APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1993, p. 1904-19100099-2240/93/061904-07$02.00/0Copyright X) 1993, American Society for Microbiology

Presence of Culturable Bacteria in Cocoons of theEarthworm Eisenia fetidat

JOSEPH E. ZACHMANN AND J. A. E. MOLINA*Department of Soil Sciences, University of Minnesota, St. Paul, Minnesota 55108

Received 12 November 1992/Accepted 1 April 1993

Viable bacteria were found to coexist with developing embryos in egg capsules (cocoons) of the earthwormEiseniafetida. Earthworms were reared under standardized conditions, and bacterial densities were measuredin distinct batches of cocoons collected weekly for 10 weeks. Cocoons weighing 12 mg contained a mean viablebacterial population of approximately 108 CFU/g of cocoons. No difference was found in viable counts obtainedfrom cocoons incubated at 15°C and cocoons incubated at 24°C. Viable bacterial numbers increased withcocoon age, while acridine orange direct counts of microbial cells were stable at approximately 109 cells per gof cocoons. Bacteria isolated from cocoons were used to develop antisera in rabbits for the production ofstrain-specific fluorescent antibodies. Fluorescent antibody and selective plating techniques were used tomonitor populations of these bacteria in earthworm bedding and to determine whether cocoons acquirebacteria from the environment in which they are formed. Cocoon isolates were readily recovered from cocoonsformed in inoculated bedding at densities of 108 CFU/g of cocoons. Bradyrhizobiumjaponicum USDA 110 andUMR 161 added to bedding were also recovered from cocoons, but at lower densities than cocoon isolates.Escherichia coli K-12(pJP4) inoculum was recovered from bedding but not from cocoons. The bacterialcomplement ofEiseniafetida cocoons is affected by inoculation of selected bacterial isolates in the worm growthenvironment.

Earthworms belong to a class of hermaphroditic inverte-brates which produce egg capsules (commonly called co-

coons) external to the body. Cocoons are the product ofspecialized epithelial cells and are shed from the anteriorportion of the worm after passing over the mouth. Embryosdevelop in a nutritive and protective fluid inside the cocoon,and hatchlings are sexually immature juveniles.Earthworms live in direct contact with and ingest a variety

of microorganisms found in soil and decomposing organicmatter (17). Symbiotic and synergistic relationships betweenearthworms and microorganisms have been postulated; how-ever, most investigations have been limited to specific mi-crobial species and their populations or fate in the earth-worm gut (17, 28) or their presence in earthworm nephridia(5).

Studies of earthworm physiology have included character-ization of gut enzymes (16, 24), yet the source and degrada-tive capability of the enzymes are debatable. The possibilitythat some enzymes, especially cellulases, might be of micro-bial origin has been considered previously (10, 15, 31).Axenic earthworms for use in physiological or ecologicalexperiments can be tedious to rear (12, 22, 35), and theresistance of many microorganisms to laboratory cultureand antibiotic treatment makes asepsis difficult to substanti-ate.

Microbial associations with earthworm organs, such as

nephridia, may be established through intergenerationaltransfer of putative symbionts via earthworm cocoons; how-ever, experiments supporting such hypotheses have notbeen conducted (23). The nature and function of observedassociations with nephridia and the manner in which bacteriacome to occupy nephridia have yet to be determined.

* Corresponding author.t Minnesota Agricultural Experimental Station Scientific Journal

Series paper no. 20510.

Incubation of cocoons at a high temperature led to de-creases in cocoon viability and the number of juvenilesemerging at hatching (32). Because earthworms, especiallymanure feeders, encounter a broad range of bacteria, andbecause bacteria have been noted in otherwise normalcocoons (23, 36), possible explanations for observed cocoon

mortality include microbial degradation of the nutritivecocoon fluid. Moreover, higher incubation temperaturesmay foster the growth of bacteria pathogenic to earthwormembryos.The earthworm Eisenia fetida significantly hastens decom-

position of organic matter and rapidly increases its weight ondiets high in cellulose (e.g., forest litter and crop residues),manures, and microorganisms (11, 19). Eisenia fetida hasbeen employed as a biological indicator of organic matterstabilization (13, 37) and environmental contamination byxenobiotic compounds (6, 19, 20). In order to better under-stand the role of earthworms in nature and their potential as

organic waste stabilizers and indicators of soil health, earth-worm-microbe interactions must be rigorously defined andquantified.For our studies, the cocoon was used as a focal point in an

investigation of earthworm microbial ecology. More infor-mation on cocoon formation and characteristics has beengiven by Laverack (16), Lee (17), and Valembois et al. (33).Externally sterile cocoons could be obtained by using themethods of Miles (18) and Rouelle (27), but it was discoveredthat cocoons of Eisenia fetida contained a diverse comple-ment of microbial phenotypes internal to the pliable chitin-like shell in which the embryos mature.

