Laboratory
Animals
http://lan.sagepub.com/
Developmental and biochemical characteristics of sterile cultures of the
blowfly Lucilia cuprina
Keith Leslie Williams, Susan Nurmi and L. M. Birt
Lab Anim 1974 8: 177
DOI: 10.1258/002367774781005814
The online version of this article can be found at:
http://lan.sagepub.com/content/8/2/177
Published by:
http://www.sagepublications.com
On behalf of:
Laboratory Animals LtdLaboratory Animals Ltd
Additional services and information for Laboratory Animals can be found at:
Email Alerts: http://lan.sagepub.com/cgi/alerts
Subscriptions: http://lan.sagepub.com/subscriptions
Reprints: http://www.sagepub.com/journalsReprints.nav
Permissions: http://www.sagepub.com/journalsPermissions.nav
>> Version of Record - May 1, 1974
What is This?
Downloaded from lan.sagepub.com at PENNSYLVANIA STATE UNIV on February 28, 2014
Laboratory Animals (1974) 8,177-187.
177
DEVELOPMENTAL
CHARACTERISTICS
AND BIOCHEMICAL
OF STERILE CULTURES OF
THE BLOWFLY LUCILIA
CUPRINA
by
KEITH LESLIE WILLIAMS*, SUSAN NURMI
and
L. M. BIRT
Department of Biochemistry,
School of General Studies,
Australian National University,
P.O. Box 4, Canberra, A.c.r.
2600, Australia
SUMMARY
A procedure is described for the routine sterile culture of large numbers
(2000 per week) of blowflies, Lucilia cuprina, through all stages of the life
cycle. Comparisons of the rate of development and basic chemical composition of the sterile and non-sterile insects revealed no significant differences.
Although it is well established that holometabolous insects are particularly
suitable for developmental studies, stock cultures of such insects bred without special precautions are almost invariably contaminated with microorganisms. For many purposes this contamination is unimportant, but when
some techniques for examining biosynthetic pathways are employed, it becomes
essential to eliminate the possibility of a significant microbial contribution to
the results. This requirement is most pressing when the biosynthetic capacities
of tissue fractions (e.g. mitochondria) are examined in vitro with isotopic
precursors, or when enzymes of relatively low activity are being estimated.
Such studies have been undertaken recently with the blowfly Lucilia cuprina
(e.g. Williams & Birt, 1971a, b; Campbell & Birt, 1972; Smith & Birt, 1972)
as part of a detailed investigation of adult development. In consequence,
we have devised a procedure by which large numbers of insects can be maintained in a microbiologically sterile condition throughout their entire life
cycle. Blowflies of the genus Lucilia have been cultured aseptically before
(Lennox, 1939), but only small numbers have been produced at anyone time
and most frequently asepsis was broken at the wandering larval stage. Other
flies have been reared in sterile culture during the larval stage (Phormia, see
·Present address: Department of Biochemistry, University of Oxford, South Parks Road,
Oxford, OXI 3QU.
Downloaded from lan.sagepub.com at PENNSYLVANIA STATE UNIV on February 28, 2014
178
K. L. WILLIAMS, S. NURMI
AND
L. M. BIRT
Wymer, Lumb & Tate, 1970; Sarcophaga, see Goodfellow & Barnes, 1971)
but there are no detailed descriptions of culture methods for raising sterile
adults. Moreover, no investigation of possible differences in gross chemical
composition between the sterile and non-sterile insects has been reported,
though it is known that intestinal bacteria are not essential for the development of Lucilia cuprina (Lennox, 1939; Hobson, 1932). Differences have,
however, been reported with some insects, for example in the activity of the
enzyme mevalonate kinase in Sarcophaga (Goodfellow & Barnes, 1971), and
(in the hemimetabolous cockroach) in concentrations of amino acids (Wharton
& Lola, 1970).
This paper describes a breeding procedure which permits the routine production of at least 2000 sterile adult Lucilia per week and which has been used
to provide the insects for a number of investigations discussed in detail elsewhere (Williams & Birt, 1971a, b; Smith & Birt, 1972; Campbell & Birt, 1972).
It also presents a brief comparison of the gross chemical composition of the
sterile and non-sterile cultures.
