APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2004, p. 293–300 Vol. 70, No.

1
0099-2240/04/$08.00⫹0 DOI: 10.1128/AEM.70.1.293–300.2004
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Census of the Bacterial Community of the Gypsy Moth Larval Midgut

by Using Culturing and Culture-Independent Methods
Nichole A. Broderick,1,2,3* Kenneth F. Raffa,1 Robert M. Goodman,2,3,4 and Jo Handelsman2,3
Departments of Entomology1 and Plant Pathology,2 the Microbiology Doctoral Training Program,3 and the Gaylord Nelson
Institute for Environmental Studies,4 University of Wisconsin, Madison, Wisconsin 53706
Received 18 March 2003/Accepted 22 September 2003

Little is known about bacteria associated with Lepidoptera, the large group of mostly phytophagous insects
comprising the moths and butterflies. We inventoried the larval midgut bacteria of a polyphagous foliivore, the
gypsy moth (Lymantria dispar L.), whose gut is highly alkaline, by using traditional culturing and culture-
independent methods. We also examined the effects of diet on microbial composition. Analysis of individual
third-instar larvae revealed a high degree of similarity of microbial composition among insects fed on the same
diet. DNA sequence analysis indicated that most of the PCR-amplified 16S rRNA genes belong to the ␥-Pro-
teobacteria and low GⴙC gram-positive divisions and that the cultured members represented more than half
of the phylotypes identified. Less frequently detected taxa included members of the ␣-Proteobacterium, Acti-
nobacterium, and Cytophaga/Flexibacter/Bacteroides divisions. The 16S rRNA gene sequences from 7 of the 15
cultured organisms and 8 of the 9 sequences identified by PCR amplification diverged from previously reported
bacterial sequences. The microbial composition of midguts differed substantially among larvae feeding on a
sterilized artificial diet, aspen, larch, white oak, or willow. 16S rRNA analysis of cultured isolates indicated that
an Enterococcus species and culture-independent analysis indicated that an Entbacter sp. were both present in
all larvae, regardless of the feeding substrate; the sequences of these two phylotypes varied less than 1% among
individual insects. These results provide the first comprehensive description of the microbial diversity of a
lepidopteran midgut and demonstrate that the plant species in the diet influences the composition of the gut
bacterial community.

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Microorganisms play important and often essential roles in and function is lacking. Several attributes of Lepidoptera lar-
the growth and development of many insect species. Endosym- vae make their bacteria of particular interest. In particular, the
bionts contribute to insect reproduction, digestion, nutrition, high alkalinity (typically pH 8 to 10) of most lepidopteran
and pheromone production (7, 13, 14, 20, 50, 53, 55). Symbiotic midguts and the diverse chemistry of the midgut generated by
relationships between insects and their gut bacteria have been the unusually broad feeding range of some species make this a
studied extensively in several systems, particularly in termites particularly challenging environment for microorganisms (5,
and aphids, which feed on wood and plant phloem content, 22, 29, 44). Furthermore, the Lepidoptera include some of the
respectively (8, 17, 31). However, relatively little is known most damaging agricultural and forest pests worldwide, includ-
about microbial associates in other insect groups, particularly ing species that overcome existing control measures, such as
those that feed on foliage. synthetic insecticides, microbial insecticides, genetic host plant
Describing relationships between insects and their associ- resistance, and genetically modified plants (60, 70). Knowledge
ated bacteria has proved challenging because many of the of the gut bacteria of the Lepidoptera and the roles they may
microbial associates are not readily culturable. The availability play in larval biology could lead to new targets for pest man-
of culture-independent tools, such as PCR, provides an oppor- agement.
tunity to detect and classify microorganisms that cannot be As an initial step toward understanding relationships be-
cultured by existing methods (74). PCR has revealed the phy- tween Lepidoptera and their midgut bacteria, we surveyed the
logeny of endosymbionts and associates of a number of insect midgut of the gypsy moth, Lymantria dispar (L.), which is an
species (1, 4, 15, 19, 33, 55, 57, 62, 64). invasive species of Eurasian origin and the most damaging
Little is known about the bacteria associated with Lepidop- defoliator of deciduous trees in North America (51). The gypsy
tera, a primarily phytophagous group that is one of the largest moth alters natural ecosystems, causes economic losses to the
insect orders, containing over 150,000 species (65). A few stud- forest industry and homeowners, elicits allergic reactions in
ies indicate that lepidopterans harbor midgut bacteria (14, 49). humans, and is highly polyphagous with a reported host range
These studies suggest the possibility that microorganisms pro- of 300 species (41). We examined the effects of diet and insect
vide essential nutrients or assist in important biochemical func- source on the microbial composition of third-instar midguts.
tions. However, an understanding of the types of microbes
present in the midgut and of their roles in insect development
MATERIALS AND METHODS
Sources and treatments of gypsy moth larvae. (i) Larvae from diverse popu-
* Corresponding author. Mailing address: Department of Entomol- lations feeding on a common substrate. Gypsy moth egg masses were obtained
ogy, University of Wisconsin—Madison, 1630 Linden Dr., 345 Russell from the culture New Jersey Standard Strain (NJSS) at the U.S. Department of
Laboratories, Madison, WI 53705. Phone: (608) 262-8735. Fax: (608) Agriculture (USDA)-Animal and Plant Inspection Service (APHIS) laboratory
262-5289. E-mail: [email protected]. at the OTIS Air National Guard Base, Cape Cod, Mass. (MA colony) and the