In this report, we describe the enumeration of bacteriapresent in maturing Eisenia fetida cocoons. The use ofimmunofluorescence and antibiotic-marking techniques todocument the appearance in cocoons of selected bacteriaadded to worm-breeding microcosms is also described.

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BACTERIA IN EISENUA FETIDA COCOONS 1905

MATERIALS AND METHODS

Bacterial strains and sources. Cocoon bacterial strainsCCO01, CCO03, and CCO05, used in bedding inoculationexperiments, are gram-negative, rod-shaped isolates ob-tained from spread plates containing the albumen-like fluidof 12- to 19-day-old Eisenia fetida cocoons. All operationswere conducted aseptically. Viable cocoons were washedand embedded in molten (52°C) 6% agar so that one side ofthe cocoon remained unsubmerged after the agar hardened.A solution containing 1% bleach (0.05% sodium hypochlo-rite) and 0.7% Triton X-100 was swabbed over each exposedcocoon surface for 1 min. A sterile swab was used to dry thecocoon, and an inoculating loop was scraped over thecocoon repeatedly. Loose cocoon scrapings were trans-ferred to culture plates as a check for surface asepsis. Asmall, longitudinal incision was made with a scalpel, andcocoon fluid was removed with a 5-,ul microcapillary tube(Drummond). The cocoon fluid was serially diluted 10-fold insterile, filtered (pore size, 0.45 ,um) phosphate-bufferedsaline (PBS), 100-,ul portions of the suspension were spreadon duplicate plates, and the preparations were incubated at28°C to obtain individual colonies. The three isolates se-lected were chosen at random from more than 50 colonytypes observed in 10 different cocoon trials. A total of 22additional isolates were collected for fluorescent-antibody(FA) cross-reactivity tests. CCO01 and CCO05 exhibitedinherent resistance to kanamycin sulfate. CC101 and CC105are spontaneous streptomycin-resistant mutants of CCO01and CCO05, respectively.Bradyrhizobium japonicum USDA 110 (culture and ho-

mologous FA) and Escherichia coli K-12(pJP4) were ob-tained from E. L. Schmidt. Escherichia coli K-12(pJP4) isresistant to Hg2e. Bradyrhizobium japonicum UMR 161(culture and homologous FA) was obtained from P. Graham.

Bacterial media and culture maintenance. Cocoon bacteriawere originally isolated on solid (1.5% agar) soil-yeast ex-tract (SY) medium (pH 6.5), which contained 3 g of yeastextract (Difco) per liter of soil extract. The soil extract wasmade from 600 g of air-dried Waukegan silt loam and 1,100ml of distilled water autoclaved for 21 min at 121°C at a

pressure of 15 lb/in2 and allowed to settle for 16 h; thesupernatant was removed and filtered twice (Whatman no. 1filter) in a Buchner funnel. SY medium was used for generalenumeration experiments and asepsis checks.For recovery studies, bacteria (strains CC001, CC101, and

CC105) in cocoons and inoculated bedding were recoveredon solid N-2-hydroxyethylpiperazine-N-2-ethanesulfonicacid (HEPES)-2-(N-morpholino)ethanesulfonic acid (MES)salts medium (8) amended with 0.1% glucose and 0.1% yeastextract (HMGY). Filter-sterilized antibiotics (Sigma Chem-ical Co., St. Louis, Mo.) were added to selective media atthe following concentrations: streptomycin, 1,000 ,ug/ml;kanamycin, 30 ,ug/ml; and cycloheximide, 100 ,ug/ml (tosuppress fungal growth during isolation of bacteria frombedding and cocoons). Escherichia coli was recovered on