MATERIALS AND METHODS
Culture of insects
All insects were grown in continuous illumination at 30°C. All operations
involving transfer of sterile insects were conducted in a bacteria-free area
(Clemco UV Products, 73 Dickson Avenue, Artarmon, New South Wales,
Australia). After the eggs have hatched larvae feed for about 3 days, then
leave the food ('wandering' larvae). At day 4-5 they become immobile and
form the 'white puparium'. Development within the puparium continues for
6 days (till day 10-11). During the latter part of this development (from
about day 5!) the insect exists as a pharate adult. The emerged fly matures
about 4 days after emergence.
Chemical analyses
Protein estimation was by the Biuret method of Gornall, Bardawill & David
(1949) or the microbiuret method of Goa (1953).
Carbohydrates (total soluble carbohydrate and glycogen) were determined
with the phenol-sulphuric acid reaction (Crompton & Birt, 1967).
Lipids were extracted from the tissue in chloroform-methanol and the extracts purified by the methods described by D'Costa & Birt (1966). Phospholipids and triglycerides were separated on columns of silicic acid (D'Costa &
Birt, 1966) and estimations of the total ester content of each fraction were
made by the method of Rapport & Alonzo (1955).
Ninhydrin-positive material was determined on the supernatant fluids obtained
by homogenizing insects in ice-cold perchloric acid (0'3 M), using the colori-
Downloaded from lan.sagepub.com at PENNSYLVANIA STATE UNIV on February 28, 2014
STERILE CULTURE OF BLOWFLIES
179
metric method of Rosen (1957); the method was essentially that described
by Smith & Birt (1972).
Amino acids were individually determined on acid supernatants with an
amino-acid analyser (Technicon Instruments Co. Ltd, 80 Talavera Road,
P.O. Box 135, North Ryde 2113, Sydney, Australia). Samples of the tissue
preparations were treated with perchloric acid (final concentration 0,3 M),
and the supernatants neutralized and applied to ion-exchange columns ('Dowex,;
Dow Chemical (Australia) Ltd, 105 Miller Street North, Sydney, New South
Wales 2060, Australia). The amino acids were eluted with 2·5 M ammonia,
the eluates evaporated to dryness and washed to remove the remaining traces
of ammonia. The samples were then dissolved in concentrated hydrochloric
acid and analysed.
RESULTS AND DISCUSSION
Development of the technique for sterile culture
Development of eggs
Eggs were obtained from non-sterile flies from a stock culture fed on liver.
They require moist conditions for hatching and development is temperaturedependent; at 100 % relative humidity, hatching occurs after 7-9 h at 37°C,
and after 12-14 h at 30°C. Eggs both develop and hatch at temperatures over
15°C, but at 10°C development commences without hatching. Evans (1934)
also noted with Lucilia sericata that development begins over a slightly larger
temperature range than does hatching.
Studies on egg survival at low temperature were undertaken in the hope
that eggs could be stored after sterilization. Storage was attempted at 0-4,
-60 and -196°C. At the lower temperatures, a number of variables was tested,
e.g. rate
of cooling
and rewarming
(Klassen
& Moline,
1965; Mazur,
1970),
addition of cryoprotectants (glycerol and dimethylsulphoxide), serum albumin,
glucose, milk and sodium citrate. All attempts were unsuccessful.
Table 1. Survival of eggs in cold storage.
Days at 0-4°C
o
1
2
3
Emergence
mean
% s.d.
74
61
31
20
15
23
26
12
Eggs were incubated for 5 h at 37°C in moist conditions, sterilized, and placed in groups
of 10 on autoclaved liver in test tubes. Tests were done in quadruplicate; 4 samples were
placed at 30°C immediately, the rest stored at 0-4°C for varying lengths of time. The
results are from 6 repetitions of the experiment.
Downloaded from lan.sagepub.com at PENNSYLVANIA STATE UNIV on February 28, 2014
180
K. L. WILLIAMS, S. NURMI
AND
L. M. BIRT
Survival after storage at 0-4°C was more satisfactory, but the stage of egg
development at which storage was commenced was critical. Newly-laid eggs
would not survive for even I day at 0-4°C, but if they were incubated at 3rC
for 5 h and then stored at 0-4°C, appreciable numbers survived for 4 days;
the mortality increased with time of storage at 0-4°C (Table 1). Regardless
of the length of the preincubation (1-7 h at 3rq there was a sharp decrease
in the survival of eggs stored at 0-4°C for longer than 4 days. Less than 5 %
survived the 5th or 6th day, and no survivors were ever detected after 7 days.