293
294 BRODERICK ET AL. APPL. ENVIRON. MICROBIOL.

Beneficial Insects Introduction and Research Laboratory, U.S. Department of labeled with the dye 6-carboxyfluorescein (6-FAM) (16, 42). All oligonucle-
Agriculture-Agricultural Research Services, Newark, Del. (DE colony). In addi- otide primers were synthesized by the Biotechnology Center at the University
tion, egg masses were collected from field sites in Wisconsin (WI field) and of Wisconsin—Madison. PCR contents for a 50-␮l volume were 25 ng of
Michigan (MI field). Wisconsin egg masses were collected on 24 March 1999 template DNA, 0.5 ␮l of 20 ␮M forward (27F) and reverse (1492R) primers,
from five trees (four oak trees [Quercus sp. L.] and one maple tree [Acer sp. L.]) 0.8 ␮l of 10 mM deoxynucleoside triphosphates (Gibco BRL, Grand Island,
on a rural property in York Township, Dane County (R11E, T9N). The Michigan N.Y.), 5 ␮l of 10⫻ Taq polymerase buffer, and 0.5 ␮l of Taq DNA polymerase
egg masses were collected 23 March 1999 from a newly established population in (2.5 U; Promega, Madison, Wis.). The thermal cycling conditions were an
a mixed oak and aspen (Quercus sp. and Populus sp.) stand in Lincoln Township, initial 3-min denaturation step at 94°C, 30 cycles at 94°C for 30 s, 55°C for
Clare County (R5W, T18N). Field-collected egg masses were kept in plastic bags 90 s, and 72°C for 2.5 min, with a final 5-min extension at 72°C. A hot-start
at 4°C from the time of collection until they were used in assays. Assays were protocol (34) was used with Taq polymerase added to each reaction after 1
timed to coincide with Wisconsin field larval emergence (around 5 May 1999). min of the 3-min initial 94°C denaturation step. All reactions were carried out
Gypsy moth larvae were reared as described in Broderick et al. (10). Briefly, in 0.2-ml reaction tubes in a Robocycler 96 (Stratagene, La Jolla, Calif.).
egg masses were surface sterilized with a solution of Tween 80 (polyoxyethylene Each PCR was examined by electrophoresis in a 0.8% agarose gel, and gels
sorbitan monooleate), bleach, and distilled water. Larvae were reared in 17-cm- were visualized by staining with ethidium bromide.
diameter petri dishes on a sterile artificial diet (USDA Hamden Formula) under (iii) T-RFLP analysis. Samples of PCR amplification products from 10 indi-
a 16 h:8 h (light:dark) photoperiod at 25°C. Colony (MA and DE) larvae were vidual larvae exposed to each diet type and from each population source were
reared in a quarantine facility at the University of Wisconsin—Madison Depart- digested (37°C for 3 h) with the restriction endonucleases AluI and HhaI (Pro-
ment of Entomology. To reduce the potential for introduction of pathogens into mega). Digestion reactions (10 ␮l total volume) contained 5 ␮l of PCR product,
the quarantine facility, field egg masses and larvae (WI and MI) were reared in 0.3 ␮l of enzyme, and 0.3 ␮l of the appropriate incubation buffer according to the
a separate campus quarantine facility at the University of Wisconsin—Madison synthesis protocol. Samples (5 ␮l) of the digestion reactions were mixed with 19
Biotron under the same conditions as above. Larvae were provided with artificial ␮l of deionized formamide and 1 ␮l of an internal standard (CST ROX 200-2000;
diet after emergence and provided a new diet every 48 h. BioVentures, Inc., Murfreesboro, Tenn.), denatured for 5 min at 95°C, and kept
(ii) Larvae from a common source feeding on multiple tree species. Gypsy on ice until they were loaded into an autosampler. The samples were analyzed by
moth egg masses from culture NJSS at the USDA-APHIS laboratory (MA capillary electrophoresis in an ABI DNA sequencer (model 310; Perkin-Elmer,
colony) were reared according to methods described previously (10, 11, 18). Foster City, Calif.) (39). Data from individual samples were analyzed with Ge-
Two-year-old white oak (Q. alba L.), larch [Larix laricina (Du Roi) K. Koch], neScan 3.1 (Perkin-Elmer) and were compared to other samples by using the
and quaking aspen (P. tremuloides Michaux) trees were obtained from the Wis- Ribosomal Database Project II (RDP II) T-RFLP Online Analysis software (46).
consin Department of Natural Resources Nursery, Hayward. Two-year-old scrub To assess the similarity of microbial composition among midguts, T-RFLP pro-
willow (Salix fragilis L.) trees were obtained from the Iowa State Nursery, Ames. files from replicate samples were aligned and analyzed by pairwise comparison
The trees were chilled at 4°C for 20 days after receipt to ensure good bud (40). Similarity values, Sab, were determined by using the equation 2 Nab / (Na ⫹
development. Following the chilling period, the trees were planted in 12-liter Nb), where Nab is the number of peaks in common between samples and Na and
pots in Sunshine Mix LC1 (Sungro Horticulture, Bellevue, Wash.) and flood Nb are the number of total peaks in each sample.
irrigated. The trees were grown in a greenhouse at 25°C under a 16 h:8 h (iv) Cloning 16S rRNA genes. Total DNA from pools of 10 larvae from all
(light:dark) photoperiod. Trees were watered every 5 days until bud develop- population and diet treatment samples was PCR-amplified separately by using
ment; following bud development the trees were watered every 2 to 3 days. the bacterial primers 27F and 1492R. Clone libraries were constructed by ligating

