Vol. 55, No. 9APPLIED AND ENVIRONMENTAL MICROBIOLOGY. Sept. 1989. p. 2200-22050099-2240/89/092200-06$02.00/0Copyright ©) 1989, American Society for Microbiology

The Cutworm Peridroma saucia (Lepidoptera: Noctuidae) SupportsGrowth and Transport of pBR322-Bearing Bacteria

JOHN L. ARMSTRONG,* L. ARLENE PORTEOUS, AND NATHAN D. WOOD

Terrestrial Microbial EcologylBiotechnology Progr-am, U.S. Envi'ironinental Protection Agency, Corvallis, Oregon 97333

Received 2 February 1989/Accepted 14 June 1989

Variegated cutworms were exposed to bean plants in microcosms sprayed with pBR322-carrying strains ofEnterobacter cloacae, Klebsiella planticola, and Erwinia herbicola. The three bacterial species exhibiteddifferential survival on leaves, in soil, and in guts and fecal pellets (frass) of the insects. High numbers ofEnterobacter cloacae(pBR322) were detected in all samples, while the other species were unable to establishresidence in the insect. To assess the impact of this colonization on site-to-site transport of microorganisms,larvae were fed plants that had been sprayed with the bacteria and then were transferred to uninoculatedplants. Cutworms were efficient carriers of Enterobacter cloacae(pBR322), as indicated by its rapid appearanceon uninoculated leaves and continued persistence in the insects for 3 days after transfer. Few Erwiniaherbicola(pBR322) and K. planticola(pBR322) were obtained from larvae after transfer, although up to 103CFU/g were detected in soil and on plants. Differences in bacterial survival and growth were confirmed byincubating frass overnight and observing the change in population numbers. The proportion of total samplesshowing at least a 25-fold increase during incubation was 68% for Enterobacter cloacae(pBR322), 39% for K.planticola(pBR322), and 0% for Erwinia herbicola(pBR322). Our results emphasize the role that cutworms andpossibly other insects have in persistence and growth of microorganisms in the environment.

The well-known abundance and variety of microorganismsin the natural flora of insect digestive tracts (4, 14, 18) raisequestions about the role insects play in the survival ofmicroorganisms in the environment. The issues are espe-cially timely in view of the surge in development of recom-binant microorganisms destined for field dispersal and thecontroversy about such activities (10, 17). Because largeinsect populations are indigenous to most field sites, it iscertain that nontarget insect species and their associatedmicroflora will be exposed to genetically engineered micro-organisms released into the environment.On the basis of available reports on numerous nonrecom-

binant bacteria and fungi, it is probable that strains releasedin field sites also will establish residence and grow in insects.For example, in one study enterococci were cultured from53% of 403 field-collected insects representing 37 taxa (13).In another study, Enterobacteriac(eae populations in hind-guts of locusts (Sclhistocerca giregar-ia) numbered 3 x 109/mlof gut, while streptococci were 10- to 100-fold less abundant(9). Gut and frass (fecal) samples from grasshoppers (Mel-anoplius sangiuinipes) yielded between 105 and 106 coloniesper mg of sample (15). Of these isolates from guts, 168 werecategorized into 11 bacterial species, including Enterococcutsspp., Serrlatia liquejaciens, Pseiudoinonais spp., and Entelo-bactier spp. Other investigators reported that hindguts offifth-instar larvae of turnip moths (Scotia segetuin) con-tained up to 4 x 105 bacteria, which typically grouped asEnterobacteriaceac and Str-eptococcius ftiecalis, but in-cluded 10 species of fungi and a species of yeast (5). Therecurrence of certain microbial groups in insects has ledsome investigators to propose the existence of "entomoge-nous" bacteria, specific forms that are common in insectsand have close ecological interactions with these host insects(11). However, use of this definition is doubtful since popu-

* Corresponding author.