MacConkey medium (Difco) amended with 30 Fg of Hg2+per ml. All bacteria were cultured at 28°C and were main-tained as frozen stocks at -80°C in 50% sterile glycerin.Cocoon production. For the initial isolation of cocoon

bacteria and the subsequent cocoon bacterial populationexperiments, 45 adult Eisenia fetida specimens (weight ofeach, 800 to 900 mg) were maintained at 24°C in a ventilated,covered, styrofoam box containing bedding consisting of 150g of nonsterile Canadian sphagnum peat moss (Magic Prod-ucts, Amherst Junction, Wis.) mixed with 20 g of instant

oatmeal and 300 ml of distilled water. After 7 days, theworms were removed, washed in distilled water, and trans-ferred to fresh bedding to begin a 7-day cocoon productionperiod. Cocoons were collected on day 7 from spent bed-ding, washed under running tap water, rinsed in distilledwater, and incubated at 24°C for 12 days on water-saturatedfilter paper in petri dishes. The incubation period and con-ditions yielded cocoons 12 to 19 days old and 3 to 5 daysshort of hatching. In some experiments, some of the cocoons(selected at random from the total population) were left tohatch after the 12-day incubation period. Results are re-ported below as numbers of cocoons collected and tested forhatching and percentages of hatching and juveniles percocoon. The remaining cocoons were examined for viability,weighed, and used for bacterial enumeration.

Enumeration of bacteria in cocoons. Except where notedbelow, all cocoons analyzed for bacterial numbers containedviable embryos or juveniles, as determined by stereoscopicobservation at a magnification of x3. Viability was deter-mined by observing embryonic growth and motion or byobserving circulating blood in the juveniles through thetransparent cocoon walls. After 12 days of incubation,cocoons to be used for bacterial enumeration were cleanedwith a cotton swab and washed under tap water. Thecocoons were then surface sterilized by vortexing them in asterile 50-ml screw-cap tube for 1 min in 20 ml of a solutioncontaining 1% bleach and 0.7% Triton X-100 (Sigma). Thesupematant was decanted, and the cocoons were rinsed infour 20-ml aliquots of sterile, filtered (pore size, 0.45 ,um),distilled water, with vortexing for 30 s for each rinse. Thefinal rinse preparations were preserved in 4% formaldehyde(vol/vol) for FA and acridine orange (AO) analyses as acheck for sterility. The cocoons were also checked forsterility by rolling them on a clean SY medium plate. Thecocoons were then pooled, placed in a single, sterile plasticbag, weighed, and homogenized in 5 ml of sterile, filtered(pore size, 0.45 Am) PBS for 3 min with a Colworth-Stomacher apparatus (model STO-80; Tekmar Co., Cincin-nati, Ohio). Twofold serial dilutions of the homogenate werespread plated in duplicate on SY medium, and the prepara-tions were incubated at 28°C and examined after 7 and 14days to determine the number of colony-forming units pergram (fresh weight) of cocoons. Results are given below aslog1o colony-forming units per gram of cocoons. The remain-ing homogenate was preserved with 4% (vol/vol) formalde-hyde, stored at 4°C, and analyzed within 1 week by using FAor AO techniques.To enumerate bacteria from individual cocoons, the co-

coons were checked for viability and number of juveniles,weighed, and processed as described above in 5 ml of PBS.Temperature and time effects on cocoon bacterial popula-

tions. To test whether cocoon incubation temperature af-fected the number of viable bacteria in cocoons, 40 cocoonsfrom one 7-day production period were divided into twogroups. Twenty cocoons were incubated and monitored forviability as described above. The remaining cocoons wereincubated at 15°C for 26 days before selection, homogeniza-tion, and plating. Spread plates were incubated at 28°C.To test whether the length of time that cocoons were

incubated affected the numbers of culturable bacteria, co-coons from one 7-day production period were divided intothree groups by age: cocoons to be incubated for 3 to 10days, cocoons to be incubated for 8 to 15 days, and cocoons

to be incubated for 12 to 19 days. Each cocoon was pro-cessed and plated in duplicate on half-strength nutrient agar(Difco). Homogenates of individual cocoons were further

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analyzed by using the AO technique to evaluate the total cellcounts per gram of cocoons.