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Larvae were provided with a sterilized artificial diet for 24 h after emergence PCR product into pGEM-T Vector system (Promega) according to the manu-
and then were divided into 40 groups of 200 larvae each. These 40 groups were facturer’s instructions and by transforming competent Escherichia coli cells
distributed among treatments such that 10 groups of larvae fed on leaves of one (DH5␣) by electroporation (61). Plasmid DNA was isolated from transformants
of the four tree species in petri dishes lined with a piece of moistened sterilized by using QIAprep Spin Plasmid Miniprep kits (QIAGEN, Valencia, Calif.). All
filter paper. For each tree species 10 trees were sampled, with one group of 200 clones in libraries of approximately 250 clones from each diet type were se-
larvae feeding on leaves collected from each tree (a total of 2,000 larvae for each quenced.
tree species). Whole leaves were provided, with the petioles inserted in a micro- (v) 16S rRNA sequencing and data analysis. Sequencing reactions were per-
centrifuge tube containing distilled water to prevent desiccation. Foliage was formed by using the BigDye reaction mix (Perkin-Elmer Corp.) according to the
replaced every 48 h until the larvae were used for experiments. manufacturer’s instructions. Purified plasmid DNA was initially sequenced by
Culture-independent methods. (i) Midgut separation and DNA extraction. using the plasmid primers T7 and SP6, which flank the insert DNA in pGEM-T,
Third-instar larvae were surface sterilized for 5 s in 95% ethanol prior to dis- and additionally sequenced with 704F and 787R to obtain full 1,500-bp se-
section. Dissecting scissors were used to cut laterally behind the head capsule, quences (37). DNA from cultured organisms was purified by using the PCR
and the gut was removed from the cuticle with larval forceps. The crop and product Pre-sequencing Kit (USB, Cleveland, Ohio) and was sequenced by using
midgut were collected and placed in a 1.5-ml microcentrifuge tube for process- either 27F, 1492R, 704F, or 787R (37). Two representatives of each colony
ing. Samples were placed in a ⫺80°C freezer. morphology and a total of 400 clones (approximately 45 to 115 per diet treat-
Gypsy moth midguts were analyzed individually in all experiments. Total ment) were analyzed. All 16S rRNA sequences (1,450 to 1,490 bp) were com-
microbial DNA was extracted from individual gypsy moth crops and midguts by piled by using the SeqMan program from the DNAStar software package
using a protocol modified from the method described by Ausubel et al. (3). (DNASTAR, Inc., Madison, Wis.) and were compared to available databases by
Microcentrifuge tubes, each containing a single gut, were thawed, and 600 ␮l of use of BLAST to determine approximate phylogenetic affiliations (2). Individual
