ISSN 0013-8738, Entomological Review, 2013, Vol. 93, No. 9, pp. 1107–1115. © Pleiades Publishing, Inc., 2013. Original Russian Text © A.A. Zagorinskii, O.G. Gorbunov, A.V. Sidorov, 2013, published in Zoologicheskii Zhurnal, 2013, Vol. 92, No. 7, pp. 825–833.
An Experience of Rearing Some Hawk Moths (Lepidoptera, Sphingidae) on Artificial Diets A. A. Zagorinskii, O. G. Gorbunov, and A. V. Sidorov Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow, 119071 Russia e-mail:
[email protected],
[email protected],
[email protected] Received May 25, 2012
Abstract—A method for rearing five hawk moth species, namely Acherontia atropos (Linnaeus, 1758), A. lachesis (Fabricius, 1798), Agrius convolvuli (Linnaeus, 1758), Daphnis nerii (Linnaeus, 1758), and Hyles livornica (Esper, 1780) (Lepidoptera, Sphingidae) on artificial diets is described. Two recipes of these artificial rearing media are proposed. Special attention is given to maintenance of adult moths in captivity and raising immature stages. DOI: 10.1134/S0013873813090029
When studying the biology of preimaginal stages of hawk moths from the European part of Russia, we are faced with the problem of rearing caterpillars of certain species during wintertime. This mainly applies to migratory species which are more frequently encountered in the second half of summer and sometimes have no diapause. Our testing of rearing methods for the caterpillars included development of artificial diets and feeding techniques. Most success has been achieved with rearing the following three hawk moth species: death’s-head [Acherontia atropos (Linnaeus, 1758)] (13 generations obtained), oleander [Daphnis nerii (Linnaeus, 1758)] (4 generations), and striped [Hyles livornica (Esper, 1780)] hawk moths (2 generations). This paper is primarily a description of their rearing methods. In addition, two more species have been successfully reared from egg to adult, the convolvulus hawk moth Agrius convolvuli (Linnaeus, 1758) and the oriental death's-head Acherontia lachesis (Fabricius, 1798), each represented by several individuals. However, it was impossible to keep them in the laboratory for generations because, due to the scarcity of the original material, all the adults appeared to be of the same sex. The rearing on artificial media have been previously reported for three species out of the five studied by us, in particular, for the death’shead (Gade, 1980), oleander (Koch and Heinig, 1977), and convolvulus hawk moth (Kiguchi and Shimoda, 1994). For the lattermost species, which is a pest of sweet potato in Japan, the rearing method has been most thoroughly elaborated. MATERIALS AND METHODS Laboratory colonies of the death’s-head, oleander, and convolvulus hawk moths originated from several
caterpillars of each species (in the case of the convolvulus hawk moth, also eggs) that were collected in Krasnodar Territory and the Crimea in autumn 2009. Eggs of the striped hawk moth were obtained from a female captured in Ethiopia during the work of the Joint Ethio-Russian Biological Expedition in the environs of the town of Ambo in 2011. Several A. lachesis eggs laid by a female captured in Vietnam were received by courtesy of V.V. Zolotuhin (Ulyanovsk State Pedagogical University) in 2010. Caterpillars collected in the field were reared on their natural host plants: caterpillars of the death’shead, on boxthorn (Lycium barbarum); those of the oleander hawk moth, on oleander (Nerium oleander). Caterpillars of all the other species hatched from eggs were immediately transferred onto an artificial medium. Pupae were kept in slightly moist sawdust at a temperature of +25...27°C and relative humidity of 50%. All the subsequent work was done under the same conditions of temperature and humidity. The photoperiod was 12 h. Composition and Preparation of Artificial Diets A recipe of the artificial diet developed for the tobacco hornworm Manduca sexta (Linnaeus, 1763) (Bell and Joachim, 1976) was taken as the basis. Considering the accessibility of the ingredients, we have modified it as shown in Table 1. In particular, casein was substituted with soy flour, and a different vitamin premix was added. The resulting composition was used for rearing of A. atropos and A. lachesis. For the striped and convolvulus hawk moths, a part of wheat
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Table 1. Compositions of artificial diets used in the study Ingredient
Artificial diet for Acherontia atropos, Daphnis nerii, and A. lachesis
Artificial diet for Agrius convolvuli and Hyles livornica
Dry mix no. 1, g Wheat germ Fodder yeast Sugar Soy flour Wesson salts Cholesterol Choline chloride Sorbic acid
80 16 28 32 8 2 1 2.8
50 16 28 32 8 2 1 2.8
Dry mix no. 2, g Powdered dried food plant* Ascorbic acid Kanamycin sulfate Streptomycin sulfate Vitamin premix**
– 4 0.07 0.26 2
30 4 0.07 0.26 2
25 3 4 1000****
25 3*** 4 1000****
Other ingredients Agar, g Formaldehyde 40%, ml Linseed oil, ml Water, ml
* Bindweed for the convolvulus hawk moth and willowherb for the striped hawk moth. ** 500 mg of the premix contain: thiamine, 4 mg; riboflavin, 6 mg; niacinamide, 60 mg; calcium pantothenate, 20 mg; pyridoxine hydrochloride, 6 mg; cyanocobalamin, 9 µg; folic acid, 54 mg; biotin, 50 µg; choline, 150 mg; inositol, 150 mg; PABA, 50 mg. *** For Agrius convolvuli only. **** For 1st–4th instar larvae. For 5th instar larvae, 700 ml of water was added.
