristics of different larval stages of Z. atratus lengths of larvae were also measured because of the small head commercialized in Korea remain unclear. In addition, the capsule size during the early larval stages. In addition, we took precise number of instars that Z. atratus larvae go through at the photographs of each instar using a digital camera (SX220 HS, optimal temperature of 25°C is almost unknown. To this end, Canon, Tokyo, Japan ). we investigated the characteristics of Z. atratus during different larval stages and determined the average number of larval instars. The results of this study will provide basic information Results & Discussion for future investigations on the physiological characteristics of different instars, as well as for more effective commercialization As shown in Table 1, the average incubation period for of Z. atratus. hatching was eight – nine d, and the duration for the first instar three or four d. Interestingly, the size of larvae was not significantly different for different incubation periods or Materials & Methods first instar durations. Between the second and 18th instars, the duration of each instar showed large variations among Breed condition individuals. The first pupation was observed at the 13th instar even though only 2% of larvae were pupated. Z. atratus adults (about 120 individuals) were raised in acrylic Previous studies have shown that the body size of larvae is boxes (58.2 cm width × 49.5 cm length × 17 cm height) at highly co-related with the pupation rate (Sehnal, 1985). The 25±3°C, 50–70% relative humidity (RH) and a photoperiod of developmental variability in tenebrionid beetle depends on two 14L:10D. In each acrylic box, wheat bran (ca. 3 cm deep) was parameters: the number of larval instars and the duration of placed as a food source, and a Chinese cabbage leaf was placed each larval instar. Previous studies have suggested that juvenile on the wheat bran layer as a moisture source. Once a mating hormones play a key role in the mealworm developmental couple was observed, they were moved to a petri dish (10 cm variability; the alimentary periods as well as the percentage of

46 47 Int. J. Indust. Entomol. Vol. 30, No. (1), pp. 45-49 (2015)

Table 1. Larval instar period, pupation rate, and pupal weight of the 13th to 18th instars, and body lengths of each larval stage of Zophobas atratus. Larval period Pupation rate Pupal weight Body length

Instar Mean (d) (%) (g) (cm) Incubation period 8.38±0.49 - - - 1 3.00±0.46 - - 0.30±0.40

2 6.95±1.74 - - 0.34±0.20

3 6.39±2.82 - - 0.44±0.27

4 9.67±3.25 - - 0.50±0.39

5 6.58±2.67 - - 0.62±0.52

6 8.81±2.90 - - 0.72±0.90

7 9.19±2.61 - - 0.84±0.94

8 10.31±5.98 - - 0.97±1.85

9 9.00±6.78 - - 1.18±2.49

10 7.83±1.48 - - 1.44±3.22

11 8.86±3.59 - - 1.65±3.46

12 9.36±2.61 - - 1.98±5.80

13 10.78±4.18 2.86±0.13 0.60±0.00 2.48±5.94

14 12.31±3.84 11.43±0.27 0.63±0.06 3.05±5.76

15 13.03±5.00 17.14±0.39 0.66±0.08 3.56±5.54

16 13.29±2.85 25.71±0.43 0.64±0.10 4.16±5.43

17 13.47±2.03 25.71±0.43 0.66±0.04 4.52±3.61 18 15.33±3.44 17.14±0.37 0.72±0.10 4.71±2.45

The values are indicated as mean ± SD.

larval molts are increased by juvenoids (Feyereisen, 1985; Connat The first pupation was observed at 13th instar. Approximately