Tris-EDTA (TE) (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) was added to each sequences of isolates and clones that were ⱖ98% identical to each other were
tube. The contents of the tube were then sonicated (50 to 60 Hz, 117 V, 1.0 A; considered the same phylotype and were combined for analysis. All sequences
Branson Ultrasonics, Danbury, Conn.) for 30 s to separate bacterial cells from were tested for possible chimeric structures by using RDP Check_Chimera (39).
the gut wall, and 537 ␮l of TE was removed and placed in a new 1.5-ml (vi) Culturing and enumeration of midgut bacteria. Twenty-third-instar larvae
microcentrifuge tube. The sample was sonicated under the same conditions for from each of the five diet treatments were dissected for culturing studies follow-
45 s to break open bacterial cells and was mixed thoroughly with 60 ␮l of 10% ing a 24-h starvation period to allow gut clearing. Midguts were each placed in
sodium dodecyl sulfate and 3 ␮l of 50 mg of proteinase K/ml and was incubated 1 ml of phosphate-buffered solution (PBS) in a 1.5-ml microcentrifuge tube. Ten
for 1.5 h at 37°C. Each tube was mixed with 100 ␮l of 5 M NaCl prior to the larvae feeding on foliage from two trees of each species were used. Individual
addition of 80 ␮l of 10% cetyltrimethylammonium bromide–5 M NaCl. The guts in PBS were sonicated for 45 s and then were dilution plated on 1/10
sample was mixed thoroughly and incubated at 65°C for 30 min. DNA was strength tryptic soy agar (TSA) at neutral pH and were incubated at 28°C.
extracted with equal volumes of chloroform-isoamyl alcohol (CIA) (24:1 [vol/ Colonies were classified based on morphological parameters of shape, color,
vol]) and phenol CIA (25:24:1 [vol/vol/vol]). DNA was precipitated with isopro- margins, elevation, and texture. Colonies of each designated morphology were
panol and recovered by centrifugation. Pellets were resuspended in 100 ␮l of TE counted after 24, 48, and 72 h. Isolates of two representatives of each colony
buffer. DNA concentration was determined by absorbance ratio at 260/280 nm, morphology were purified for individual colonies, and cellular morphology of
and the DNA suspension was stored at ⫺20°C until it was used for PCR and each isolate was examined with 1,000⫻ magnification. DNA was extracted with
further analysis. the EasyDNA kit (Invitrogen, Carlsbad, Ohio) from 14-h-old cultures of each
(ii) PCR amplification. Bacterial 16S rRNA was amplified by PCR from isolate in 1/2-strength tryptic soy broth. DNA was quantified, amplified, and
total DNA by using primers 27F and 1492R (37). For terminal-restriction sequenced as described above.
fragment length polymorphism (T-RFLP) analysis, the 5⬘ primer (27F) was Ten individual third-instar midguts from larvae feeding on artificial diet were
VOL. 70, 2004 BACTERIAL DIVERSITY OF GYPSY MOTH MIDGUT 295