germ from the basic recipe was substituted with dried food plants: willowherb (Chamerion angustifolium) and bindweed (Convolvulus sp.), respectively. Also, formalin was excluded from the artificial diet for the striped hawk moth. Caterpillars of the oleander hawk moth were reared on artificial diet without the food plant; however, first instar larvae refused to eat it. Therefore, fresh or frozen periwinkle (Vinca minor) leaves were grinded in a mortar with a small amount of water, and the resulting liquid mixture was applied with a brush over the surface of the artificial diet. This promoted feeding in the larvae. From the third instar onwards, they fed on the artificial diet without addition of periwinkle. In order to prepare the feeding medium, agar was added to an adequate amount of cool water; this suspension was then well stirred and brought to the boil.
After that, dry mix no. 1 was put into the agar solution while continuously stirring the latter. Once the temperature of the medium went down to 57°C, dry mix no. 2 was added along with adequate amounts of formalin and linseed oil. The resulting product was poured into plastic containers and kept in a refrigerator at +2-3°C for no longer than 2 weeks. Methods of Maintenance of Adult Moths in Captivity Methods of maintenance of adult hawk moths are described for D. nerii, H. livornica, and A. atropos, because laboratory colonies of only these species have been kept for a long time. The biology and behavior of the former two species are rather similar while those of the death’s-head hawk moth are strikingly different. Two schemes of adult maintenance are thus given below, one for A. atropos and the other for D. nerii and H. livornica. ENTOMOLOGICAL REVIEW Vol. 93 No. 9 2013
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Fig. 1. A feeding female of Acherontia atropos.
A. atropos. The death’s-head hawk moth has a unique biology, various aspects of which have been studied by a few researchers (Harbich, 1978, 1978a, 1980, 1981; Moritz et al., 1981). Adults were kept in 60×60×60 cm cubic wooden cages with two large ventilation grids in the back wall and in the top, half of the latter also being the lid. Over the upper ventilation opening, there was a 20-watt fluorescent lamp connected to a timer producing 12 h:12 h light:dark cycle. The alternation of night and day is very important for moths of this species to maintain their activity. The interior space of the cage was arranged as follows. A plant for oviposition, the Jerusalem cherry (Solanum pseudocapsicum), was put into the middle. The walls were lined with egg cartons or polystyrene foam. The absence of space for flight does not hamper reproduction in this species; moreover, it reduces damage to the wings. The bottom of the cage was covered with a layer of slightly moist sawdust which was replaced on the as-needed basis. Two open Petri dishes with daily changed water were put into the cage as drinking pans. Natural sealed honey bee combs were also placed in cages for feeding the moths. Combs were installed in such a way that moths could creep on their ENTOMOLOGICAL REVIEW Vol. 93 No. 9 2013
surface constantly without being trapped in honey. Moths usually find this nourishment easily and feed by themselves (Fig. 1). However, sometimes they reject such food, probably due to certain properties of a given sort of honey. In this case, moths were fed artificially as follows. A 50% honey solution was prepared, the moth was immobilized in the hand, its proboscis was unfolded with an entomological pin and dipped into the solution. After that, the moth immediately started to feed. There were up to 20 adult moths per cage at a time. It is advisable that males be less numerous than females due to their extremely high, as compared with other species, sexual activity which often damages females or hampers oviposition. Eggs were collected daily, transferred onto moistened filter paper in a Petri dish and incubated at a temperature of +25...27°C (Fig. 2). D. nerii and H. livornica. These species, in contrast to A. atropos, require vast space for flight. To supply this need, a large cage was constructed of fine mesh and a wire frame with bottom dimensions of 80×80 cm and a height of 140 cm. The oleander hawk moth, with its exceptionally powerful flight machinery, breaks its
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Fig. 2. A. atropos eggs, collected and ready for incubation.