et al., 1991). In addition, Z. atratus is a model organism to study 85.70% of pupation was observed between the 15th and 18th the relationship between external events and endocrine factors instars. The largest proportion of pupae (25.71%) was observed that regulate the onset of insect metamorphosis (Riddiford, 1976). at the 16th and 17th instars. In other words, most of the larvae The lack of uniformity among larvae between the second and in this experiment exhibited 15 to 18 instars in their life cycle. 18th instars may be caused by variations in nutrition. A study T. molitor larvae from old parents showed shorter larval period on Manduca sexta by Nijhout (1975) revealed that the number (Tracey, 1958). Moreover, the growth rate of offspring from of instars increased under poor nutritional conditions. However, old parents was delayed (Fiore, 1960). Therefore, we suggest it remains to be verified whether this phenomenon occurs in Z. that further studies should investigate larvae from the same atratus or not. Furthermore, the symptoms caused by pathogens age groups to reduce the effects caused by the parents’ age. are highly similar to those of poor nutritional conditions in Z. Further studies using a wide range of rearing temperatures might atratus. It was also revealed that the gregarious nature of T. elucidate the effects of rearing temperature on the number of molitor enhanced its ability to resist pathogens (Barnes and Siva- larval stages. Jothy, 2000). Therefore, further studies are required to investigate Body length of Z. atratus instars increased gradually, and whether the duration of each instar is influenced by nutritional reached the maximum body length in the 18th instar. Mortality status, pathogen activity, or larval behavior. was mostly observed between the second and fifth instars; one

46 47 Sun Young Kim et al. Developmental characteristics of Zophobas atratus (Coleoptera: Tenebrionidae) larvae in different instars

Fig. 1. Different larval instars. Photographs were taken after the exuvium was observed. larva died in the 17th instar. Based on these results, we conclude with enough isolated space successfully pupated (Tschinkel, that more intensive care during the early stages of Z. atratus 1981). Cannibalism of pharate pupae by active larvae may be larvae leads to successful pupation when the larvae are reared the selective factor that evolved to the inhibition of pupation individually. by crowding, higher larval growth rates, and higher fecundity The first instar was white, and gradually turned brown from (Tschinkel, 1978; Tschinkel, 1981). or after the second instar. In particular, the anterior and posterior In this study, we determined the incubation period of eggs, ends were darker than the middle of the body. Except for the the duration of the first instar, and the average number of larval color change, no significant morphological differences were instars in Z. atratus. Our results will provide a basis for further detected in Z. atratus larvae (Fig. 1). We also checked the cuticle studies on this species, as well an insight into an important color, which typically changes based on the population density protein source in commercial feeds for various animals. (Applebaum and Heifetz, 1999), and has been correlated with resistance in a lepidopteran (Reeson et al., 1998). We observed more severe cannibalism in pupae or larvae that Acknowledgments were close to molt in Z. atratus under grouped conditions. In addition, successful pupation requires enough space for the each This research was supported by the Bio-industry Technology last instar larva. Previous study showed that successful pupation Development Program, Ministry of Agriculture, Food and Rural was affected by population density because only Z. atratus larvae Affairs (Project No.312026-3).

48 49 Int. J. Indust. Entomol. Vol. 30, No. (1), pp. 45-49 (2015)