TABLE 1. Bacterial phylotypes identified by culturing and culture-independent analysis of third-instar gypsy moth midguts based on 16S
rRNA gene sequence analysis

Database Detection in gypsy moth fed ona:

Sequence identy and Genus (ⱖ95%
Bacterial division matches (ⱖ98% Artificial
identification method identity) Larch Willow Aspen White oak
identity) diet

Identified by culturing
and culture-
independent
analysis
NAB1 ␥-Proteobacteria Pseudomonas P. putida 1.8 ⫻ 105 1.1 ⫻ 107 3.6 ⫻ 105 3.5 ⫻ 105
NAB2 ␥-Proteobacteria Pseudomonas 2.2 ⫻ 106
NAB3 ␥-Proteobacteria Enterobacter Uncultured soil 4.8 ⫻ 106 7.0 ⫻ 107 8.9 ⫻ 107
bacterium
F42326.1
NAB4 ␥-Proteobacteria Pantoea P. agglomerans 2.5 ⫻ 105 3.7 ⫻ 104 1.5 ⫻ 107
NAB5 ␥-Proteobacteria Serratia S. marcescens 4.6 ⫻ 104 7.5 ⫻ 108 4.0 ⫻ 104
NAB6 low G⫹C gram positive Bacillus 4.0 ⫻ 108
NAB7 low G⫹C gram positive Staphylococcus S. lentus 8.1 ⫻ 104 9.1 ⫻ 106 4.5 ⫻ 106
NAB8 low G⫹C gram positive Staphylococcus S. cohnii 2.9 ⫻ 106 4.7 ⫻ 106 1.3 ⫻ 107
NAB9 low G⫹C gram positive Staphylococcus S. xylosus 1.3 ⫻ 106 1.07 ⫻ 105
NAB10 low G⫹C gram positive Paenibacillus 2.6 ⫻ 106
NAB11 low G⫹C gram positive Enterococcus E. faecalis 1.5 ⫻ 108 1.9 ⫻ 106 3.6 ⫻ 108 1.4 ⫻ 104 2.1 ⫻ 107
NAB12 low G⫹C gram positive Enterococcus 4.8 ⫻ 107 4.1 ⫻ 106 2.4 ⫻ 105
NAB13 Actinobacterium group Rhodococcus 1.2 ⫻ 107
NAB14 Actinobacterium group Microbacterium 5.2 ⫻ 108 2.2 ⫻ 106
NAB15 CFBb group 7.4 ⫻ 104

Identified by culture-
independent
analysis only
NAB16 ␣-Proteobacteria Agrobacterium ⫹ ⫹ ⫹
NAB17 ␥-Proteobacteria Enterobacter ⫹ ⫹ ⫹ ⫹ ⫹
NAB3 ␥-Proteobacteria Enterobacter ⫹

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NAB18 ␥-Proteobacteria ⫹ ⫹
NAB19 ␥-Proteobacteria ⫹ ⫹
NAB20 low G⫹C gram positive ⫹ ⫹ ⫹
NAB21 Actinobacterium group Micrococcus ⫹
NAB22 Actinobacterium group ⫹
NAB23 CFB group ⫹
a
For cultured cells the numbers represent the average population of phylotype based on colony morphology in 20 individual larvae per treatment (CFU/milliliter
of gut material). For noncultured samples, a plus indicates presence in guts of insects in diet treatment.
b
CFB, Cytophaga/Flavobacteria/Bacteroides.

