Vol. 101. No. 2. March & April 1990
123
IMPROVED CULTURE TECHNIQUES FOR MASS
REARING GALLERIA MELLONELLA (LEPIDOP-
TERA: PYRALIDAE ) 1
Frank A. Eischen^, Alfred Dietz^
ABSTRACT: Supplementing an artificial diet with 5% pollen, honey, or beeswax signifi-
cantly increased surv ival of adult Galleria mellonella. Surv ival on the basic diet averaged
27.4%. When honey, pollen, or wax was added, survival was 44.7%. 80.8%, and 89.6%, respec-
tively. Developmental time for moths fed diets containing 5% pollen or a combination of
pollen, honey, and wax was shorter by approximately 2-5 days. Newly emerged virgin
females which had fed as larvae on a 100% pollen, honey, and wax diet weighed 36% more
than females derived from larvae fed the artifical diet. A phagostimulatory hypothesis
is suggested.
Additionally, 78% of prepupae accepted cut plastic soda straws as puparial sites and
spun their cocoons in them. This greatly facilitated the handling of individual prepupae
and pupae and should be a convenience to both research programs and commercial wax
moth producers.
The greater wax moth. Galleria meIIonelIa(L.\ is easily reared and is
often used as a laboratory animal. However, our initial attempts to
establish a culture from wild stock resulted in low survival rates (ca. 30%)
when an artificial diet [Stoneville(SV) developed by Linger al. 1979] was
used. However, pilot studies indicated that survival was good (ca. 95%)
on a diet composed wholly of their naturally occurring larval food. i.e.
pollen, honey, and beeswax. Inspection of the artificial diet (King et al.
1979) did not reveal nutritional shortcomings. Previous studies have
shown that small amounts of beeswax improved growth rates (Beck
1960, Dadd 1966, Young 1964). Further, Dadd (1966) observed higher
survival rates during portions of larval development when beeswax w r as
added to an artificial diet. These findings suggested that small amounts
of natural food could play an important nutritional role in adult sur-
vival. We report here the results of a study done to determine why sur-
vival differed so strikingly on artificial diet compared with a diet com-
posed of natural ingredients.
MATERIALS AND METHODS
Six diets were tested. Their composition by weight was as follows: 1)
100% Stoneville(SV), 2) 95% SV + 5% pollen(P), 3) 95% SV + 5% hon-
ey(H), 4) 95% SV + 5% beeswax(W), 5) 95% SV + 5% PH W, 6) 100% PH W.
| Received June 19, 1989. Accepted September 23. 1989.
“Dept. Of Entomology, Washington State University. Pullman, WA 99164
^Dept. of Entomology. University of Georgia. Athens, GA 30602
ENT. NEWS 101(2): 123-128, March & April, 1990
124
ENTOMOLOGICAL NEWS
The Stoneville diet consists largely of baby foods (Gerber®mixed and
high protein cereals), gycerol, wheat germ, water, sucrose, and vitamins
(see YAngetat. 1979 for details). Its protein composition is about 13%. The
pollen-honey-wax diet was made by mixing bee-collected pollen (63%;
see Dietz 1 982 for plant species represented) with chipped honey comb
(37%). The honeycomb (cappings) contained approximately 50% honey.
The protein content of this diet is estimated to be about 13%. Twenty
grams of diet were placed in 100ml glass rearing jars, sealed and kept
frozen until used. Fortified diets were prepared by placing 19gofSV diet
in the rearing jar, and then lgof either pollen, honey, or wax was sprink-
led or dripped onto its surface.
Moths were obtained by removing overwintering prepupae from a
dead honey bee hive in Athens, Georgia. They were incubated at 30°C.
Adults emerged in about two weeks and mated inter se. Eggs were collected
between strips of pleated wax paper. Three days after laying, egg masses
were divided into small clusters of 10-1 5 eggs. These clusters were placed
on filter paper in petri dishes and incubated at 30°C + 2°C. Just before
hatching, eggs were examined and clusters containing at least 10 larvae
that appeared normal were selected for testing. Excess larvae were des-
troyed by puncturing the eggs with a needle. Eggs were then placed in
small aluminum foil baskets ( 1 6mm x 10mm) and randomly assigned to
diets. Twenty replications of each diet were performed. After addition of
the eggs, the rearing jars were sealed with a solid metal screw lid contain-
ing a rubber interseal. Rearing jars were held at 32°C + 1 °C, 40 + 10%
RH. and 12:12LD. Seven days after infestation, egg clusters were re-
moved and percent hatch determined. Also, the solid lids were replaced
with a similar lid containing a 9mm hole. This hole was covered with a
piece of transparent tape, sticky side out. Several small holes were
punched in the tape with a small pin to allow air exchange. When the
first prepupae were observed, this tape was replaced with a circular piece
of aluminum screen. At this stage, rearing jars were inspected daily. As
adults appeared they were removed, sexed, and their emergence date
noted. The first twenty adult virgin females that emerged from the SV
and the pollen-honey-wax diets were weighed. Sixty days from the time
of egg laying, the contents of the rearing jars were examined. Cocoons
were counted and pupal mortality noted.