Sterilization
of eggs
Eggs were routinely incubated in 100 % relative humidity for 5 h at 37°C;
they were separated in either excess a-1M sodium hydroxide or 1 % sodium
sulphide. Groups of about 20000 were treated at one time and, after being
dispersed,
were washed free of alkali or sulphide with sterile distilled water.
They were soaked for 5 min in a sterilization medium composed of mercuric
chloride (0·5 g), concentrated hydrochloric acid (1,25 ml), sodium chloride
(6'5 g), and absolute ethanol (250 ml), made up to I litre with distilled waterusing approximately 1 ml of eggs with 30 ml of fluid. The liquid was decanted
and the eggs soaked in 50 % ethanol for 10 min; they were then washed several
times with sterile distilled water (in some later experiments, freshly prepared
I % sodium hypochlorite was used instead; this was equally satisfactory as a
sterilizing agent and had the advantage that it was not essential to remove
it before the eggs were deposited on the sterile food material).
Preparation of sterile medium
Sheep liver was freed from connective tissue, cut into 10 mm cubes and
reduced to a creamy homogenate in a blender. The homogenate was poured
into 100 ml glass jars to a depth of about 20 mm. Metal lids were screwed on
loosely and the jars autoc1aved at 121°C for 20 min. After being cooled, the
jars were stored at 0-4°C. Batches of jars were prepared at weekly intervals.
Initiation of larval development
Sufficient sterilized eggs were used to ensure that 100-200 larvae emerged
in each jar; the lids were replaced loosely to ensure adequate aeration. The
jars were either placed in the incubation room at 30°C, or returned for cold
storage at 0-4°C and then transferred at daily intervals to the incubation room
for hatching and development.
Growth of larvae and pupae
The duration of each stage of the life-cycle of the sterile insects was identical
to that of non-sterile insects grown on raw liver. Sterile larvae fed voraciously
Downloaded from lan.sagepub.com at PENNSYLVANIA STATE UNIV on February 28, 2014
STERILE CULTURE OF BLOWFLIES
181
metal lid
Fig. 1. The sequence of manipulations in sterile breeding of
Lucilia cuprina.
A. Culture
jar
contammg
autoc\aved liver and sterilized
eggs, capped with a looselytitted metal lid;
B. Culture jar attached by
means of a perforated double
lid to another jar containing
a bed of dry-sterilized sand to
collect wandering larvae before pupariation;
C. Sterilizing dish containing
dry-sterilized sand in which
larvae pupariate after transfer
from the double jar;
A
sterile
liver
culture jar
D. Sterile petri dish for storage of puparia.
double lid
B
_~
_.~
f·:?~~;:>·;·~~
\:.:j';~";)
-larvae,
sterile
-
liver
.....
•...~
..
sterile
sand
....
c
sterile
D
II
pupae,
-S7
sterile
~
petri
tray
r;dish
--dl
Downloaded from lan.sagepub.com at PENNSYLVANIA STATE UNIV on February 28, 2014
182
K. L. WILLIAMS,
S. NURMI
AND
L. M. BIRT
and soon liquefied the autoc1aved liver, which initially had a texture like moist
bread.
After about 3 days at 30°C, the lids were removed from the jars and
sterile 100 ml glass bottles with 'double lids' (Fig. 1) previously dry-sterilized
for 1that
180°C and containing sand to a depth of approximately 20 mm, were
screwed onto the bottles containing the growing larvae.
The double lids were
perforated to permit larvae of the wandering stage to escape from the medium
into the dry sand. Larval movement frequently moistened the sand, thus
preventing pupariation;
nevertheless the procedure successfully removed larvae
from the liquefied medium.
They were separated from the moist sand by
sieving through a dry-sterilized sieve, and transferred to fresh dry-sterilized
sand in a flat dry-sterilized tray with a metal lid. Each tray held 200-400
larvae.
Insects were collected as they pupariated and were transferred to
sterile plastic petri dishes.
Aseptic growth of adult flies
Groups of (less than 50) newly-emerged sterile flies were transferred from
the sterile petri dish (cooled to O°C to immobilise the flies) into a dry-sterilized
900 ml glass jar with a metal lid containing a mixture of sand and silica gel
(about 5: 1) to a depth of 20 mm. Immediately before adding the sterile flies,
a previously autoclaved 20 ml beaker containing cotton wool moistened with
0,5 M sucrose was placed in the jar. The interior of the jars remained dry
for a week. Growth could be sustained for longer periods by transferring
flies to a second jar.