lations associated with insects often reflect the microflora ofthe outer environment (4). For example, when gnats (Ciili-coides v(lriipennis) were fed Escherichia coli and Serratiaiinarcescens, the bacteria were detected for 8 days afterinsects were transferred to sterile food (16). In feedingexperiments with mayfly larvae (Epheinera danica), theAeromonas hzVdrophila population in the diet declined innumbers as food passed through the gut, while the Flavo-bac teriulm sp. population in the diet increased (3). Theresearchers concluded that there was differential survival ofthese bacteria in mayflies. The decrease in A. hydrophilainumbers reflected preferential digestion, whereas persist-ence and growth of the Flaohacteril,n sp. meant that it hadcolonized the hindgut. Anderson and Bignell (1) observed a10- to 100-fold increase in total bacteria after passage ofbacteria through the guts of millipedes (Glomeri.s inarginatai)and attributed the increase to rapid growth in the midgut. Instudies with variegated cutworms (Per-idromti saucia HRb-ner), other researchers recovered approximately 106 totalbacteria per gut and 105 total bacteria from each frass pelletwhen guts and pellets were plated on a tryptone-yeastextract medium (2). These larvae were then fed plantssprayed with Pseuidomonas cepacia(R388: :Tn 1721), whichcould be identified unambiguously owing to the plasmid-encoded trimethoprim and tetracycline resistance genes.Within 24 h, the investigators observed about 106 Pseuldo-monas cepacia(R388::Tn/721) per gut, while none weredetected in frass.

In this paper, we present microcosm studies with varie-gated cutworm larvae that were fed specific plasmid-bearingbacteria to (i) clarify the potential role of insects as carriersof bacteria, (ii) evaluate the site-to-site transport of bacteriain the environment, and (iii) determine whether cutwormdigestive tracts can provide nutrients for growth of somebacteria. The results emphasize the dynamic interactionsexisting between these insect larvae and the environmentalmicroflora.

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PERIDROMA SA UCIA TRANSPORTS pBR322-CARRYING BACTERIA 2201

MATERIALS AND METHODS

Bacterial strains and culture conditions. Bacteria used wereEnterobacter cloacae (isolated from frass of P. saucia thatwas collected from a local peppermint field), Erii'inia herbi-cola 112Y (S. Beer, Cornell University, Ithaca, N.Y.), andKlebsiella planticola ATCC 33531 (from radish roots; Amer-ican Type Culture Collection, Rockville, Md.). Spontaneousrifampin-resistant (Rif) mutants were transformed (12) withplasmid pBR322, which codes for tetracycline resistance(Tcr). Bacteria were cultured in Luria-Bertani (LB) medium(12) amended with three antibiotics, rifampin (100 p.g/ml),tetracycline (15 p.g/ml), and cycloheximide (25 jig/ml) (SigmaChemical Co., St. Louis, Mo.), to inhibit growth of indige-nous fungi. In preparation for a spray, bacteria were grownin broth for 18 h at 30°C with shaking, washed twice bycentrifugation (10 min at 5,000 x g at 4°C) in sterile water,and suspended at 106 to 108 CFU/ml in sterile water. Toenumerate bacteria, samples were diluted serially in 10 mMTris buffer (pH 7.5), spread on LB agar containing antibiot-ics, and incubated for 20 h at 30°C. Colony numbers frompairs of plates were averaged in calculating CFU.

Plant growth conditions. Thirty bean seeds (cv. Blue LakeBush; Denson Feed & Seed, Corvallis, Oreg.) were plantedin potting mix (1 part Sunshine Mix [Fisons Western Corp.,Vancouver, British Columbia, Canada] to 1 part perlite) in aplastic tray (27 by 55 by 5 cm) lined with a polyethylene bag(2). Plants were raised under 40-W fluorescent lamps (Syl-vania Cool White, F40/CW) on an 18-h-light/6-h-dark cycle.Each tray was fertilized every 48 to 72 h with 1 liter ofphytotron nutrient solution (7), without hampol copper andsequestrene cobalt. Plants were used in an experiment afterthe appearance of the second trifoliate leaves (2 to 3 weeks).Three days before an experiment, trays were placed in amicrocosm chamber to expose plants to the experimentalenvironment. During an experiment, plants were wateredwith distilled water.

Insect growth conditions. Variegated cutworm larvae werereared from eggs on diet 9000 (Bioserve Inc., Frenchtown,N.J.), as described elsewhere (2). Bactericidal chemicals inthe diet were removed from insects prior to experiments byfeeding them fresh plants for 2 days.Microcosm design. Trays containing plants were housed in

a 1.0- by 0.75- by 0.75-m box (8) with a Pyrex glass top andPlexiglas or glass walls, which rested on a polyethylene boxmeasuring 1.0 by 0.75 by 0.55 m. Each chamber wasirradiated with a 1,000-W Sylvania metal halide lamp on an18-h-light/6-h-dark cycle. Air was pumped through the cham-ber and exhausted through a HEPA filter (Astrocel; Ameri-can Air Filter, Louisville, Ky.). Ambient air was maintainedat 22 + 1°C during the dark period and 23 ± 1°C during thelight period. Temperatures within microcosm units were 28to 30°C during the light period and 23°C during the darkperiod. During experiments, trays were placed 50 cm abovethe floor on a rack supported by bricks in the chamber.