Fig. 3. An oleander hawk moth feeding.
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Fig. 4. Individual rearing of A. atropos caterpillars.
Fig. 5. Group rearing of A. atropos caterpillars.
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wings in captivity very easily. This may even hamper reproduction in the species as moths have to be active for a few days before they are ready for mating whereas poorly flying males usually do not mate. The cage was then designed in a special way in order to minimize damage to the wings. Ribbons of soft paper (crumpled newspapers were used) were hung vertically along the cage walls at intervals of 3 cm. The top of the cage was made of thin film painted in wide sharp black stripes. Moths that see paper ribbons and stripes above almost never collide with the walls but fly in circles within the cage space. Besides, a low level of illumination in the room the cage is in is also important. The presence of a window is sufficient; if there is none, dim artificial light from a low-powered LED would be necessary. A feeder for moths was installed, made of a white or light-blue plastic bottle cap and filled with 10% fructose or honey solution (Fig. 3). It is important that this feeder be installed in the upper third of the cage. In the middle there was a stage with a food plant: oleander or periwinkle branches set in water for the oleander hawk moth and grapevine for the striped hawk moth. Each cage contained up to 10 individuals (5 males and 5 females) of the oleander hawk moth or up to 20 individuals of the striped hawk moth. The cage was moisturized with a spray bottle of water every evening. Eggs were collected daily, as in A. atropos. Methods of Rearing Preimaginal Stages Individual rearing. This rearing method was used for all the species except H. livornica. The caterpillars were allocated in plastic boxes with ventilation holes. From the first to the third instar, the box volume was 150 ml; for the fourth and fifth instars, 500 ml (Fig. 4). A wooden stick whose length corresponded to the greater diagonal of a box was also put in so that a caterpillar could climb on it. The absence of such a support made it difficult for a caterpillar to molt. Food was replaced and the box cleaned as often as required, usually every 4–5 days. Food containing a natural plant was replaced once in 2–3 days. Group rearing. Rearing in groups was attempted for A. atropos, D. nerii, and H. livornica caterpillars. The first instar appeared to be the most difficult period in group rearing on an artificial diet. As a rule, caterpillars did not start feeding immediately and tended to gather in some parts of the cage, often provoking mass mortality. In order to prevent such events, several rearing techniques were used, the most successful of
which was the following. Eggs which were about to hatch during the next day were transferred to Petri dishes filled with entangled plastic thread 0.7 mm wide. Strips of artificial diet were laid on top. Hatched caterpillars dispersed along the thread and fed on these strips until the middle of the third instar. Each dish housed 20 caterpillars. Starting from the middle of the third instar, caterpillars were transferred to plastic containers measuring 30×40 cm at the bottom and 10 cm in height with a wavelike folded plastic lattice inside (Fig. 5). Pieces of artificial diet were laid over the lattice, and then caterpillars were allocated by 30 individuals per container. A similar method for maintaining last-instar caterpillars on a plastic lattice is sometimes used for rearing silkworms on artificial diets. It was also used by other researchers for rearing the convolvulus hawk moth (Kiguchi and Shimoda, 1994). If there were few caterpillars in a container, an unfolded piece of lattice was simply laid on the bottom. Prior to pupation, a layer of sterilized sawdust 0.5 cm thick was poured underneath the lattice to absorb excess moisture excreted by caterpillars before pupal molt. Caterpillars lived under such conditions till the end of the feeding period. Pupation. Caterpillars of all the species studied were put singly into 500 ml plastic containers with several ventilation holes for pupation. Tissue paper was also placed inside to absorb moisture excreted by the pupating caterpillar. Individuals ready for pupation, which was reflected in their coloration or behavior, were taken away every morning. A. atropos and A. lachesis caterpillars became brick-red and those of D. nerii maroon in color. A. convolvuli and H. livornica caterpillars did not change their coloration before pupation noticeably; however, as