dissected, sonicated, and stained with 4,6-diamino-2-phenylindole (DAPI) to Variation in bacterial composition among individual larvae.
determine the total number of bacterial cells per gut (72). For each individual
Comparisons of the T-RFLP profiles of the bacteria in repli-
midgut, twenty randomly selected fields were counted by epifluorescence micros-
copy (Olympus America Inc., Melville, N.Y.). cate larvae from either the same population source or the same
Nucleotide sequence accession numbers. The compiled 16S rRNA sequences diet treatment showed a high similarity index of between 0.96
have been deposited in the GenBank database under accession numbers and 1.00 (Tables 2 and 3). These kinds of similarities were also
AY39005 to AY39035.
reflected in the types of colony morphologies obtained in the
culture-dependent analyses. The majority of isolates examined
RESULTS were found in ⬎90% of the larvae examined and all were
isolated from ⬎50% of the larvae in a given treatment.
Bacterial diversity in third-instar gypsy moth midguts. Eval- To identify factors associated with variation in microbial
uation of the midgut bacteria of gypsy moth larvae using a
composition, we compared PCR-amplified 16S rRNA genes
combination of 16S rRNA analysis of the cultured bacteria and
isolated from the midguts of gypsy moth from lab and field
culture-independent PCR amplification of 16S rRNA se-
populations by using T-RFLP analysis. The T-RFLP profiles of
quences led to the identification of 23 phylotypes (Table 1).
Approximately 65% of these phylotypes are newly reported the MA colony and WI field samples and those of the DE
sequences (⬍98% identity to any of the bacterial 16S rRNA colony and MI field all fed artificial diet were very similar
sequences within GenBank). The majority (70%) of these bac- (Table 2). Moreover, these pairs differed only slightly (less
terial sequences fell within the low G⫹C gram-positive and than 6%) from each other (Table 2), indicating that egg source
␥-Proteobacteria divisions. Actinobacterium, Cytophaga/Flexi- did not have a significant effect on the bacteria of the gypsy
bacter/Bacteroides, and ␣-Proteobacteria accounted for 17, 9, moth midgut. Given these similarities, only the MA colony
and 4% of the sequences, respectively. population was used to examine the variation in bacterial di-
296 BRODERICK ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 2. Pairwise comparisons for similarity of T-RFLPs from the terial composition of the midgut, 16S rRNA sequences derived
midgut of gypsy moth from lab and field populations feeding on an from midguts were analyzed in silico by using the RDP II
artificial diet
T-RFLP Analysis Program (43) and were compared to actual
Coefficient Coefficient between populations T-RFLPs from the same samples. In general, 16S rRNA se-
Population within MA DE quences showed the predicted peak pattern, and all sequences
population WI field
colony colony could be accounted for in the T-RFLP. However, this analysis
also demonstrated that multiple fragments were found at the
MA colony 0.996
DE colony 0.997 0.952 same peak position regardless of the restriction enzyme, espe-
WI field 0.996 0.994 0.958 cially among similar 16S sequences. These results are consis-
MI field 0.998 0.955 0.992 0.953 tent with previous indications of the resolution limits of T-
RFLP analysis in assessing the full extent of diversity (23).
However, these results also indicate that T-RFLP analysis is
useful as a first step to assess sample diversity and identify
treatments or conditions for further investigation.
versity due to diet. Diet greatly affected the T-RFLP profiles
(Fig. 1), as indicated by the lower similarity values of T-RFLP
profiles from different diets (Table 3). DISCUSSION
Comparison of bacterial diversity revealed by culturing and
These results demonstrate that the microbial diversity of
culture-independent analysis. Culturing yielded 15 distinguish-
the gypsy moth midgut is relatively simple, with 15 phylo-
able types of bacteria across all diet treatments from gypsy
types at its most complex and 7 phylotypes at its simplest.
moth midguts based on cellular and colony morphological
The relative simplicity of this community is especially ap-
traits; 93% of these isolates could be assigned to a genus, while
parent compared to other gut environments, such as the
only 47% could be assigned to a species by using 16S rRNA
human intestine and termite hindguts, which contain at least
analysis (Table 1). DAPI staining and microscopic enumera-
500 and 50 phylotypes, respectively (54, 76). We expect that
tion indicated that there were 4 ⫻ 108 viable bacterial cells/gut.
the census presented here is fairly complete given that we
The phylotypes indicated by culture-independent methods ex-
can account for more than 40% of the viable cells by cul-
hibited greater divergence and diversity than phylotypes recov-
turing techniques, and all of the peaks in the T-RFLP anal-
ered by culturing. In addition to the 15 phylotypes recovered by
ysis are accounted for in the PCR-generated 16S rRNA
culturing, 9 phylotypes were indicated based on 16S rRNA
sequences. However, there may well be minor species whose
analysis in gypsy moth midguts across all diet treatments; only