Introduction of bacteria into microcosms. Approximately100 ml of a suspension containing 1 x 107 to 5 x 107 bacteriaper ml was sprayed on plants until run-off by using a mistingbottle (rinsed with ethanol, followed by sterile water) in anegative-air-flow hood equipped with an Astrocel HEPAfilter. The plants were then transported to microcosm cham-bers. After each experiment, contents of trays were auto-claved for 1.25 h, and the inner surfaces of microcosm boxeswere disinfected.

Introduction of insects into microcosms. Within 2 h afterspraying, fourth- or early fifth-instar cutworms were placed

on plants. Polyethylene bags were fastened near the top ofwire frames (2) with clamps to prevent escape of insects. Instudies of transport of bacteria by cutworms, larvae were fedspray-inoculated bean plants for 1 day, collected with for-ceps, and transferred to a microcosm containing uninocu-lated plants.Sample collection and processing. Two types of experi-

ments were performed to study the appearance and persis-tence of bacteria associated with insects. Survival experi-ments consisted of microcosms sprayed with bacteria andsampled for bacterium populations on plants, in soil, and incutworm foreguts and frass. During these experiments,samples were collected on days 0 (3 to 4 h after spraying), 1[exception: samples were not collected for Enterobactercloacae(pBR322) on day 1], 3, 7, and 10. Experiments werelimited to 10 days so that larvae could be collected beforepupation. In site-to-site transfer experiments, cutwormswere fed sprayed plants for 24 h and then transferred tounsprayed bean plants. During these experiments, sampleswere collected on days 0, 1, 2, and 3 after the transfer.

In both types of experiments, plant samples (weighingabout 1.5 to 3.5 g) consisted of three bean leaves: onecotyledon, one first trifoliate leaf, and one second trifoliateleaf. Leaves were collected aseptically at random, placed insterile plastic bags, and blended for 1 min in 12.5 ml of 10mM Tris buffer (pH 7.5) in a Stomacher blender (model 80;Tekmar Co., Cincinnati, Ohio). Random soil samples(weighing about 0.4 to 1.0 g) were collected aseptically witha spatula from the soil surface. These samples were vortexedfor 1 min in 18- by 150-mm screw-cap tubes containing 5 mlof 10 mM Tris buffer (pH 7.5) and five glass beads (5 mm indiameter). To obtain a larval gut, we aseptically removed thehead with scissors and used forceps to pull out both the largeforegut and the attached thinner, mid-hindgut sections. Gutsamples were not used if there was evidence of ecdysis (i.e.,absence of leaf matter in the foregut). In the survivalexperiments, two guts (each weighing 0.1 to 0.4 g) werepooled and vortexed for 1 min in an 18- by 150-mm screw-cap tube containing five glass beads (5 mm in diameter) and2.5 ml of 10 mM Tris buffer (pH 7.5). In the transferexperiments, each sample consisted of a single gut. Frasssamples were collected aseptically with forceps as the pelletswere excreted by insects. Two pellets (each weighing 5 to 50mg) were placed in a 1.5-ml Microfuge tube, weighed,dispersed for 30 s with a Pellet Pestle (Kontes, Vineland,N.J.) in 0.5 ml of 10 mM Tris buffer (pH 7.5), and vortexedfor 30 s. At least five isolated colonies cultured from eachsample were identified with API 20E strips (Analytab Prod-ucts, Plainview, N.Y.). The presence of pBR322 was verifiedby alkaline (pH 12.6) lysis extraction of DNA (6), followedby electrophoresis (12).Regrowth studies. Bean leaves were sprayed as described

above and fed to larvae for 20 h. Frass samples were

collected aseptically as they were excreted by larvae. Pelletswere then cut in half, and both pieces were weighed. Initialbacterial numbers were obtained by processing one-half ofthe pellet and plating the suspension on selective media as

described above. The other half of each pellet was placed on

a freshly cut, uninoculated bean leaf in a petri dish contain-ing water-soaked Whatman IM paper. The dishes were

sealed with Parafilm. After 20 h at 30°C, the frass pelletswere removed from the leaves and processed as describedabove to obtain final bacterial numbers after incubation.