Recovery of inoculated bacteria from bedding and cocoons.To determine whether cocoons acquire bacteria from thegrowth environment in which the worms mate, adult wormswere incubated at 24°C for 7 days in 250-ml glass jarscontaining 16 g of sieved (pore size, 2 mm) peat moss, 4 g ofAvicel (microcrystalline cellulose; FMC Corp.), and 40 ml ofbacterial suspension washed with PBS or 40 ml of PBS (forcontrols). Each microcosm contained six worms and a singletreatment. Suspensions of strains CCO01, CC101, andCC105, Bradyrhizobium japonicum strains, and Escherichiacoli were added at rates of 105 to 108 CFU/g (dry weight) ofbedding. At 1 h after inoculation and again on day 7,duplicate subsamples (wet weight, 1.0 g) of well-mixedbedding were removed from each microcosm and suspendedin 19.5 ml of PBS. Each sample was vortexed for 2 min andallowed to settle for 30 min. The supematants (10 ml) fromCCO01, CC101, CC105, and Escherichia coli treatmentswere serially diluted 10-fold and spread plated in duplicateon HMGY containing appropriate amendments for recoveryof culturable bacteria. The results are expressed below as

loglo colony-forming units per gram of bedding. The remain-ing homogenates from all treatments were preserved for FAanalysis.Cocoons were collected at the end of 7 days and processed

as described above. Bedding homogenates from the CCO01,CC1O1, and CC105 treatments were serially diluted 10-foldand plated in duplicate on selective HMGY. Escherichia coliwas recovered on selective MacConkey medium. Homoge-nates from all treatments were preserved for FA analysis.AO analysis of cocoon homogenates. To determine AO

counts for homogenates obtained from general enumerationexperiments, 0.3 ml of a filtered (pore size, 0.2 p,m) stocksolution of 0.01% AO (Sigma) in 2.0 mM Tris (pH 8.0)preserved with 0.2% (vol/vol) formaldehyde was added to 1ml of a dilution of homogenate yielding between 10 and 50cells per microscopic field under UV incident light. Eachdiluted suspension was incubated for 5 min. AO-stainedsamples were impinged on black polycarbonate filters (poresize, 0.2 ,um; Nuclepore Corp., Pleasanton, Calif.) by using8 ml of sterile, filtered, distilled water as a carrier. Fifty fieldsper filter were counted on duplicate filters. The results are

expressed below as loglo number of cells per gram ofcocoons.

Antigen, antisera, and FA preparation. For the FA analysisof cocoon homogenates and bedding, antigens for CCO01,CCO05, and CCO03 were prepared from cells grown in SYliquid medium for 3 to 4 days. Somatic cell antigens forinjection into young adult rabbits were prepared by heatingcell suspensions in a boiling water bath for 1 h (CCO01 andCCO05) or by treating cell suspensions in 2% (vol/vol)formaldehyde for 1 h (CC003). Antigen suspensions wereused to produce antisera by the method of Schmidt et al.(29). The injection schedule was the same as that of Belserand Schmidt (1) as modified by Robert and Schmidt (26).FAs were prepared by procedures described elsewhere (26).FA specificity was tested against the prepared antigen,

more than 20 different gram-positive and -negative bacteriarandomly isolated from microcosm suspensions, 30 differentgram-negative bacteria randomly isolated from cocoons, andthe following soil and nonsoil microorganisms: Acetobacterspp., Achromobacter spp., Aeromonas hydrophila, Azoto-bacter spp., Bacillus megaterium, Bacillus pumilis, Brady-rhizobium japonicum, Caulobacter spp., Clostridium spp.,Erwinia spp., Escherichia coli, Gluconobacter spp., Photo-

TABLE 1. Numbers of bacteria in groups of cocoons producedby 45 adult Eisenia fetida worms in bedding changed weekly

No. of cocoons:_*___ No. of Log10

Week Tetdfr ace juveniles per CFU/ofCollected Thested. for Analyzed' Hatched uvn1sprCUobCletdhatch'ng Anlzdcocoon cocoonsb