Testing for sterility
The sterility of cultures was tested on blood agar plates incubated at 37°C
for 48 h. Any culture showing contamination
was discarded.
The cultures
were tested after sterilization of eggs, with a sample of disrupted eggs, and
when the double lid was attached to the culture jar, with a sample of the
medium (preliminary tests showed that the medium was always contaminated
if larvae were contaminated).
They were tested again at pupariation,
with
a crushed pupa; in addition, at this stage, a pupa was crushed on to nutrient
agar and incubated at 25°C for 72 h. This test was used to detect the infrequent bacterial contaminants (probably airborne) which are unable to grow
at 37°C. Routinely about 70 % of cultures at this stage were sterile. A final
test was made after emergence of the adult flies, when a sample fly from each
group used for experiments was tested.
Comments on the method
The stage of egg development at the time of sterilization was critical for
survival (Lennox, 1939; Mackerras & Freney, 1933). The resistance of newlylaid eggs was poor, while eggs that were almost ready to hatch were much more
resistant.
Although the sterilization treatment was slightly different from that
Downloaded from lan.sagepub.com at PENNSYLVANIA STATE UNIV on February 28, 2014
STERILE CULTURE OF BLOWFLIES
183
of Lennox (1939), similar values for egg survival were obtained. Eggs incubated for 5 h at 37°C before sterilization survived the treatment satisfactorily.
Happily this period also promoted optimal survival during storage at 0-4°C.
Using the methods outlined, eggs could be held at 0-4°C for up to 3 days.
Since egg survival varies with time of storage (Table 1), more eggs were used
in the containers stored for longer periods so that about 150 larvae emerged
in each bottle.
The most serious problems encountered during the operations were environmental. To maintain sterility, containers must be completely enclosed.
Thus, in the presence of moist food, the atmosphere was saturated with water
vapour, resulting in ideal conditions for larval growth but unfavourable conditions for pupariation. The development of a simple method for transferring
larvae to dry conditions was essential for the routine aseptic growth of the
fly.
A more difficult problem was that of gas exchange at the larvae stage. Blowfly larvae produced ammonia and carbon dioxide in lethal quantities (Hobson,
1932) so that adequate aeration had to be provided. The 100 ml bottles were
apparently the largest containers which could be used without forced aeration,
since attempts to grow similar numbers of larvae in 900 ml jars without forced
aeration produced unacceptably high mortality. Even with aeration by
diffusion in the 100 ml bottles, continual vigilance was necessary to ensure
that aeration remained adequate. The use of plastic rather than metal lids
was unsatisfactory, since they allowed only limited aeration by 'binding' to
the glass as the larvae liquefied the liver (plastic double lids were satisfactory).
Although the lids were screwed on loosely it was not necessary to stand the
jars in a sterile environment. Even very loose lids produced essentially no
contamination problems unless they were so loose that larvae could escape.
In general the larvae tried to escape only when cultures were overcrowded.
Biochemical properties of the sterile cultures
Homogenized liver was used as the medium supporting larval growth to
preserve the closest possible similarity between the conditions for development
of the sterile and non-sterile cultures. Thus, it was obviously desirable to
confirm that the sterile rearing did not alter the overall pattern of utilization
of carbohydrates, lipids and nitrogenous compounds, compared with those
previously established (Figs 2-5). All the values presented in Figs 2-5 have
been calculated on the assumption that the fresh weight of an insect in the
puparium is 30 mg.
Carbohydrates
The levels of both total soluble carbohydrate and glycogen (Fig. 2) varied
in a similar fashion in developing adults, whether sterile or non-sterile (Crompton & Birt, 1967).
Downloaded from lan.sagepub.com at PENNSYLVANIA STATE UNIV on February 28, 2014
K. L. WILLIAMS, S. NURMI
184
u
CIl
AND
L. M. BIRT
2
II)
c:
..••.
CIl
III
0
U
:::l
01
CIl
0
E
::J..
0
J
Age (days)
2
of.
s
7
Fig. 2. Variations in the level of soluble carbohydrate in sterile and non-sterile insects. Values
have been calculated on the assumption that the fresh weight of an insect is 30 mg. Age of
insects is expressed as days after pupariation (day 0). Total soluble 0-0 (sterile); .-.