Data analysis. Bacterial numbers were transformed tologarithm (log) CFU per gram (fresh weight) of sample(leaves, soil, guts, and frass). When bacteria were detected

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2202 ARMSTRONG ET AL.

in all replicate samples collected on a given day, means werecalculated by using data from all samples. Bars about meansrepresent standard errors of means. If bacteria were notdetected in all replicates on a given day, the mean wascalculated by using data from only those replicates whichwere positive for bacteria. When no bacteria were detectedin any of the replicates, the mean of the detection thresholdwas plotted. Means based on less than all replicates areflagged in the graphs, with the ratio of the number of samplesin which bacteria were detected over the total number ofsamples analyzed.

Survival and site-to-site transfer experiments were per-formed twice. To test for significant (P < 0.05) differencesbetween replicate studies of leaf and soil samples, linearmodels [log CFU = a + (b x day); a = y intercept, b =slope] with different slopes and intercepts were fit to eachexperiment. t tests for differences between the slopes fromthe two experiments were then calculated. Next, a linearmodel with a common slope but different y intercepts was fitto the two experiments for each species. We used t tests toidentify differences among the slopes calculated for the threespecies.

RESULTS

Survival of bacteria on foliage and in soil in the presence ofcutworms. The microcosm methods were used to studyingestion and survival of plasmid-bearing bacteria in herbiv-orous insects feeding on bean plants spray inoculated withthe bacteria. Numbers of bacteria in the leaf canopy and insoil were tracked over the duration of the experiments. BothEnterobacter cloacae(pBR322) and Erwinia herbicola(pBR322) appeared to persist in high numbers on leaves (Fig.1A), while K. planticola(pBR322) numbers dropped about10,000-fold. K. planticola(pBR322) survived better in soilthan it did on leaves, showing a 10-fold drop in numbers byday 10 (Fig. 1B). Erwinia herbicola(pBR322) survived betteron leaves than in soil.

Figure 2 illustrates the appearance of bacteria in guts andfrass pellets of insects after the spray at the beginning of theexperiment shown in Fig. 1. High numbers of Enterobactercloacae(pBR322) were detected in these samples on the thirdday after spraying: 107 CFU/g of gut and 6 x 108 CFU/g offrass. Populations of this organism were consistently large inall samples that were collected during the experiment. Thiscontrasted markedly with Erwinia herbicola(pBR322), whichsurvived poorly in larval digestive tracts and feces. Forexample, on the third day after the spray, Erwiniaherbicola(pBR322) was present at 4 x 10' CFU/g of foliage(Fig. 1A) and 105 CFU/g of soil (Fig. iB), when an average

of only 37 CFU/g were detected in two of three gut samplesand none were detected in three frass samples (threshold ofdetection = 93 CFU/g). Furthermore, on days 7 and 10,Erwinia herbicola(pBR322) was present in one of three andzero of three gut samples, respectively, while none wereobserved in frass. When all data for both experimentalreplicates were pooled, this organism was detected in six ofthe 24 gut samples (trial 1, 3/12; trial 2, 3/12). It was alsopresent in 8 of 24 frass samples (trial 1, 2/12; trial 2, 6/12). K.planticola(pBR322) populations in guts and frass tended tooccur at levels intermediate to those of Enterobacitercloacae(pBR322) and Erwinia herbicola(pBR322). K.planticola(pBR322) occurred in 13 of 24 gut samples (trial 1,5/12; trial 2, 8/12) and in 16 of 24 frass samples (trial 1, 7/12;trial 2, 9/12). Identities of isolates from plant, soil, gut, andfrass samples were confirmed by testing isolated colonies

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FIG. 1. Survival of pBR322-carrying bacteria on leaves (A) andin soil (B). Separate microcosms containing bean plants weresprayed with suspensions of each bacterial species, followed byintroduction of cutworm larvae. Symbols indicate means of the logCFU per gram that were obtained from three foliage or soil samplescollected on each sampling date. Bars indicate standard errors aboutmeans.

with API 20E analytical strips. Also, pBR322 was detectedin all isolates identified.