1 51 0 25 NDC ND 9.12 58 0 28 ND ND 8.83 41 18 17 89 2.4 8.64 37 18 18 89 1.8 8.65 48 24 24 92 2.3 8.36 42 20 19 90 2.3 8.47 41 0 38 ND ND 8.58 38 10 15 89 2.3 8.69 64 15 30 89 2.4 9.010 41 41 0 93 3.5 NDa The cocoons were viable and 12 to 19 days old before surface sterilization,

homogenization, and spread plating in duplicate.b Cocoons pooled and homogenized as a group, with no replicates.c ND, not determined.

bacterium spp., Pseudomonas fluorescens, Rhizobiumphaseoli, Rhodopseudomonas spp., Rhodococcus spp., Sac-charomyces cerevisiae, Serratia marcescens, Streptomycesspp., Vibrio spp., and Xanthomonas campestris.FA analysis of cocoons and bedding. FA staining of bacteria

in preserved cocoon or bedding suspensions was performedby analyzing 2 ml of an appropriate dilution of suspension,using procedures developed for quantitative autecologicalimmunofluorescence and black polycarbonate filters (poresize, 0.45 ,um) with rhodamine-gelatin background stain andstrain-specific FA (29). Suspensions were steam heated at80°C for 20 min to enhance FA staining.

RESULTS

Cocoon production. None of the 45 worms used to initiatethe study died or showed signs of loss of secondary sexualcharacteristics (presence of clitellum) during the course ofthe experiments. To limit variability, cocoon production wascontrolled, and bedding was not disturbed for 7 days. Earliertrials indicated that cocoon production levels decreased withmore frequent disturbance. Nevertheless, cocoon produc-tion varied considerably from week to week, thus alteringthe number of cocoons available for various tests (Table 1).Forty-five adult Eisenia fetida worms produced 1.0 + 0.2cocoon per worm per week (mean ± standard deviation for10 sample weeks). During 7 weeks for which hatching datawere collected, the level of nonviable cocoons never ex-ceeded 11% of the total number of cocoons produced.During the same 7 weeks, the average number of juvenilesemerging from viable cocoons was 2.4 ± 0.5 worms percocoon. The juveniles appeared to be normal and wereroutinely used to repopulate laboratory stock cultures ofearthworms, growing to reproductive age in 4 to 6 weeks.Enumeration of culturable bacteria in cocoons. For 9 weeks

of the initial study, cocoons from a single production periodwere homogenized as a group for bacterial enumeration(Table 1). The average cocoon weight was 12 ± 0.4 mg(mean ± standard deviation for 22 randomly selected co-coons). Subsequently, a cocoon weight of 12 mg was used inall calculations. For 9 sampling weeks, the average loglobacterial level was 8.7 ± 0.3 CFU/g of cocoons.

Filtered cocoon homogenates from weeks 5 through 9were also examined by using strain-specific FAs for CCO01,

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TABLE 2. Filter counts (FA analysis) for isolates CCO01, CCO03,and CCO05 compared with total viable plate counts

for pooled cocoon homogenatesa

Log1o no. of FA-reactive cellsWeek per g of cocoonsb Loglo CFU/g

of cocoonscCCOO1 CC003 CCOO5

5 7.7 - + 8.3d6 + - - 8.47 + + - 8.58 + + - 8.69 - - + 9.0

a Cocoon homogenates were prepared from cocoons collected duringweeks 5 to 9 in the experiment shown in Table 1.

, no FA-reactive cells per 100 microscopic fields; +, fewer than 10FA-reactive cells per 100 microscopic fields, corresponding to less than 3 x105 cells per g of cocoons.

c Data from Table 1.d FA-positive colonies from dilution plates were identical in morphology to

the original isolate CC001 and were present at a level of log1o 7.5 CFU/g ofcocoons.

CCO03, and CCO05 (Table 2). Not more than 2 ml ofundiluted cocoon homogenate could be filtered in less than 1h. Filtering greater amounts of homogenate hindered micro-scopic examination because of increased background fluo-resence from cocoon albumen and worm tissues. Therefore,our analysis was limited to ca. 0.05 mg of cocoon materialper 2 ml of homogenate.