Survival and emergence data were analyzed with a 1-way ANOVA,
and differences among treatments evaluated with Duncan’s multiple
range test. Student's t-test was used to determine differences in adult
female weights.
To confirm that the supplemented Stoneville diet was suitable for a
mass rearing program, we fed the Stoneville+5% fresh frozen pollen to
ca. 6,000 hatching G. mellonella larvae (eggs were weighed, not counted).
Vol. 101, No. 2, March & April 1990
125
As prepupae emerged from the diet and began spinning light cocoons,
they were removed and placed in 3.81 glass jars containing cut plastic
soda straws (2 X 0.5cm; Sweetheart R ). About 70 prepupae were added to
jars containing 200-300% excess of cut straws (scattered horizonally on
the bottom of the jar), which were covered with a single circular piece of
toweling. Jars were kept in a darkened incubator (same conditions as in
above dietary trials).
RESULTS
The addition of 5% of either pollen, honey, or beeswax (PH W) to the
Stoneville(SV) diet resulted in a significant increase in adult survival
(Table 1). Adult survival on the basic diet averaged 27.4%. The addition of
honey increased survival to 44.7% (P < 0.05). Fortification with either
pollen, wax, or a combination of pollen, honey, and wax produced sur-
vival rates that did not differ significantly from the control diet (89.2%).
Males reared on the SV+5% pollen, SV+PHW. and control diet
eclosed in a significantly shorter time (ca. 3-5 days, P < 0.05) than moths
on the other three diets (Table 1). Average time to first emergence was
slightly shorter for males (0.2- 1 .3 days) than that required by females, but
this difference was not significant. Newly emerged virgin females weighed
on average less when reared on the SV diet, than on the control diet ( 1 14
and 156mg, respectively; P <0.001). During the mass rearingtrial 78% of
prepupae spun their cocoon inside the cut soda straws. The adult sur-
vival rate for larvae fed the Stoneville+5% pollen diet during this trial
was estimated to be about 84%.
DISCUSSION
The striking increases in survival caused by small amounts of pollen,
honey, or beeswax was not the result of an altered physical consistency,
nor the addition of essential nutrients (Haydak 1936, 1940. Allegret 1964;
Dadd 1966; Marston and Campbell 1973). Dadd (1966) suggested that
the inclusion ofbeeswax served as a source of metabolic water. However,
the basic Stoneville diet contains about 17% water and supports good lar-
val growth (King etal. 1979). The 300% increase in survival with the addi-
tion of 5% beeswax to this diet suggests that beeswax performed an
additional function.
Eisehen et al. (unpublished) found in a preliminary test that newly
hatched larvae preferentially chose artificial diets to which alcoholic
extract of pollen was added. This suggests that pollen provided phago-
stimuli attractive to G. mellonellci. Other observations support this view.
Balazs (1958) reported that newly hatched larvae preferred honeycomb
126
ENTOMOLOGICAL NEWS
to an artificial diet, even though this diet contained honey and beeswax.
He also noted that older larvae fed the same artificial diet chose hon-
eycomb when given the opportunity. Haydak ( 1936) reported that larvae
attacked old combs in those areas that contained pollen. We have on
numerous occasions observed newly hatched larvae that have been
given the Stoneville diet wander about the inner walls of their rearing
container when food was nearby. Newly hatched larvae given a honey-
pollen-wax diet were rarely seen away from the food. The natural history
of this moth also lends support to a phagostimulatory hypothesis. Adult
females oviposit in and around bee colonies; generally in crevices where
eggs are protected from the bees (Paddock 1918, Nielsen and Brister
1977). Once hatched, the larvae must search for food. Though distances
may be short, it would be adaptive to be able to locate food (which also
serves as shelter) rapidly in the hostile interior of a honey bee colony.
The shorter times to eclosion of moths fed diets containing pollen
indicate that even small amounts of pollen are effective in promoting
development. Since pollen was localized on the upper surface of the diet
mass, it seems unlikely that its consumption would have been uniformly
distributed during larval growth. If the phagostimulatory hypothesis is
true, then consumption and consequent beneficial effects oceured dur-
ing the earliest instars. From this it follows that an early pollen meal may
supply a nutrient, perhaps protein, that allows faster development, while
apparently wax and honey do not.
Prepupal acceptance of soda straws greatly facilitated the handling
of prepupae and pupae. Large numbers of known age groups can be held
in relatively small containers. This is convenient when storing prepupae
under refrigerated conditions (ca. 15.5°C). We find that chilled, lightly
spun prepupae are easy to use by fishermen, a slight squeeze on the straw
prompts the prepupae to crawl out. Because male prepupae emerged
from the diet slightly before females, the first harvest of prepupae results
in a high percentage of males. This is an added benefit when separation
of sexes is desired. Since these tests, we have successfully reared many
thousand G. mellonella using this technique.