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presence is obscured because they were not cultured by our
four of these phylotypes could be assigned definitively to a
methods and were not detected by either T-RFLP analysis
genus.
or 16S rRNA sequence analysis. Diet had a significant im-
Effect of diet on bacterial composition. Two phylotypes, En-
pact on midgut microbial diversity, as shown by culturing
terococcus faecalis and an uncultivated Enterobacter sp.
and sequence analysis of 16S rRNA genes amplified directly
(NAB17) were found in all larvae, regardless of treatment. In
from larval midguts. Molecular, culture-independent meth-
comparing the relative diversity of gut bacteria associated with
ods revealed that many of the amplified 16S rRNA genes
each diet treatment, we found that 8 bacterial phylotypes were
from gypsy moth midguts consisted of sequences not previ-
detected in only one of the diet treatments (Table 1). Larvae
ously described. Perhaps the most surprising result of this
feeding on larch contained the greatest diversity of bacterial
study was the culturing of numerous bacterial species whose
phylotypes (15, of which 11 were cultivated). Larvae feeding on
16S rRNA sequences were as yet undescribed in public
aspen contained the highest proportion of uncultivated bacte-
databases and, in some cases, diverged deeply from known
rial phylotypes of all the diet treatments. Among the 13 phy-
species or genera.
lotypes identified from this group, over 50% of the phylotypes
Molecular characterization of cultured isolates and culture-
were obtained by using culture-independent methods only. On
independent methods identified E. faecalis in all midguts.
average, 51% of phylotypes in each diet treatment were pre-
While the 16S rRNA sequences from these isolates match most
viously reported sequences.
closely to E. faecalis, preliminary analysis revealed that these
Comparison of T-RFLP profiles and 16S rRNA sequences.
isolates differ physiologically (data not shown) from the clinical
To assess the ability of T-RFLP analysis to describe the bac-
isolates of E. faecalis reported previously (21, 52). However,
other E. faecalis isolates from environmental samples consis-
tently diverge from the clinical type strains in certain physio-
TABLE 3. Pairwise comparisons for similarity of T-RFLPs from the
midgut of gypsy moth fed various diets
logical characteristics, including colony color, carbohydrate
metabolism profiles, and growth temperature profiles (26, 47).
Coefficient Coefficient between diet groups Further analysis is required to determine the implications of
Diet within Artificial these differences for species definition within the genus En-
group Larch Aspen Willow
diet terococcus (63).
Artificial diet 0.993 The novelty of phylotypes found in the gypsy moth midguts
Larch 0.962 0.382 was anticipated, based on the insect’s physiology. While the
Aspen 0.975 0.139 0.029 caterpillar lives in a common terrestrial macroenvironment as
Willow 0.968 0.077 0.053 0.083 an external phytophage, the microenvironment in which these
White Oak 0.989 0.045 0.028 0.038 0.091
midgut bacteria reside is chemically extreme. Most insect mid-
VOL. 70, 2004 BACTERIAL DIVERSITY OF GYPSY MOTH MIDGUT 297