References Nakakita H (1982) Effect of larval density on pupation of Tribolium freeman Hinton (Coleoptera: Tenebrionidae). Appl Entomol Zool 17, Animal and Plant Qurantine Agency. [Online] Insect pests and pet 209-215. . http://www.qia.go.kr/plant/pest/plant_insec_rule.jsp#this. Nijhout HF (1975) A threshold size for metamorphosis in the tobacco Applebaum SW, Heifetz Y (1999) Density-dependent physiological hornworm, Manduca Sexta (L.). Biol Bull 149, 214-225. phase in insects. A Rev Entomol 44, 317-341. Park HC, Jung BH, Han TM, Lee YB, Kim SH, Kim NJ (2013) Aribi N, Quennedey A, Pitoizet N, Delbecque JP (1997) Ecdysteroid Taxonomy of introduced commercial insect, Zophobas atratus titres in a Tenebrionid beetle, Zophobas atratus: effects of grouping (Coleoptera; Tenebrionidae) and a comparision of DNA barcoding and isolation. J Insect Physiol 43(9), 815-821. with similar tenebrionids, Promethis valgipes and Tenebrio molitor in Barnes AI, Siva-Jothy MT (2000) Density-dependent prophylaxis in the Korea. J Seric Entomol Sci 51(2), 185-190. mealworm beetle Tenebrio molitor L. (Coleoptera: Tenebrionidae): Quennedey A, Aribi N, Everaerts C, Delbecque JP (1995) cuticular melanization is an indicator of investment in immunity. Proc Postembryonic development of Zophobas atratus Fab. (Coleoptera: R Soc Lond B 267, 177-182. Tenebrionidae) under crowded or isolated conditions and effects of Connat JL, Delbecque JP, Glitho I, Delachambre J (1991) The onset of juvenile hormone analogue applications. J Insect Physiol 41(2), 143- metamorphosis in Tenebrio molitor larvae (Insecta, Coleoptera) under 152. grouped, isolated and starved conditions. J Insect Physiol 37(9), 653- Reeson AF, Wilson K, Gunn A, Hails RS, Goulson D (1998) 662. Baculovirus resistance in the noctuid Spodoptera exempta is Feyereisen R (1985) Regulation of juvenile hormone titer: synthesis. In phenotypically plastic and responds to population density. Proc R Soc Comprehensive Insect Physiology, Biochemistry and Pharmacology Lond B 265, 1787-1791. (EdsKerkut GA and Gilbert LI) Vol 7, pp. 391-429. Pergamon Press, Riddiford LM (1976) Hormonal control of insect epidermal cell Oxford. commitment in vitro. Nature 259, 115-117. Fiore C (1960) Effects of temperature and parental age on the life cycle Sehnal F (1985) Growth and life cycles. In Comprehensive Insect of the dark mealworm, Tenebrio obscurus Fabricius. J N Y Entomol Physiology, Biochemistry and Pharmacology (EdsKerkut GA and Soc 68(1), 27-35. Gilbert LI) Vol 2, pp. 1-86. Pergamon Press, Oxford. Hsia WT, Kao SS (1987) Application of head width measurements for Tracey SKM (1958) Effects of parental age on the life cycle of the instar determination of corn earworm larvae. Plant Prot Bull (Taiwan mealworm, Tenebrio molitor Linnaeus. Ann Ent Soc America 51, R.O.C) 29, 277-282. 429-432. Jabir MDAR, Razak SA, Vikineswary S (2012) Nutritive potential and Tschinkel WR, Willson CD (1971) Inhibition of pupation due to utilization of super worm () meal in the diet of nile crowding in some tenebrionid . J Exp Zool 176, 137-146. tilapia (Oreochromis niloticus) juvenile. Afr J Biotechnol 11, 6592- Tschinkel WR (1978) Dispersal behavior of the larval tenebrionid 6598. beetle, Zophobas rugipes. Physiol Zool 51, 300-313. Kotaki T, Nakakita H, Kuwahara M (1993) Crowding inhibits pupation Tschinkel WR (1981) Larval dispersal and cannibalism in a natural in Tribolium freemani (Coleoptera: Tenebrionidae): effects of isolation population of Zophobas atratus (Coleoptera: Tenebrionidae). Animal and juvenile hormone analogues on development and pupation. Appl Behavior 29, 990-996. Entomol Zool 28, 43-52. Tyschchenko VP, Sheyk Ba A (1986) Photoperiodic regulation of Ludwig D (1956) Effects of temperature and parental age on the life larval growth and pupation of Tenebrio molitor L. (Coleoptera: cycle of the mealworm Tenebrio molitor L. Ann Ent Soc Am 49, 12- Tenebrionidae). Ent Rev 65, 35-46. 15. Urs KCD, Hopkins TL (1973) Effect of moisture on growth rate and Murray DRP (1968) The importance of water in the normal growth of development of two strains of Tenebrio molitor L. (Coleoptera, larvae of Tenebrio molitor. Entomologia Exp Appl 11, 149-168. Tenebrionidae). J Stored Prod Res 8, 291-297.

48 49