These findings should be of value to those who wish to preserve the
genetic variation observed in wild stock or to increase the yield of G.
mellonella in newly-established cultures (Bush 1973). However, cultures
that have been reared for many generations on a particular artificial diet
and consequently undergone selection for it may not show greatly im-
proved survival. Nevertheless, under some conditions it could be pru-
dent as well as cost effective to supplement artificial wax moth diets with
small amounts of properly stored pollen or unprocessed honeycomb.
Vol. 101, No. 2, March & April 1990
127
Table 1 . Developmental characteristics of Galleria mellonella reared on an artificial diet for-
tified with honey, pollen, or beeswax
Diet
% adult
survival
(X±SE)
days to
1st cf
emergence
(X±SE)
days to
1st 9
emergence
(X±SE)
adult
9
weight
(X±SE)
Stoneville
27.4 ±4.7 a 1
42.7 ± 1.1 a
43.9 ± 0.8 a
1 14.2 ± 5.3 mg 2
Stoneville +
5% honey
44.7 ± 6.0 b
43.3 ± 1.3 a
43.5 ± 0.8 a
—
Stoneville +
5% wax
89.6 ± 3.8 c
42.8 ± 0.7 a
43.7 ± 1.0 a
—
Stoneville +
5% pollen
80.8 ± 4.1 c
39.7 ± 0.8 b
41.0 ± 0.9 b
—
Stoneville + 5%
honey, pollen, wax
82.5 ± 4.7 c
39.2 ± 0.8 b
39.4 ± 0.8 c
Honey, pollen, wax
89.2 ± 2.6 c
37.6 ± 0.7 b
37.8 ± 0.7 cd
156.0 ± 5.8
^ Means in the same column followed by the same letter do not differ significantly at the 5%
level according to Duncan's multiple range test.
“Means weights are significantly different (P < 0.001). Female weights on other diets were
not recorded.
ACKNOWLEDGMENTS
We thank R.D. Akre and CAV. Bersiford for reviewing the manuscript. J. Fields helped
with the statistical analysis. This investigation was supported in part by Cooperative
Agreement 25-2 1-RC293-078 between the University of Georgia (A. Dietz, principle inves-
tigator) and the Honey Bee Breeding, Genetics, and Physiology Laboratory, USDA-ARS,
Baton Rouge, LA.
LITERATURE CITED
Allergret, P. 1964. Interrelationship of larval development, metamorphosis and age in a
pyralid lepidopteran. Galleria mellonella(L .), under the influence of dietetic factors.
Experimental Gerontology 1: 49-66.
Balazs A. 1958. Nutritional and nervous factors in the adaption of Galleria mellonella to
artifical diet. Acta Biol. Acad. Sci. Hung. 9: 47-69.
Beck, S.D.1960. Growth and development of the greater wax moth Galleria mellonella( L.)
(Lepidoptera: Galleriidae). Trans. Wis. Acad Sci., Arts Lett. 49: 137-148.
Bush,G.L. 1975. Genetic variation in natural insect populations and its bearing on mass-
rearing programmes. In pp. 9-17: IAEA/FAO Panel Proc. Ser., Controlling Fruit Flies
by the Sterile Insect Technique, Vienna, 1973.
128
ENTOMOLOGICAL NEWS
Dietz, A., R. Krell, and M.S. Brower. 1982. Pollination and our seashores, pp. 57-66. In:
Proc. 10th Pollination Conference, Southern Illinois University, July 1982.
Dadd, R.H. 1966. Beeswax in the nutrition of the wax moth. Galleria mellonella (L.). J.
Insect Physiol. 12: 1479-1492.
Haydak, M.H. 1936. Is wax a necessary constituent of the diet of wax moth larvae? Ann.
Entomol. Soc. Am. 29: 581-588.
Haydak, M.H. 1940. The length of development of the greater wax moth. Science. 91:
525.
King, E.G., G.G. Hartley, D.F. Martin, J.W. Smith, T.E. Summers, and R.D. Jack-
son. 1979. Production of the tachinid Lixophaga diairaeae on its natural host, the sugar-
cane borer, and on its unnatural host, the greater wax moth. U.S.D.A., S.E.A. Advances in
Agricultural Technology Southern Series No. 3.
Marston, N. and B. Campbell. 1973. Comparison of nine diets for rearing Galleria
mellonella. Ann. Entomol. Soc. Am. 66: 132-136.
Nielsen, R.A. and D. Brister. 1977. The greater wax moth: Adultbehavior. Ann. Entomol.
Soc. Am. 70: 101-103.
Paddock, F.B. 1918. The beemoth or waxworm. Texas Agric. Exp. Stat. Bull. 231: 3-38.
Young, R.G. 1964. Digestion of wax by the greater wax moth. Galleria mellonella(L.). Ann.
Entomol. Soc. Am. 57: 325-327.
ANNOUNCEMENT
July 31-Aug. 3, 1990. International Symposium on Biotic Stresses of Barley in Arid and
Semi-Arid Environments. Huntley Lodge, Big Sky, Montana. Contact: Jack Riesselman,
Dept of Plant Pathology, Montana State University, Bozeman, MT 59717. Phone: 406-994-
5149 FAX: 406-994-6579.
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