Results of statistical calculations showed no significantdifferences between the two replicate experiments withEnterobacter cloacae(pBR322) on plants, in soil, in guts, orin frass. Likewise, there were no differences between exper-iments with Erwinia herbicola(pBR322) on plants or in soil.Results of experiments with K. planticola(pBR322) popula-tions in soil did not differ. However, there was a significantdifference between the replicate studies of K. planticola(pBR322) on plants (slope, standard error: trial 1, -0.35,0.05; trial 2, -0.27, 0.04; P = 0.0005), although the overalltrend was similar for both experiments. Since Erwiniaherbicola(pBR322) and K. planticola(pBR322) were not al-ways detected in gut and frass samples, the data sets wereincomplete and slopes were not compared.Data from each pair of replicate experiments were pooled

to calculate slopes for intergeneric comparisons of die-off onplants [slope, standard error: E. cloacae(pBR322), -0.11,0.059; Erwinia herbicola(pBR322), -0.057, 0.036; K.planticola(pBR322), -0.31, 0.051]. No significant differ-ences in slopes were noted between Enterobactercloacae(pBR322) and Erwinia herbicola(pBR322). Althoughthere appeared to be a qualitative difference in slopesbetween Enterobacter cloacae(pBR322) and K. planticola(pBR322) (Fig. 1), the difference could not be demonstratedwith linear modeling of the data. However, Erwiniaherbicola(pBR322) and K. planticola(pBR322) had signifi-cantly different slopes (P = 0.004). In soil, estimates of

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PERII)ROMIA SA UCIA TRANSPORTS pBR322-CARRYING BACTERIA 2203

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FIG. 2. Survival of pBR322-carrying bacteria in cutworm guts(A) and in frass (B). Separate microcosms containing bean plants.were sprayed with suspensions of each bacterial species. followedby introduction of cutworm larvae. Symbols indicate means of thelog CFU per gram that were obtained from three gut or frass samplescollected on each sampling date. Fractions indicate the number ofsamples in which bacteria were detected over the total number ofsamples analyzed. When this ratio is 0/3. the mean of the detectionthreshold is plotted. Bars indicate standard errors about means.

slopes for the three species did not differ significantly [slope.standard error: Enzterobcutcer (/ot(w(i(pBR322). -0.087,0.067; Erwinia herbi(olI(pBR322), -0.24, 0.055; K. pl(/liti-(ola(pBR322), -0.18, 0.038].

Site-to-site transfer of bacteria associated with cutworms.Microcosms also were used to assess the role of cutworms ascarriers of bacteria. Larvae were fed inoculated foliage for24 h and then transferred to uninoculated plants. Figure 3shows the population levels of the three bacterial species inguts, on leaves, and in soil on the day of transfer and on thefollowing 3 days. As expected, when insects were fed leavesinoculated with approximately 107 to 108 CFU/g, they ac-quired about 106 CFU of Enterobacer (/louUae(pBR322) perg of guts that were transported to the receiving microcosm(Fig. 3A). As shown in Fig. 3B, more than 103 CFU ofEnterobhacter Ioa(cae(pBR322) were detected in each gramof foliage on day 1, increasing to 5 x 104/g by day 3. Thepopulation in soil reached about 106 CFU/g by day 3. Nosignificant differences were noted between replicate experi-ments involving Enterobaucter (c1oa(wae( pBR322) on leaves, insoil, or in guts. Since Erwinial1therbi(olai(pBR322) and K.plaintimola(pBR322) were detected infrequently in plant andgut samples, data from replicate experiments with thesebacteria were not fit to linear models for statistical compar-isons of slopes. Instead, the results are summarized in Table1, which permits qualitative comparisons of efficiency ofuptake and transfer of bacteria in the replicates. For exam-ple, in both experiments using Enterobatcwr (cloucae

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FIG. 3. Site-to-site transfer of pBR322-carrying bacteria associ-cited with cutworms during trial 1. Separate microcosms weresprayed with suspensions of each bacterial species. followed byintroduction of cutworm larvae. After one day, larvae were trans-ferred to microcosms containing unsprayed plants. Symbols indicatemeans of the log CFU per gram that were obtained from six gut (A),three leaf (B). or three soil saimples (C) collected on each samplingdate. Fractions indicate the number of samples in which bacteriawere detected over the total number of samples analyzed. When thisratio is 0/3 or 0/6. the mean of the detection threshold is plotted.Bars indicate standard errors about means.