Colonies from spread plates containing the correspondinghomogenate were tested by using all available FAs. OnlyCCO01 reactive cells were present at detectable levels onfilters and on spread plates. In addition, all colonies withmorphology similar to CC001 colony morphology reactedpositively to homologous FA. There were no cross-reactionsbetween FAs developed for the various cocoon isolates orwith any of the other soil and nonsoil microorganisms tested.CCO03 and CC005 reactive cells were observed on filters atlevels below our defined detection limit (<10 FA-reactivecells per 100 microscopic fields at the lowest practicaldilution of 2 ml of undiluted homogenate). For some ho-mogenate samples, a single cell was observed in 100 micro-scopic fields on duplicate filters; we could not justify calcu-lating homogenate concentrations in these samples given thelow statistical reliability associated with such an observa-tion.

Subpopulations of cocoons produced during weeks 8 and 9were homogenized individually (i.e., one cocoon per dilutionseries) to obtain data on bacterial density for cocoons. Anarrow range of bacterial counts, which bracketed the valuesobserved with pooled cocoons, was observed (Table 3). Themean log1o level of bacteria per gram of cocoons did not varysignificantly between weeks or with increasing sample size.We observed no relationship between the number of colony-

TABLE 3. Number of colony-forming units per gram of cocoonsas a measure of variability among cocoons obtained during

weeks 8 and 9 and analyzed individually

No. of cocoons Loglo CFU/g of cocoonsWeek'analyzed Mean + SD Range

8 7 8.7 ± 0.5 8.2-9.49 15 8.7 ± 0.5 7.8-9.4

a Weeks 8 and 9 of the experiment shown in Table 1.

f'O

c

0

0

IL)

0

0)

vw0)

8

4

26 9 12 15

Average Cocoon Age (days)

FIG. 1. Changes in density of microbial cells in homogenates ofmaturing Eisenia fetida cocoons as determined by AO counts (0)and spread plating (0). Subpopulations of a larger pool of cocoonswere selected for homogenization at three stages of maturity. Eachdatum point represents the mean + 1 standard deviation (verticalbars) for six cocoons.

forming units per gram of cocoons and cocoon weight,number of juveniles, or the presence of visibly circulatingblood.Temperature effects on cocoon viability and bacterial pop-

ulations. Incubation at 24°C favored cocoon viability, butlimited embryonic development within viable cocoons, com-pared with incubation at 15°C. Stereoscopic observation of20 cocoons incubated at 15°C for 26 days indicated that thelevel of viability was 50%. This contrasted with the 90%level of viability in the control group of 20 cocoons incubatedat 24°C and examined after 12 days. Viable cocoons incu-bated at 15°C were beyond the early embryonic stages byday 26 and contained juveniles with visibly circulating blood.In contrast, 50% of the cocoons incubated at 24°C were inthe early embryonic stage of growth by day 12, and only 25%of these cocoons had visibly circulating blood by day 12. The10 viable cocoons incubated at 15°C were homogenized as a

group and had log10 8.8 CFU/g of cocoons. This value issimilar to the values obtained for the control group incubatedat 24°C and for cocoon populations sampled in the enumer-

ation experiments performed at 24°C.Time effects on cocoon bacterial populations. Since cocoons

were collected after a 7-day production period, and since wecould not determine the exact age of a randomly selectedcocoon, only an average age could be assigned to thecocoons sampled (Fig. 1). The numbers of culturable bacte-ria associated with cocoons increased after approximately 8to 10 days of incubation and then appeared to stabilize atvalues near those obtained in earlier experiments (log10 8.0CFU/g of cocoons). The AO counts, in contrast, were higherthan the viable counts within each group and showed littleincrease with time (log1o 9.0 cells per g of cocoons). Nocorrelation was found between cocoon bacterial counts andcocoon mass, stage of juvenile development, or number ofjuveniles per cocoon.

Recovery of inoculated bacteria from bedding and cocoons.

Cocoon isolate CC001, mutants CC101 and CC105, andBradyrhizobium japonicum strains were recovered fromworm bedding and viable cocoons produced in inoculatedbedding (Table 4). Control preparations (bedding with

T

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TABLE 4. Bacterial enumeration and levels of recovery of selected bacteria from Eisenia fetida cocoonsformed in inoculated, nonsterile beddingeLog1o no. of cells per g of bedding" Log1o no. of cells per g of

Inoculum Added on Recovered on cocoons as determined ofcocoonsgday 0 day 7 by FA analysis' fcoon