well as the caterpillars of the former three species, they exhibited different behavior by the end of the fifth instar: the caterpillars began to wander across the container bottom, a pattern that indicated their readiness for pupal molt. RESULTS AND DISCUSSION In the course of our work, considerable speciesspecific differences in behavior and growth parameters of caterpillars were revealed, as well as those in the biology of adult moths. Adult longevity under our conditions appeared to be the greatest in A. atropos, as compared with the other species, and amounted to 18–30 days in males and 25–40 days in females. Occasional individuals, usually females, lived for a month and a half. Mating in this ENTOMOLOGICAL REVIEW Vol. 93 No. 9 2013
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Table 2. Survivorship of hawk moth caterpillars under different conditions, % Acherontia atropos, a sample of 200 individuals
Daphnis nerii, a sample of 100 individuals
Hyles livornica, a sample of 100 individuals
From hatch to the end of the 1st instar, individual rearing
92
82
No data
From hatch to the end of the 1st instar, group rearing
69
28
95
From the beginning of the 2nd to the end of the 5th instar, individual rearing
89
88
No data
From the beginning of the 2nd to the end of the 5th instar, group rearing
82
35
81
Developmental period
Table 3. Developmental times of caterpillars and pupae, days Acherontia Acherontia Daphnis nerii, Hyles livornica, Developmental atropos, atropos, privet, artificial diet, artificial diet, stage artificial diet, 22 individuals 100 individuals 100 individuals 200 individuals Caterpillar Prepupa Pupa
17.60 ± 0.31
24.00 ± 2.30
Acherontia lachesis, 1 individual
Agrius convolvuli 3 individuals
15.7 ± 0.89
22 ± 2.10
19
18, 18, 19
4 ± 0.11
No data
3 ± 0.23
4 ± 0.29
4
4, 4, 4
18.2 ± 2.91
No data
16 ± 0.89
16 ± 0.93
18
16, 16, 17
Note: For the former three species, mean ±SD is given; for the latter two, individual values.
species, in contrast to the rest of those studied, occurred repeatedly, often 2–3 times a night for one female. Mating began on the second night after male emergence from pupae and could be observed almost anytime throughout the night. Some couples remained in copula until the following night. Although one- and two-day-old females clearly resisted mating attempts, it was no hindrance to males. A description of behavior of these moths, detailed enough, can be found in Harbich, 1978. On the whole, the mating process and other specificities of adult behavior in A. atropos are very uncommon. Oviposition in females is preceded by a considerable delay which lasts, according to our observations, from 5 to 18 days. The average value of 9 days is mentioned in the literature (Friedrich, 1986). Most certainly, it was this feature which led to the opinion that death’s-head hawk moths emerging in the European part of Russia are sterile (Derzhavets, 1984). We believe that this is hardly probable. Fertilized females, ready for oviposition, approached the host plant, alighted on it, and laid eggs on the leaves’ lower surface. Eggs in considerable numbers were also found on the cartons and cage walls, most often near the drinking pan and in moistest corners. The overall fertility of females was 150–280 eggs. Usually there were 25–35 eggs laid by a female during one night. We ENTOMOLOGICAL REVIEW Vol. 93 No. 9 2013
attempted to optimize the oviposition process by making use of the “artificial leaf” method employed in the rearing of convolvulus and tobacco hawk moths (Shimoda and Kiuchi, 1998). Plant extract was prepared from potato leaves. This method is much more convenient than the usage of a living plant; however, in the case of the death’s-head hawk moth, it gave no positive results. Mating and oviposition in oleander and striped hawk moths occurred somewhat differently. Females of the oleander hawk moth exhibited attractive behavior from the second night onwards. In the evening, just after the light had been switched off, males and females fed actively, whereas matings occurred, as a rule, between 2 and 4 a.m. The duration of attractive behavior was prolonged significantly if the female remained unmated. Females that had not been fertilized by the tenth day of their life emitted pheromones even during the daytime. In contrast to the death’shead, female moths of this species mated only once. As for the striped hawk moth, its behavior was largely similar to that of the oleander hawk moth. However, matings were observed throughout the whole night, as well as in the morning. Adult longevity in both species was 15–25 days.