(pBR322), the organism was present in all gut samplesimmediately after transfer on day 0, and in all gut, plant, andsoil samples on days 1, 2, and 3 after transfer. However, it isapparent that Er)vinia herbitola(pBR322) and K.pJlnlti(o0la(pBR322) did not survive ingestion and transportto plants as effectively as Enterobacter (I1oa-cae(pBR322).On the day of transfer, 8 of a total of 12 gut samples fromboth experiments contained Erwinisiia herbic ola(pBR322),while K. planthiola(pBR322) was observed in only 1 of 12 gutsamples. Numbers of Erwvinia herhicola(pBR322) depositedon leaves after the transfer were also low. This species wasdetected in 7 of the 18 plant samples, but K. planiti-

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TABLE 1. Transport of pBR322-carrying bacteria associatedwith cutworms after transfer to unsprayed bean plants

No. of samples withorganism/total no.'

Test organism Trial Guts oi.geSlTrilo ut Foliage Soil

Day Days (days (davs0 1-3 1-3) 1-3)

Enterohacter (loacae(pBR322) 1 6/6 18/18 9/9 9/92 6/6 18/18 9/9 9/9

Erivinia herbicolo(pBR322) 1 4/6 5/18 4/9 9/92 4/6 4/18 3/9 9/9

K. planticola(pBR322) 1 1/6 0/18 0/9 8/92 0/6 2/18 0/9 7/9

" Results from each trial are tabulated as fraction of total samples contain-ing the test organism on day 0 or on days 1 to 3 after transfer. Cutworm larvaewere fed for 1 day on bean plants spratyed with one of three test organisms.Larvae were then transferred to unsprayed plants. Bacteria in gut. foliage. andsoil samples were enumerated on days 0, 1, 2, and 3.

kola(pBR322) was not seen in any plant samples (thresholdof detection, ca. 30 CFU/g). In contrast to the low numbersof these two species on leaves, there were detectable num-bers in most soil samples taken during the three days aftertransfer (Fig. 3C and Table 1).Regrowth of bacteria in insect frass. Differential survival of

bacteria in cutworm guts (Fig. 2A) suggested that lowrecovery of Erwinia herbicola(pBR322) and K. planticola(pBR322) from frass (Fig. 2b) could have been due toselection against them in the digestive tract prior to expul-sion of frass. We therefore determined whether frass pelletscould support growth of the strains by comparing the initialnumbers of CFU per gram with the numbers after 20 h ofincubation at 30°C. In 34% of 29 samples (Fig. 4), Enttero-bacter cloacae(pBR322) numbers increased more than 500-fold, with an initial mean log (CFU/g) equal to 5.8 and a

postincubation value of 7.9. In three samples, the changewas over 6,000-fold. This species decreased in numbers in

only one sample. Changes in K. planticola(pBR322) popula-

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FIG. 4. Final numbers of bacteria in frass after 20h of incubation

at 30'C as compared with initial numbers detected immediately after

sampling. The mean log CFU per gram was calculated by using all

samples containing bacteria before or after the incubation period.Abbreviations: EC, Enterobac-ter- cIowace(pB R3221) KP. K. ploi,tti-c-ola(pBR322); ER, Erwinia Iwhebiola(pBR322). None detected

means that isolates of the three species were not detected inenumerations for initial or final populations.

tions were not as extreme. In 15% of the samples, numbersof these bacteria increased over 500-fold, while none wererecovered from 21% of the samples either before or after theincubation. For Er/vinia Iherbicola(pBR322), 21% of thesamples exhibited an increase of only 25-fold or less, whilethe organism was not detected in 59% of the samples.

