Oecologia (1988) 77:140-142
9 Springer-Verlag1988
Short communication
Surfactant-producing microorganisms isolated from the gut
of a EucMyptus-feeding sawfly, Perga affinis affinis
C.P. Ohmart 1, J.R. Thomas 1, and B. Bubela 2, ~r
1 CSIRO Division of Forestry and Forest Products, PO Box 4008, Canberra, ACT 2600 Australia
2 BAAS Becking Geobiological Laboratory, Canberra ACT, Australia
Summary. Microorganisms which produce surfactants were
isolated from the gut of a eucalypt-feeding sawfly. It is
possible that surfactants in the guts of some defoliators
may be microbiologically-produced.
Key words: Surfactants - Eucalyptus - Perga affinis affinis
It has been suggested that a large number of plant second-
ary metabolites are deleterious to insect herbivores and,
as a result, some general theories have evolved regarding
the chemical defenses of plants against herbivores (Feeny
1976; Levin 1976; Rhoades 1979; Rhoades and Cates
1976). Some studies, however, have shown that secondary
metabolites have no deleterious effects on certain insect her-
bivores (Bernays 1981, 1982). Surfactants present in the
gut contents of some insect herbivores can reduce the pre-
cipitation of dietary proteins by tannins and it has been
suggested that the gut of many insect herbivores have deter-
gent properties (Martin and Martin 1984). Further, due
to the presence of these surfactants, tannins do not interfere
with digestion or lower the nutritive quality of foliage for
insect herbivores (Martin et al. 1987). Eucalyptus foliage
contains large amounts of terpenes (essential oils) and tan-
nins which potentially reduce the quality of the foliage for
insect herbivores. However, eucalypt-feeding insects do not
appear to be adversely affected by dietary oils or tannins
(Fox and Macauley 1977; Morrow and Fox 1980; Ohmart
et al. 1985) and eucalypts have at least as many associated
herbivores as do other trees (Morrow 19~77; Ohmart et al.
1983). Physiological mechanisms used by herbivorous in-
sects to process these chemicals have not been determined.
It is well known that certain microorganisms produce sur-
factants (Korsaric 1987). Surfactants produced by bacteria
have enabled a microbiological approach to be investigated
for enhancing recovery from natural oil reservoirs (Bubela
1985). We hypothesized that some eucalypt-defoliating in-
sects may contain microorganisms in their digestive tracts
which produce surfactants. These surfactants may prevent
tannins from interferring with digestion by lowering the
interfacial tension between essential oils and gut fluids, en-
hancing the insect's ability to deal with these compounds.
* Deceased
Offprint requests to: C.P. Ohmart
Oecologia
Material and methods
Twelve groups of Perga affinis affinis Kirby (Hymenoptera:
Pergidae) larvae were collected over a 5-month period from
colonies located within a 35 km radius of Canberra, Austra-
lian Capital Territory. Larvae were anaesthetized with chlo-
roform and the abdomen opened in a laminar flow chamber
using sterilized equipment. The hind gut was then opened
and a small quantity of gut contents was removed and in-
oculated onto a sterile agar medium (Table 1). Incubation
of initial cultures from the gut was done anaerobically with
no success. All subsequent cultures were aerobically incu-
bated. After microbial colonies fon~aed on the agar plates
(3-7 days at room temperature) a loop sample of a pure
microbial colony was inoculated into a sterile liquid medi-
um (Table 1). After 3-5 days incubation at room tempera-
ture, surfactant production was assessed by measuring the
reduction in interfacial tension (IT) of the liquid medium
against a standard liquid (hexadecane). An unioculated me-
dium was also incubated and its IT measured as a controt.
Interracial tension measurements were made using the
"drop-weight" method of Harkins and Brown (1919) modi-
fied so that the fluid being measured for IT was dispensed
into hexadecane from a precision syringe (500 ul), the
plunger of which was operated by a micrometer head. The
Table 1. Composition of the medium upon which the gut micro-
flora of P.a. affinis were cultured (Liquid medium is simply the
above medium without the addition of agar)
5 g/t K2HPO4
2 g/1 KHzPO4
10 g/1 glucose
5 g/l yeast extract
20 g/1 agar
plus 1 ml of the following solutions
Solution A Solution B
1 g/1 FeC13 5.3 g/t00 ml CaCIz "2HzO
0.4 g/1 ZnSO4-7H20 5 g/100 ml MgCI2-6H20
0.1 g/1 CuSO~. 5H20
0.1 g/1 MnC12 "4H20
0.08 g/l CoC12-6HzO
0.024 g/1 NiCI2 -6H20
plus 1 ml of 0.15 g/100 ml solution of EDTA (ethylenediamine-
tetra-acetate disodium salt)
 I41