FIG. 1. T-RFLP profiles of third-instar midguts of gypsy moth feeding on diverse diets. Each profile is representative of 10 individual larvae. Downloaded from https://journals.asm.org/journal/aem on 13 July 2025 by 2603:7000:d1f0:b800:d5ec:fe3c:4146:e67d.

guts are generally neutral to acidic (pH 4 to 7), while lepidop- gut pH, detoxifying plant allelochemicals, and maintaining the
teran midguts are typically pH 8 to 10. However, values as high midgut microbial community structure. Midgut pH is impor-
as 12.4 have been reported for gypsy moth (5, 22, 29). In tant from the perspective of both the microbes and the insect.
addition, the diverse plants on which this insect feeds contain A predominant member of the midgut microbial community,
an array of allelochemicals, such as phenolics, tannins, and E. faecalis, is commonly found at higher pH (8 to 9) and
terpenoids, many of which are toxic to microorganisms (28). acidifies its environment through its metabolism (45). This
Therefore, this extreme environment may have selected for could confer some advantage to the insect host, as some mi-
unusual phenotypes, resulting in unusual phylotypes. crobial toxins of lepidoptera, such as Bacillus thuringiensis
The microbial midgut inhabitants seem likely to benefit the toxin, are activated by alkaline conditions (75). Thus, E. fae-
insect host, based on observations of other insect systems (7, 9, calis might help protect this insect from B. thuringiensis toxin by
14, 31). Examples of such benefits may include modifying mid- decreasing the midgut pH. Previous work has shown that lar-
298 BRODERICK ET AL. APPL. ENVIRON. MICROBIOL.

vae that are more susceptible to B. thuringiensis toxin have effects on the larvae (69, 70). However, host plant switching
smaller populations of E. faecalis in their midguts (11). Bacte- can alter the performance of gypsy moth parasitoids and could
ria may also contribute to their own survival by managing likewise affect the composition of the bacterial community
midgut pH to enhance their own growth or exclude competing (36). The relative stability of the gut bacterial community is
microorganisms. While numerous studies have investigated mi- also unknown. Further analysis of this system, including iden-
crobial diversity in neutral and acidic environments (6, 24, 27, tification of the source of microbial inoculum in insects fed on
32, 56, 66), few have examined bacteria in highly alkaline sterilized artificial diet, use of methods, such as metagenomics
environments, particularly in nonaquatic ecosystems (25, 30). (59), that link function to phylogeny, and improved culturing
Further analysis of these bacteria may identify adaptations that techniques will advance our understanding of the role of mi-
permit them to function in this extreme environment, and crobes in the gypsy moth midgut.
whether they have a role in maintaining it.
The broad range of phytochemicals consumed by this ACKNOWLEDGMENTS
polyphagous herbivore may present a challenge for both the We thank Frank Martin (USDA-APHIS), Roger Fuester (Beneficial
gypsy moth and its associated bacteria. Toxic compounds may Insects Introduction and Research Laboratory, USDA), Dave Schu-
select for bacteria that can metabolize them, and these bacteria macher (WI-DATCP), and Roger Mech (MI-DNR) for their assis-
tance in obtaining gypsy moth egg masses. We also thank Trenten
may degrade ingested compounds that are otherwise toxic to Marty (WI-DNR) for providing the trees. We are grateful to Madeline
the insect (41). For example, the 16S rRNA gene sequence of Fischer and Angela Kent (University of Wisconsin—Madison) for
one cultured isolate from the gypsy moth (NAB13) was most their assistance with T-RFLP analysis.
similar to a Rhodococcus species, and recent work identified an This work was funded by the Wisconsin Department of Natural
Resources, McIntire-Stennis Projects 4054 and 4529, the Howard
enzyme produced by R. erythropolis DCL14 (73) that degrades Hughes Medical Institute, and Hatch Projects 4038 and 5234 from the
monoterpenes, a widespread class of phytochemicals that can College of Agricultural and Life Sciences at the University of Wiscon-
be toxic to many insect larvae and adults (38). The gypsy moth sin—Madison.
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