CC0oo 8.3 7.9 8.9 + 0.5 (5) NDeCCoo1 5.3 7.4 8.8 ± 0.7 (6) NDCC101 8.2 7.8 8.2 + 0.6 (5) 8.0CC105 6.7 8.3 8.8 ± 0.7 (6) 9.0Bradyrhizobium japonicum USDA 110 8.7 8.8 6.4 + 0.6 (6) NDBradyrhizobium japonicum UMR 161 7.8 8.1 6.8 ± 0.1 (2) NDEscherichia coli K-12(pJP4) 6.9 6.0 ND (6) BLDfNone BLD BLD BLD (6) BLD

a Adult worms were added on day 0; cocoons were collected on day 7 and were incubated for 12 days.b As determined by FA analysis of bedding homogenate, except for Eschenchia coli, which was recovered on Hg2+-amended MacConkey agar. Selective

plating data verified the filter counts of antibiotic mutants.c FA analyses were performed on individual homogenates. Values are means ± standard deviations. The numbers in parentheses are the numbers of cocoons

analyzed.d Numbers of colony-forming units determined for pooled homogenates from individual cocoons. Identities were verified by staining smears with homologous

FAs.e ND, not determined.f BLD, below the limit of detection (log1o 5.5 cells per g of cocoons when anti-CCO01, -CCO05, and -Bradyrhizobiumjaponicum FAs were used; log1o 3.7 CFU/g

of cocoons when selective plating for Escherichia coli was used).

worms but no inoculum) contained undetectable levels ofthese bacteria.The numbers of CCO01 added to bedding increased by 2

log units when the preparations were inoculated with log105.3 CFU/g of bedding, whereas inoculation with a higherlevel of bacteria did not result in population increases withtime. The numbers of CC105 added to bedding increased by1.6 log units when the preparations were inoculated withlog10 6.7 CFU/g of bedding. Escherichia coli populations inbedding did not increase from an initially low inoculationlevel during the 7-day incubation period. In the presence ofactively feeding and reproducing worms, the bedding wasable to sustain levels of CCO01, CC101, CC105, andBradyrhizobiumjaponicum strains near log1o 8.0 CFU/g overa 7-day period.Of the bacteria inoculated into bedding, those of cocoon

origin (CC101 and CC105) were recovered from cocoons ingreater numbers than the Bradyrhizobium japonicum strainsor Escherichia coli. Escherichia coli was not detected incocoons when bedding was inoculated with log1o 6.9 CFU/gof bedding. The limit of detection for Eschenchia coli waslog10 3.7 CFU/g of cocoons. The variation in bacterial countsamong cocoons containing strain CCO01 or CCO05 orBradyrhizobium japonicum strains, as determined by FAanalysis, was similar to the variation observed in earlierexperiments (Table 3). CC101 and CC105 reached levels incocoons comparable to the levels found in bedding. Thesedensities were within the range of densities reached in thegeneral enumeration experiments (Table 1).

DISCUSSION

Few studies have examined the relationship betweenbacteria and cocoons or cocoon albumen. Most recentinvestigations of the putative bacterial symbiosis with earth-worm nephridia have used only qualitative methods (30, 34),and the proposed model of intergenerational transfer ofsymbionts via cocoons remains a hypothesis. Cocoons havebeen injected with laboratory-grown suspensions of bacteriaisolated from nephridia, with no pathogenic effect; yet therelationship between the injected bacteria and the bacteria

observed in embryonic nephridia was based solely on micro-scopic morphology as determined by Gram staining (23).Our study is the first quantitative survey of general bac-

terial numbers in viable earthworm cocoons. In addition, byusing FAs specific to bacteria originally isolated from co-coons, we have shown that the milieu in which cocoons areformed is one possible source of their bacterial complement.Cocoon isolates grew to densities in albumen similar to thosesupported in bedding. In contrast, bedding inoculum ofnoncocoon origin was recovered from cocoons in lowernumbers (Bradyrhizobium japonicum) or was not recovered(Escherichia coli).

Direct pathogenesis to earthworms has been studied forsoil and nonsoil bacteria by antigen injection into the coelo-mic fluid in the body cavity (7). The immunological re-sponses elicited led to investigations of other earthwormsera. Agglutination by cocoon albumen of bacteria patho-genic to Eisenia fetida has been observed in vitro, but littleis known concerning the in vivo effects of albumen lectins onmicrobial ecology (see reference 33 for a review).