(non-sterile); glycogen 0-0 (sterile); .-.
(non-sterile).
6
-
5
u
CIl
III
c:
4
..••.
~
CIl
III
/\.--.
:\~=/~.
e
3
CIl
CIl
0
E
2
0
::J..
e-
0-"-
phospholipid
-lot
~Q--
~o
_e_o,:e:---
e
0
0
2
3
4
5
(j
7
Age (days)
Fig. 3. Variations in the levels of lipids in sterile and non-sterile insects. Values have been
calculated on the assumption that the fresh weight of an insect is 30 mg. Age of insects is
expressed as days after pupariation (day 0). 0-0 (sterile); e-. (non-sterile).
Downloaded from lan.sagepub.com at PENNSYLVANIA STATE UNIV on February 28, 2014
STERILE
CULTURE
185
OF BLOWFLIES
Lipids
Similar patterns of usage of triglyceride (Fig. 3) during the most active
phase of adult development (days 4 to 6 after pupariation)
were found for
both types of culture (D'Costa & Birt, 1966). However, during the first 2
days after pupariation,
triglyceride was accumulated
and subsequently degraded in the sterile insects, whereas in the non-sterile insects the reverse was
true. This was probably a consequence of differences in the growth conditions
which were controlled more precisely with the sterile cultures.
The amounts of phospholipid in the 2 types of culture were similar at all
stages of adult development.
Nitrogenous compounds
The amounts of protein and total free amino acid (Figs 4 and 5) remained
constant within the accuracy of the determinations throughout adult development (values for the levels of amino acids and proteins in non-sterile insects
were obtained from Birt & Christian (1969) assuming a mean molecular weight
of the amino acid residues of ]25; similar results have been obtained by direct
analysis).
Levels of individual free amino acids in the sterile fly were similar
5
___
4
u
:3
Q)
oJ)
0
_o-o-o----o
v/
°
.-.----.-----.-----."
•
c
"E 2
Cl
o
b
7
Fig. 4. Variations in the levels of protein in sterile and non-sterile insects. Values have been
calculated on the assumption that the fresh weight of an insect is 30 mg. Age of insects
is expressed as days after pupariation (day 0). 0-0 (sterile); e-. (non-sterile).
Downloaded from lan.sagepub.com at PENNSYLVANIA STATE UNIV on February 28, 2014
186
K. L. WILLIAMS, S. NURMI
L. M. BIRT
AND
4
3
()~()~.)~)""o
u
ell
III
C
"-CIl
2
~~
____
X ____
x ____
___
x
X
X ••••••••
X
0
E
~
•
1
o
o
1
•
•
2
4
3
Age (days)
•
5
6
7
Fig. 5. Variations in the levels of ninhydrin-positive material in sterile and non-sterile insects.
Values have been calculated on the assumption that the fresh weight of an insect is 30 mg.
Age of insects is expressed as days after pupariation (day 0). 0-0 (sterile); x-x (nonsterile). Amino acids (non-sterile insects) .-e.
to those of the non-sterile insects. There were slight variations in the amounts
of ninhydrin-positive material during the development of both types of culture,
but the patterns were similar, i.e. there was a decline after pupariation and a
rise in late pharate adult development (Fig. 5). Values for the non-sterile
insects were obtained from Pinch & Birt (1962) and Howells & Birt (1964).
These analyses showed that the sterile cultures contained somewhat larger
amounts of soluble carbohydrate, protein and ninhydrin-positive material,
but were slightly poorer in triglyceride. The differences may reflect the more
homogeneous food supply available to the larvae, and the more careful control
of larval numbers in relation to the food mass in the culture vessels and the
temperature of growth. However, there seems to be no significant difference
in the pattern of usage of 3 classes of compounds during adult development.
The results suggest strongly that previous generalizations about catabolism
during pharate adult development can be applied to sterile insects also (compare Crompton & Birt, 1967).
CONCLUSION
The procedures for mass breeding of sterile insects described in this paper
produce cultures of Lucilia cuprina which do not appear to differ significantly
Downloaded from lan.sagepub.com at PENNSYLVANIA STATE UNIV on February 28, 2014
STERILE CULTURE OF BLOWFLIES
187
from non-sterile insects in the rate of their development throughout the life
cycle or in the pattern of use of nutrient reserves during the formation of the
adult fly. Thus, results from experiments done with both types of culture
can be used in conjunction with each other in discussions of the biochemistry
of metamorphosis in Lucilia.
