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Propagation and Storage of 
Neoaplectana carpocapsae Weiser 
Using Amyelois transitella (Walker) Adults 



U.S. Department of Agriculture 
Science and Education Administration 

Advances in Agricultural Technology . AAT-W-3/February 1979 


ABSTRACT 


Describes a technique for using adult Lepidoptera in 
the simple, economical rearing of Neoaplectana carpocapsae 
and other entomogenous nematodes. The resultant invasive 
stage nematodes are stored without mechanical aeration at 
6°C in compact, stackable, plastic petri dishes. 

KEYWORDS: Entomogenous nematodes, Neoaplectana 
carpocapsae; propagation and storage; 
Lepidoptera adult hosts, navel orange- 
worm adults. 


A free copy of this publication is available from the Stored- 
Product Insects Research Laboratory, 5578 Air Terminal Drive, 
Fresno, Calif. 93727. 


Science and Education Administration, Advances in Agricultural Technology, 
Western Series, No. 3, February 1979 


Published by Agricultural Research (Western Region), Science and Education 
Administration, U.S. Department of Agriculture, Berkeley, Calif. 94705 


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PROPAGATION AND STORAGE 0? A NEOAPLECTANA CARPOCAPSAE WEISER 
USING AMYELOIS TKAflSITELLA (WALKER) ADULTS 

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By James E. Lindegren, Darlene F. Hoffmann, 
Susan S. Collier and Rodney D. Fries 1 


INTRODUCTION 


\ / 

The insect parasitic nematode Neoaplectana carpocapsae Weiser has been 
reared on artificial diets (l , 2, 4, 5, 6, 9) 2 as well as in lepidopteran larvae 
(2 , 5)- The low rearing cost of 2 cents per million nematodes reported by 
Bedding (l), as compared with $1 per million reported by Poinar (7), favors the 
use of artificial diets for the rearing of this nematode. Rearing methods uti- 
lizing insect hosts described by Dutky et al.(2) and Poinar (s), however, are 
useful for isolating and maintaining new and possibly obligate insect parasitic 
nematodes, as well as for rearing limited numbers of nematodes for laboratory 
or small scale field tests. Work in this area has prompted the following modi- 
fications of the simplified rearing method described by Poinar ( 3 ). 


NEMATODE REARING 


Navel orangeworm, Amyelois transitella (Walker), adults reared on a modi- 
fied bran diet, 3 described by Finney and Brinkman ( 3 ), are substituted for last 
instar wax moth larvae as an insect host for n. carpocapsae . The adults inac- 
tivated by storage at 4°C for 1 h are then added to 100- by 15-mm plastic petri 
dishes containing four filter papers' 4 premoistened with 4 ml 0.1 percent forma- 
lin solution, prepared with 37.8 percent formaldehyde and deionized water and 
one drop of concentrated (about 15,000) invasive stage nematodes (fig.l). 3 The 
adults are then incubated at 27°C for 48 h (fig. 2). The bottom of the exposure 


Research entomologist and biological laboratory technicians, respectively, 
Stored-Product Insects Research Laboratory, 5578 Air Terminal Drive, Fresno, 
Calif. 93727. 

2ltal ic numbers in parentheses refer to Literature Cited, p. 3. 

3 The raw bran used in this diet was autoclaved to prevent insect pathogen 
contamination and a solution of Nutritional Biochemical Corporation Vitamin Diet 
Fortification Mixture was added to help replace heat labile vitamins lost during 
sterilization. 

^No. 1 qualitat ive 7-cm-diameter Whatman. 

^Figures follow at the end of the report. 


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petri dish, containing the dead parasitized A. transitella adults, is then 
placed inside a 150- by 25-mm plastic petri dish, containing 25 ml of 0.1 
percent formalin solution. Invasive stage nematodes are then trapped in the 
formalin solution as they move out of the exposure petri dish (fig. 3). The 
trapped nematodes are harvested in glass settling flasks (fig. 4) 1 week after 
the initial host exposure and biweekly thereafter for 4 to 6 weeks. 

Two ml of concentrated nematodes (about 3 x 10^ per ml) are then trans- 
ferred with a disposable pipette to a 150- by 15-mm plastic petri dish con- 
taining 25 ml of 0.1 percent formalin solution. The nematodes are then stored 
at 6°C (fig. 5) until needed. 

Formalin solution must be added periodically to replace evaporated moisture. 
The solution level should be 2 to 3 cm deep to allow for oxygen exchange. Con- 
centrations of N, carpocapsae invasive stages, up to 4 million per petri dish, 
have, to date, remained about 94-percent viable for 9 months under these storage 
conditions. An overall view of this procedure is presented in figure 6. 