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Oviposition in D. nerii and H. livornica females was delayed, just as in A. atropos. This hold-up lasted 7–10 days in the former species and 3–6 in the latter. The moths laid their eggs primarily on the host plant, although considerable numbers of eggs were also found at the feeder. Total fecundity in females of these species was significantly higher than in the death’shead. Larger females of the oleander hawk moth could produce over a thousand eggs, usually about a hundred a night. The striped hawk moth fecundity was mainly 300–500 eggs. The egg development time was the shortest in the oleander hawk moth, being only 4 days at the mentioned temperature and as short as 3 days with the temperature rising to 30°C. Under the same conditions, egg development in the death’s-head and the striped hawk moths lasted 5 days. Hatching success was 98.2% in the oleander, 97.5% in the striped, and 62.5% in the death’s-head hawk moth. Such a high percentage of dead eggs in the latter species is not quite understood. Probably it was not because a part of females had remained unfertilized. Even after multiple matings with different males, the percentage of live caterpillars emerging from the eggs did not exceed the given value. Furthermore, about a half of those eggs that failed to hatch showed signs of embryonic development. Presumably, the non-viability of a considerable number of eggs was caused by mechanical damage while breaking them away from the host plant. However, there were also many dead eggs among those laid on cartons, i.e., which we did not take off the substrate. The first instar is critical in the rearing of caterpillars. The main difficulty associated with group rearing of the oleander hawk moth was cannibalism which, however, was not observed when caterpillars fed on plants. Therefore we primarily used the individual method of rearing for this species. Caterpillars of all the species except the striped hawk moth did not start feeding for more than a day. This does not occur when caterpillars are placed onto their natural host plants. They also show pronounced positive phototaxis and negative geotaxis, aggregating in the upper and more illuminated parts of the cage. This often resulted in caterpillars getting fatally trapped in their own web. The cage therefore should not be very high and illumination should be even while rearing first-instar caterpillars. It appeared very effective to fill the cage volume with plastic thread as it minimized contacts between caterpillars and prevented them from gathering
in the same patches. Survivorship data for the caterpillars of three species reared by the methods described above are given in Table 2. Table 3 summarizes the data on the duration of development of individuals reared singly (or, for the striped hawk moth, in groups). The highest developmental rate was found in the oleander hawk moth. Death’s-head caterpillars generally developed a bit slower, and notably slower on their natural host plant in South Europe, privet (Ligustrum vulgare), in comparison with artificial diet. The striped hawk moth caterpillars took the longest time to develop. This was probably caused by the non-optimal composition of the factitious feeding medium, for the representatives of the Hyles genus are commonly characterized by very fast larval development. However, we do not have any data on developmental rates of caterpillars of this species on their natural food plants. CONCLUSION The death’s-head hawk moth appeared to be the most suitable for maintenance in the laboratory of all the species reared by us. The death’s-head may be used as an alternative to the tobacco hawk moth (Manduca sexta), one of the most popular laboratory insects which is quarantine-listed and thus cannot be brought into certain countries. The rearing process of A. atropos is slightly simpler in comparison with the striped and the oleander hawk moths as the caterpillars do not require addition of a host plant to the feeding medium (hence a longer storage life of artificial diet) and can tolerate high density. Besides, death’s-head caterpillars are larger and develop quite rapidly, which is important in terms of laboratory rearing. Their possible disadvantages as of laboratory objects include adult feeding on honey and delayed oviposition in females. ACKNOWLEDGMENTS The authors are deeply grateful to O. A. Tkachev (The State Darwin Museum) for his help with the caterpillars, V. V. Zolotuhin for sharing his A. lachesis material, E. Yu. Tkacheva and M. V. Berezin (The Moscow Zoo) as well as V. B. Chernyshov (Moscow State University, Faculty of Biology, Department of Entomology) for their valuable advice on rearing the insects. ENTOMOLOGICAL REVIEW Vol. 93 No. 9 2013
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7. Harbich, H., “Zur Biologie von Acherontia atropos (Lep.: Sphingidae). 3,” Entomol. Z., Frankf. a. M. 90, 11–13 (1980). 8. Harbich, H., “Zur Biologie von Acherontia atropos (Lep.: Sphingidae). 4,” Entomol. Z., Frankf. a. M. 91, 57–62 (1981). 9. Kiguchi, K. and Shimoda, M., “The Sweet Potato Hornworm, Agrius convolvuli, as а New Experimental Insect: Continuous Rearing Using Artificial Diets,” Zool. Sciеnce 11, 143–147 (1994). 10. Koch, J. and Heinig, S., “Daphnis nerii ein Labortier? (Lep., Sphingidae),” Entomol. Z., Frankf. a. M. 87, 57–62 (1977). 11. Moritz, R.F.A., Kirchner, W.H., and Crewe, R.M., “Chemical Camouflage of the Death’s Head Hawkmoth (Acherontia atropos L.) in Honeybee Colonies,” Naturwissenschaften 78, 179–182 (1991). 12. Shimoda, M. and Kiuchi, M., “Oviposition Behavior of the Sweet Potato Hornworm, Agrius convolvuli (Lepidoptera: Sphingidae), as Analysed Using an Artificial Leaf,” Jpn. J. Appl. Entomol. Zool. 33 (4), 525–534 (1998).