REFERENCES
Blrt, L. M. & Christian, B. (1969). Changes in nitrogenous compounds during the metamorphosis of the blowfly, Lucilia cuprina. Journal of Insect Physiology 15, 711.
Campbell, A. C. & Birt, L. M. (1972). Studies on the appearance of soluble a-glycerophosphate dehydrogenase activity during the development of the sheep blowfly Lucilia. Insect
Biochemistry 2, 279.
Crompton, M. & Birt, L. M. (1967). Changes in the amounts of carbohydrates, phosphagen
and related compounds during the metamorphosis of the blowfly, Lucilia cuprina. Journal
oIInsect Physiology 13, 1575.
D'Costa, M. A. & Birt, L. M. (1966). Changes in the lipid content during the metamorphosis
of the blowfly Lucilia. Journal of Insect Physiology 12, 1377.
Evans, A. C. (1934). Studies on the influence of the environment on the sheep blowfly Lucilia
sericata Meig. 1. Influence of humidity and temperature on the egg. Parasitology 26,
366.
Finch, L. R. & Birt, L. M. (1962). Amino acid activation during the pupal development of
the fly Lucilia cuprina. Comparative Biochemistry and Physiology 5, 59.
Goa, J. (1953). A microbiuret method for protein determination, Scandinavian Journal of
Clinical and Laboratory Investigation 5, 218.
Goodfellow, R. D. & Barnes, F. J. (1971). Mevalonate kinase from the larvae of the fleshfly, Sarcophaga bullata. Insect Biochemistry 1, 271.
Gornall, A. G., Bardawi II, C. S. & David, M. M. (1949). Determination of serum proteins
by means of the biuret reaction. Journal of Biological Chemistry 177, 751.
Hobson, R. P. (1932). Studies on the nutrition of blowfly larvae. II. Role of the intestinal
flora in digestion. Journal of Experimental Biology 9, 128.
Howells, A. J. & Birt, L. M. (1964). Amino acid-dependent pyrophosphate exchange during
the life cycle of the blowfly Lucilia cuprina. Comparative Biochemistry and Physiology
11,61.
Klassen, W. & Moline, S. W. (1965). The freezing of mosquito larvae. Cryobiology 2, 30.
Lennox, F. G. (1939). Studies of the physiology and toxicology of blowflies. Part 1. The
development of a synthetic medium for aseptic cultivation of larvae of Luci/ia cuprina.
Australian Council for Scientific and Industrial Research Pamphlets 90.
Mackerras, M. J. & Freney, M. R. (1933). Observations on the nutrition of maggots of
Australian blowflies. Journal of Experimental Biology 10, 237.
Mazur, P. (1970). Cryobiology: the freezing of biological systems. Science, New York 168,
939.
Rapport, M. M. & Alonzo, N. (1955). Identification of phosphat idyl choline as the major
constituent of beef heart lecithin. Journal of Biological Chemistry 217, 199.
Rosen, H. (1957). A modified ninhydrin colorimetric analysis for amino acids. Archives of
Biochemistry and Biophysics 67, 153.
Smith, E. & Birt, L. M. (1972). Proteolytic activity during the metamorphosis of the blowfly Lucilia cuprina. Insect Biochemistry 2, 218.
Wharton, D. R. A. & Lola, J. E. (1970). Blood conditions and lysozyme action in the
aposymbiont cockroach. Journal of Insect Physiology 16, 199.
Williams, K. L. & Birt, L. M. (1971a). Incorporation in vitro of HC leucine into the mitochondrial protein of Lucilia cuprina, 1. Basic requirements. European Journal of Biochemistry 22,87.
Williams, K. L. & Birt, L. M. (1971b). Incorporation in vitro of HC leucinc into the mitochondrial protein of Lucilia cuprina. II. Energy requirements. European Journal of Biochemistry 22, 96.
Wymer, L. T., Lumb, R. H. & Tate, L. G. (1970). Techniques for maintaining a stock of
blowfly Phormia regina. In Experiments in phYSiology and biochemistry (ed. G. A. Kerkut),
vol. 3, pp. 365-375. New York: Academic Press.
Downloaded from lan.sagepub.com at PENNSYLVANIA STATE UNIV on February 28, 2014
Downloaded from lan.sagepub.com at PENNSYLVANIA STATE UNIV on February 28, 2014