RESULTS AND DISCUSSION 


The average nematode production per adult from the 1,325 navel orangeworm 
adults sampled biweekly for 6 weeks is 7,000 with 91 percent of the total nema- 
tode production occurring within the first 3 weeks of the sampling period. 

As a result of a multidisciplinary approach to control the navel orange- 
worm, a multimillion dollar pest of almonds in California, about 6,000 navel 
orangeworm adults are produced per week at this laboratory. The nematode pro- 
duction capability is therefore about 42 million nematodes per week. 

Navel orangeworm adults and other adult Lepidoptera either salvaged as by- 
products from insect rearing or possibly collected from light traps represent a 
low cost food source for rearing entomogenous nematodes. At this facility, 
entomogenous nematodes have been reared on navel orangeworm adults since 1976. 
Nematodes successfully reared with this sytem include the Mexican and DD136 
strain of Neoaplectana carpocapsae , N. glasseri Steiner, and Heterorhabd it is 
hel iothidis (Khan, Brook, and Hirschmann). 


SUMMARY 


Adult Lepidoptera provide a previously unutilized food source for the 
rearing of N. carpocapsae and other entomogenous nematodes. 

The advantages of this rearing and storage system are: (1) It is econo- 
mical if cost free adult Lepidoptera are utilized, (2) it provides a clean cul- 
ture of invasive stages, (3) it is adaptable to the isolation and maintenance 
of other entomogenous nematodes, and (4) it provides for the concentrated sim - 
plified storage of the harvested invasive stage nematodes. 


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LITERATURE CITED 


(1) Bedding, R. A. 

1967. New methods increase the feasibility of using Neoaplectana spp. 

(Nematode) for the control of insect pests. Proc. Int. Colloq. 
Invert. Pathol.: 250-254. 

(2) Dutky, S. R. , Thompson, J. V. , and Cantwell, G. E. 

1964. A technique for the mass propagation of the DD-136 nematode. 

J. Insect. Pathol. 6: 417-422. 

(3) Finney, G. L. , and Brinkman, D. 

1967. Rearing the navel orangeworm in the laboratory. J. Econ. Ent . 60: 
1109-1111. 

(4) Glaser, R. W. , McCoy, E. E. , and Girth, H. B. 

1942. The biology and culture of Neoaplectana chresima t a new nematode 
parasite in insects. Proc. Roy. Soc. Queensl. 28: 123-126. 

(5) Hansen, E. L. , Yarwood, E. A., Jackson, G. T. , and Poinar, G. 0., Jr. 

1968. Axenic culture of Neoaplectana carpocapsae in liquid media. 

J. Parasitol. 54: 1236-1237. 

(6) House, H. L. , Welch, H. E. , and Cleugh, T. R. 

1965. Food medium of prepared dog biscuit for the mass production of the 

nematode DD-136 (Nematode: Steinernematidae) . Nature (London) 
206: 847. 

(7) Poinar, G. 0., Jr. 

1971. Use of nematodes for microbial control of insects. Pp. 181-203. 

H. D. Burges and N. W. Hussey, eds., In Microbial Control of 
Insects and Mites, Academic Press, New York. 861 pp. 

(8) Poinar, G. 0., Jr. 

1975. Entomogenous nematode, a manual and host list of insect-nematode 
associations. E. J. Brill, Leiden, The Netherlands. 317 pp. 

(9) Poinar, G. 0., Jr. and Thomas, G. M. 

1966. Significance of Achromobactor nematophilus Poinar and Thomas 

(Achromobacteraceae : Eubacteriales ) in the development of the 

nematode, DD-136 ( Neoaplectana sp. Steinarnematidae) . Parasito- 
logy 56: 385-390. 


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Figure 1. — Adding N. carpocapsae invasive stages to exposure petri dish. 



Figure 2. — Dead parasitized naval 

orangeworm adults 48 h after exposure 
to nematodes. 


Figure 3. — Invasive stage nematodes 
migrating from parasitized naval 
orangeworm adults into formalin 
solution. 



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Figure 4. — Invasive stage nematodes 
trapped in formalin solution are 
settled for storage. 


Figure 5. — Concentrated invasive stage 
nematodes (about 6 million per petri 
dish) stored at 6°C. 



Figure 6. — Overview of nematode rearing procedure: A , Exposure dishes with 
about 15,000 nematodes and 4 ml 0.1 percent formalin solution can be stored 
at 6°C until needed; B, exposure dishes 48 h after the addition of navel 
orangeworm adults; C, bottom half of exposure dish in 150- by 25- mm trapping 
dish with invasive stage nematodes in 0.1 percent formalin solution; D, 
settling flask with nematodes starting to collect at the bottom; E , dispos- 
able pipette for transferring concentrated nematodes; F, formalin (0.1 per- 
cent) solution dispenser. 


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