DISCUSSION

Our results substantiate those of others who observedingestion and differential survival and die-off of bacteria ininsect guts (1-4, 9). However, our use of antibiotic resis-tance markers and a specific plasmid offered a degree ofcertainty in isolate identification that was not possible in theother studies. When variegated cutworms were fed beanplants sprayed with pBR322-carrying bacteria, we recoveredthese bacteria from the gut contents and frass of the insects.Statistical comparisons of slopes generated by linear model-ing of the survival experiment results, as well as of theresults of site-to-site transfer experiments, were useful inshowing good reproducibility of the replicates of experi-ments. However, the statistical analyses generally failed toreveal differences in persistence of the three species in soiland on leaves. One exception was the significantly fasterdie-off of K. planticola(pBR322) on leaves compared withthat of Erit'inia herbicola(pBR322) on leaves. Differencesamong species were more obvious in comparisons of bacte-ria on leaves and in soil with those in cutworm digestivetracts (Fig. 1 and 2). Enterobacter cloalcae(pBR322) exhib-ited a high degree of survival in guts and frass, showing100-fold higher counts in the latter. Since this strain wasinitially isolated from frass of field-collected cutworms,these results were not unexpected. Its prolonged persistencein soil also reflects its natural compatibility with this envi-ronment. However, its marked adaptability to bean foliagesuggests that it possesses properties favoring persistence ina wide variety of environments. In contrast, both Erwiniaherbicola(pBR322) and K. planticola(pBR322) survived rel-atively well in soil and on plants but poorly in guts and frass.For example, when populations of the epiphytic organismErivinia herbicola(pBR322) were still >10' CFU/g of leaf,they were near experimental detection limits in gut and frasssamples. Levels of the soil organism K. planticola(pBR322)decreased sevenfold in soil by day 7, while populations onleaves dropped 10,000-fold. K. plannticola(pBR322) numbersin guts and frass reached 10' to 104 CFU/g on day 1, whenpopulations on leaves and in soil were still large, butdecreased to near-threshold levels by day 3. This could havebeen due to digestion of gut contents and/or replacement ofgut contents with freshly ingested leaf matter carrying pro-gressively lower numbers of K. planticola(pBR322). Therewas no evidence that the strain could establish residence inthe gut. This suggested that these bacteria were digested orselected against through competition with normal flora orthat they failed to grow under conditions prevailing in thegut.The site-to-site transfer experiments showed that large

numbers of bacteria could be introduced into an unsprayedenvironment by cutworms fed inoculated foliage (Fig. 3). Asanticipated from results of the ingestion and survival exper-iments described earlier, the efficiency of transport into andcolonization of the new environment differed among thethree species. The effective contamination of unsprayedleaves and soil by cutworms carrying Enterobacterc/loaca((pBR322) reaffirms the efficiency of this microorgan-ism in surviving and proliferating. This organism contrasted

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PERII)ROMA SAUCIA TRANSPORTS pBR322-CARRYING BACTERIA 22)05

with both K. planticola(pBR322) and Erwinia herhicola(pBR322), whose numbers on leaves were always low aftertransfer, although they did rise to 103 CFU/g in soil. Theseobservations support our contention that these two bacterialspecies do not have a selective advantage within the insect.The poor survival of K. planticola(pBR322) on leaves prob-ably contributes to its low numbers after transfer of theinsects. The possibility that the lower numbers of Ekroiniuiherbicola(pBR322) and K. planticola(pBR322) on leaves wasdue to minimal activity of larvae over leaf surfaces isunlikely, since there was evidence of cutworm activity onleaves (i.e., ragged edges and frass pellets). Also, the rapidincrease of Entcerobacter cloacae(pBR322) populations onleaves meant that larvae were active on leaf surfaces duringthese experiments.

Surface sterilization of insects has been a standard prac-tice for studying bacteria in the gut. We chose to useuntreated larvae to avoid inadvertent killing of gut bacteria.Thus, it was possible that bacteria used in the spray inocu-lum could enter gut and frass samples as a result of contam-ination from surface populations during sample collection.Tests indicate that surface contamination is minimal. Forexample, Pseiidloinonas cepacli(R388: :Tnl 721) was sprayedinto microcosms and the insects were collected immediately,before they could feed (2). Whole insects had 2 x 104 timesmore Pseuidomonais (epacia(R388::Tn1721) than gut satm-ples had, and none of the sprayed bacteria were detected infrass samples. Since we did not surface sterilize larvae, it isuncertain how many bacteria were transported via gutcontents versus transport via the surface of the insect.However, regardless of the mode of transfer, the resultsillustrate the active role of cutworms as carriers of microor-ganisms.The studies on bacterial growth in individual frass pellets

were performed to determine whether the three bacterialspecies could grow in frass in the absence of complicatingfactors such as reinoculation of gut contents through contin-ued ingestion of contaminated foliage. Both Enterobactercloacae(pBR322) and K. planticola(pBR322) exhibited amarked ability to grow in frass. This result contrasted withthe ability of Erwinia herbicola(pBR322), which showedpoor survival in fecal pellets. Thus, insect frass can serve asa nutritive environment for some bacteria, thereby facilitat-ing their survival in the field.We suggest that all these methods are useful as test

systems to study survival of recombinant bacteria prior totheir introduction into the environment. For example, aspecific recombinant strain would be sprayed in microcosmscontaining representative plants and soil from the intendedsite of dispersal. A variety of insects indigenous to the sitealso would be introduced. If the recombinant organism is anefficient colonizer, such as Enterobacter cloalcae, furtherconsiderations concerning its longevity in the field may bewarranted before release. Also, the ability of a microorgan-ism to proliferate in frass after deposition of a pellet points tothe potential for opportunism within favorable environmentsthat stimulate bursts in population size. Finally, comparativesurvival studies of an organism in terrestrial microcosms

would be informative for an overall evaluation of the abilityof a species to persist in the field.