d r o p was forced t h r o u g h a smooth, circular glass tip, o f surfactant-producing microorganisms m a y be contained in

a k n o w n radius 1.
Results
Surfactant-producing microorganisms were isolated from
10 o f the 12 groups o f larvae collected (Table 2). In 7 o f
these groups the isolates p r o d u c e d average drops in IT o f
more than 20 millinewtons (Table 2) indicating that the sur-
factants were very effective. In m a n y cases several different
species o f microorganisms, differentiated by colony mor-
phology, were cultured from a single larval gut. Because
of the w o r k involved in subculturing and testing a single
m i c r o o r g a n i s m for surfactant p r o d u c t i o n it was not possi-
ble to test all organisms cultured on the agar plates. Three
types o f surfactant-producing microorganisms, differen-
tiated by colony m o r p h o l o g y , were cultured from isolates
during the study. Two types were rarely isolated and were
not subcultured for identification. M o r e than 95% o f the
surfactant-producing microorganisms isolated from the
sawfly larvae were a single filamentous yeast species. When-
ever this yeast species was isolated from a larva it was the
species selected for subculturing and it always p r o d u c e d
surfactants. If it was not isolated then a n o t h e r m i c r o o r g a n -
ism was selected for subculture. It is possible that some
larvae did n o t test positive for surfactant-producing micro-
organisms simply because the wrong m i c r o o r g a n i s m was
selected for subculturing.
It is possible that surfactants within insect herbivores
could result from microbial activity and there are there pos-
sible ways by which this could occur. Firstly, surfactants
p r o d u c e d by yeasts a n d / o r bacteria resident on leaf surfaces
are ingested by insects when the leaf is consumed. Secondly,
the insect gut could be operating similarly to a chemostat
in relation to the surfactant-producing microorganisms. F o -
liage u p o n which the microorganisms are resident is con-
sumed by the insect. As this material is passed through
the gut the microorgansims release surfactants into the gut.
The microorganisms are then passed out o f the gut in faeces.
M i c r o o r g a n i s m s are constantly ingested and egested a n d
produce surfactants as they pass through the gut. Thirdly,
t Detailed explanation of modified drop-weight method available
from authors
special tissues within the insect gut.
M a r t i n and K u k o r (1984) suggested that ingested
microbes m a y metabolize potential toxins present in insect
diets while Jones (1984) discussed several cases where p l a n t
allelochemicals inhibited insect-microbial systems. Little
work, however, has been done on the gut microbiology
o f foliage-feeding insects (M.M. Martin, personal c o m m u -
nication). A great deal o f w o r k needs to be done to fully
explore the possibility that surfactants in the guts o f insect
herbivores are microbiologically-produced.
Acknowledgements. We thank Dr. R. Milner for valuable help and
advice on initial lab work and, along with Dr. K.M. Old, for
useful comments on earlier versions of the manuscript.
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Table 2. Interracial tension (IT) (_+ 1 s.d.) measurements of microbial cultures from the guts of P. a. affinis larvae

Collection site Date No. of larvae No. of larvae IT (mN)

(1987) examined from which
surfactant Inoculated cultures Control
produced
Surfactant Nonsurfactant
producing producing

Wanniassa, ACT Site 1 1/1o 5 2 4;2 (0.8) 33.1 (2.1) 33.9

Wanniassa, ACT Site 2 28'10 8 8 12.2 (7.0) 0 33.0
Yarralumla, ACT Site 1 28q0 8 3 8,8 (0.8) 32.2 (2.4) 33.0
Fisher, ACT Site 1 t3/ll 9 0 0 32.3 (2.4) 33.3
Curtin, ACT 13'11 8 7 10.7 (1.6) 29.6 33.3
Yarralumla, ACT Site 2 13ql 8 7 14.2 (2.1) 34.2 33.3
Braidwood, NSW 27ql 8 2 22.3 (3.9) 31.6 (0.5) 33.1
Bungendore, NSW 27'11 8 5 23.7 (3.7) 34.6 (1.3) 33.1
Macks Reef Rd, Site 1 27ql 8 8 11.1 (1.5) 0 33.1
Macks Reef Rd, Site 2 27ql 5 0 0 33.6 (0.7) 33.0
Fisher, ACT Site 2 21q2 5 5 7.9 (2.8) 0 32.4
142
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Received April 14, 1988