In our experiments, AO counts of bacteria in cocoonhomogenates collected from immature and mature cocoonsindicated that the population level was constant with time.Because viable counts of bacteria increased with time, it ispossible that only a fraction of the microbial population isviable at a given stage of cocoon development. Also, aportion of the bacterial complement enumerated by AO maybe recalcitrant to laboratory culture or may be nonviablebecause of agglutination by albumen lectins. Experiments inwhich AO direct viable counts are used (see reference 2)would help better define this aspect of cocoon microbiology.Cocoon isolates inoculated into worm microcosms

reached densities in cocoons comparable to those found ingeneral enumeration experiments. Thus, there are methodsof controlling the viable bacterial complement associatedwith cocoons produced in microcosms. The level of controlwas greatest with the bacteria originally isolated from thecocoons, slightly less with Bradyrhizobium japonicumstrains, and least with Eschenchia coli K-12(pJP4). It ispossible that plasmid pJP4, which confers Hg2" resistance,was lost or altered during Escherichia coli residence in the

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BACTERIA IN EISENL4 FETIDA COCOONS 1909

cocoons. This might explain why Eschenichia coli wasrecovered from bedding and not from cocoons; however,there is considerable evidence of Eisenia fetida antagonismtoward Escherichia coli and several other bacteria (3, 4, 9).In nature, Eisenia fetida may select for compatible bacteriaand against other bacteria on the basis of the positive ornegative roles that they play in the invertebrate life cycle.Our cocoon production rate of approximately one cocoon

per worm per week (Table 1) is low compared with previ-ously published production rates of 5.5 cocoons per wormper week (14), indicating that a limiting factor may haveexisted under our growth conditions. The hatching rates andnumbers of juveniles emerging per cocoon were similar tothe values in other published reports (14, 25) despite differ-ent growth media and experimental variables. Our cocoonweights were near those predicted by Hartenstein et al. (14)for Eisenia fetida, with approximately 1 mg of cocoonsproduced per 100 mg of live adults. In all experiments, weassumed that cocoons determined to be viable by ourmethods would have hatched, having never seen evidence tothe contrary.

In a study examining temperature effects on Eisenia fetidacocoons incubated on moist filter paper with compost (32),the level of hatching increased from 30% at 25°C to 88% at10°C. An opposite trend was observed in our study and mayreflect the microbial ecology or biochemistry of the mediumin which the cocoons were produced or incubated. Thenumbers of viable bacteria in cocoons were similar whetherthe preparations were incubated at 24 or 15°C, indicating thateither there was no microbiological role in cocoon mortalityor there was a microbiological role determined by other,unobserved microorganisms.The similar numbers of recoverable antibiotic-resistant

marked strains and FA-reactive cells observed in the com-plex, bacterium-rich environments of earthworm beddingand cocoon albumen provided indirect evidence of the highlevel of specificity of the polyclonal antibodies employed.On the basis of the results of Valembois et al. (33) and our

results, a model of cocoon-microbe interactions can bepostulated as follows. Bacteria are incorporated, randomlyor via worm-controlled mechanisms, into cocoons as viablecells. Albumen or other physicochemical processes act uponselected cells in a bacteriostatic or bacteriolytic fashion.Certain bacteria escape such action or are permitted toremain viable by selective action. Some bacteria multiplyunder the unique growth conditions provided by the cocoonenvironment or are freed of bacteriostatic inhibitions bybiochemical or physical processes.

In our experiments, bacteria were enumerated in viablecocoons of the earthworm Eisenia fetida. The populations ofculturable bacteria sampled from cocoons increased withtime, and the variability among cocoons was within 1 log unitwhen the data were stabilized by log transformation. Cocoonincubation temperatures which extended cocoon gestationtime did not affect the number of bacterial cells recovered.Select bacteria were obtained from the growth environmentof Eisenia fetida and grew to densities in cocoons similar tothose observed in bedding. Other bacteria, such as Esche-nchia coli, may have been selected against by the adultearthworms or the cocoon environment. The exact nature ofthe observed phenomena and the role of cocoon bacteria inthe Eisenia fetida life cycle are not known.

ACKNOWLEDGMENTThis research was supported by a grant from the Greater Minne-

sota Corporation.

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