ACKNOWLEDGMENT

We thank Dave Zirkle for consulting on the statistical analyses.

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gut contents and faeces of the litter-feeding millipede Glonerisma1Cir'gitaitlI (Villers). Soil Biol. Biochem. 12:251-254.

2. Armstrong, J. L., G. R. Knudsen, and R. J. Seidler. 1987.Microcosm method to assess survival of recombinant bacteriaassociated with plants and herbivorous insects. Curr. Microbiol.15:229-232.

3. Austin, D. A., and J. H. Baker. 1988. Fate of bacteria ingestedby larvae of the freshwater mayfly. Eplcwnelwrai- danica. Microb.Ecol. 15:323-332.

4. Brooks, M. A. 1963. The microorganisms of healthy insects, p.215-250. In E. A. Steinhaus (ed.). Insect pathology, vol. 1.Academic Press, Inc.. N.Y.

5. Charpentier, R., B. Charpentier, and 0. Zethner. 1978. Thebacterial flora of the midgut of two Danish populations ofhealthy fifth instar larvae of the turnip moth. Scotia segeulntil. J.Invert. Pathol. 32:59-63.

6. Crosa, J., and S. Falkow. 1981. Plasmids. p. 266-282. In P.Gerhardt. R. G. E. Murray. R. H. Costilow. E. W. Nester,W. A. Wood. N. R. Krieg, and G. B. Phillips (ed.), Manuail ofmethods for general bacteriology. American Society for Micro-biology. Washington. D.C.

7. Downs, R. H., and H. Hellmers. 1975. Environment and theexperimental control of plant growth. Academic Press. Inc..N.Y.

8. Gile, J. D. 1983. 2.4-D-Its distribution and effects in a ryegrassecosystem. J. Environ. Quality 12:406-412.

9. Hunt, J., and A. K. Charnley. 1981. Abundance and distributionof the gut flora of the desert locust, Schistocerca gregaria. J.Invert. Pathol. 38:378-385.

10. Liang, L. N., J. Sinclair, L. Mallory, and M. Alexander. 1982.Fate in model ecosystems of microbial species of potential usein genetic engineering. Appl. Environ. Microbiol. 44:708-714.

11. Lvsenko, 0. 1963. The taxonomy of entomogenous bacteria, p.1-20. In E. A. Steinhaus (ed.), Insect pathology, vol. 2. Aca-demic Press. Inc.. N.Y.

12. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory.Cold Spring Harbor, N.Y.

13. Martin, J. D., and J. 0. Mundt. 1972. Enterococci in insects.AppI. Microbiol. 24:575-580.

14. Martin, M. M., and J. J. Kukor. 1984. Role of mycophagy andbacteriophagy in invertebrate nutrition, p. 257-263. In M. J.Klug and C. A. Reddy (ed.), Current perspectives in microbialecology. American Society for Microbiology. Washington, D.C.

15. Mead, L. J., G. G. Khachatourians, and G. A. Jones. 1988.Microbial ecology of the gut in laboratory stocks of the migra-tory grasshopper. Melinopli.s sai,guiinipes (Fab. ) (Orthoptera:Acrididae). Appl. Environ. Microbiol. 54:1174-1181.

16. Parker, M. D., D. H. Akev, and L. H. Lauerman. 1978.Persistence of enterobacteriaceae in female adults of the bitinggnat Cilicoidev.s 'air-iipenilnis. J. Med. Entomol. 14:597-598.

17. Rissler, J. F. 1984. Research needs for biotic environmentaleffects of genetically engineered microorganisms. Recomb.DNA Tech. Bull. 7:20-30.

18. Steinhaus, E. A. 1941. A study of the bacteria associated withthirty species of insects. J. Bacteriol. 42:757-790.

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