Journal of Invertebrate Pathology 78, 109 –118 (2001)

doi:10.1006/jipa.2001.5047, available online at http://www.idealibrary.com on

Chemical Defense in the Egg Masses of Benthic Invertebrates: An

Assessment of Antibacterial Activity in 39 Mollusks and 4 Polychaetes
Kirsten Benkendorff,* ,† ,1 Andrew R. Davis,* and John B. Bremner†
*Department of Biological Sciences and †Department of Chemistry, University of Wollongong, New South Wales 2522, Australia

Received April 30, 2001; accepted July 30, 2001

terial populations range from 10 3 to 10 6 per milliliter in

Many marine invertebrates deposit benthic egg the ocean, with as many as 10 9 per milliliter in marine
masses that are potentially vulnerable to microbial in- sediments (Austin, 1988). Furthermore, bacteria have
fection. To help counter this threat these species may been found to rapidly colonize a wide range of biotic
have evolved some form of chemical protection for their and abiotic substrata in the ocean (Zobell and Allan,
encapsulated embryos. In this study the egg masses from
1935; Davis et al., 1989; Wahl et al., 1994). In response
7 marine mollusks were tested for antibacterial activity
to this selective pressure, a wide range of sessile ma-
against 4 marine pathogens: Enterococcus sericolicida,
Vibrio anguillarum, Vibrio alginolyticus, and Vibrio
rine organisms have evolved chemical defense mecha-
harveyi. Extracts from all of these egg masses were nisms, which involve the production of secondary me-
found to inhibit the growth of at least 1 marine bacte- tabolites with antimicrobial activity (e.g., Burkholder
rium at concentrations that approximate the natural and Burkholder, 1958; McCaffrey and Endean, 1985;
concentration of extract in the egg masses. The egg Walls et al., 1993; Wahl et al., 1994; Koh, 1997).
masses of 39 mollusks and 4 polychaetes were then Many marine mollusks and polychaetes have
tested for antibacterial activity against 3 human patho- evolved a reproductive strategy that involves the dep-
genic bacteria; Escherichia coli, Staphylococcus aureus, osition of fertilized embryos in benthic egg masses.
and Pseudomonas aeruginosa. Activity was detected in These egg masses appear to be subject to selective
the egg masses from 34 species, including 2 polychaetes pressures similar to those of many of the sessile inver-
and mollusks from two classes and 18 families. Antibac- tebrates that are known to use chemical defense. Egg
terial activity in molluskan egg masses was found to masses provide a rich source of nutrition and most
extend across the marine, estuarine, freshwater, and species do not provide parental protection for their
terrestrial environments. Both gelatinous egg masses developing embryos, although their development can
and tough egg capsules were found to inhibit microbial take many months (e.g., Havenhand, 1993). The egg
growth, suggesting that physical protection alone may masses are composed primarily of proteins and poly-
not be sufficient to protect the eggs. Antimicrobial activ-
saccharides (Hunt, 1966; Flower et al., 1969; Karuso,
ity was observed in the fresh egg masses but not in the
1987), making them highly suitable for the settlement
well-developed egg masses of a subset of species. The
results of this study indicate that a wide range of inver-
of microorganisms. These egg masses must be ade-
tebrates use chemical defense to protect their early quately protected to ensure the long-term survival of
stage embryos against bacterial infection. © 2001 Academic the species. However, there have been no comprehen-
Press
sive studies on the antimicrobial properties of benthic
Key Words: antibacterial; egg mass; mollusk; invertebrate egg masses. Nevertheless, toxic com-
polychaete; Enterococcus sericolicida; Vibrio anguilla- pounds have been reported in the eggs of some free-
rum; Vibrio alginolyticus; Vibrio harveyi; Escherichia spawning marine invertebrates (e.g., Gil-Turnes et al.,
coli, Staphylococcus aureus; Pseudomonas aeruginosa. 1989; McClintock and Vernon, 1990; Coll, 1992; Gil-
Turnes and Fenical, 1992). Elevated chemical defence
of reproductive tissue has also been documented for a
INTRODUCTION broad range of taxa, including plants (Thompson,
1982), bryozoans (Harvell, 1984), and terrestrial inver-
The benthic marine environment can effectively be tebrates (Eisner et al., 1996).
described as a soup of microorganisms. Typically, bac- Benthic egg masses are generally thought to play a
protective role during the embryonic development of
1
To whom correspondence should be addressed. Fax: 61 242 marine invertebrates (e.g., Thorson, 1950; Mileik-
214135. E-mail: [email protected]. ovsky, 1971; Smith et al., 1989). The energy expendi-

109 0022-2011/01 $35.00

Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.
110 BENKENDORFF, DAVIS, AND BREMNER

ture associated with encapsulation appears to be high, tested. A subset of marine mollusks was tested for
suggesting that encapsulation must have substantial antimicrobial activity at two stages of embryonic de-
survival value (Pechenik, 1979). Furthermore, the in- velopment to test the prediction that freshly laid eggs
stantaneous mortality rates of eggs retained in benthic have a greater requirement for chemical protection
egg capsules appear to be much lower than the esti- than well-developed embryos.
mated mortality rates for planktonic development
(Strathman, 1985). Thorson (1950) suggested that the
METHODS
benthic egg masses of marine invertebrates could pro-
vide protection against infection, although his asser-
Collection of Egg Material
tion does not appear to have ever been tested. How-
ever, several compounds with antibacterial activity The egg material from a total of 39 mollusks (33
have been isolated from the egg masses of some marine marine, 3 estuarine, 2 freshwater, and 1 terrestrial)
mollusks (Kamiya et al., 1984, 1988; Matsunaga et al., and 4 polychaetes were collected for antimicrobial test-
1986; Yamazaki, 1993; Benkendorff et al., 2000a). ing (Table 1). Voucher specimens and/or photographs
It seems unlikely that newly fertilized eggs will des- are held in a reference collection at the University of
ignate some of their limited nutritive reserves to the Wollongong. Most of the specimens were collected from
production of defense compounds. It is more likely that intertidal reefs along the New South Wales coast, near
freshly deposited egg masses are enclosed with antimi- Wollongong (34°30⬘, 151°50⬘) Australia. However, the
crobial compounds derived from the adults. Conse- egg masses from 3 gastropod and 1 polychaete species
quently, we predict that antimicrobial metabolites are were collected from the Mediterranean coast, Spain
more likely to be found in the freshly laid egg masses (Table 1). These specimens were freeze-dried prior to
than in the well-developed or hatching egg masses. The being shipped to Australia. The egg masses were ex-
gelatinous egg masses of the sea hare Aplysia juliana amined and classified either as gelatinous when the
have been shown to lose antimicrobial activity during eggs were embedded in a jelly matrix or as tough
embryonic development (Kamiya et al., 1988). A form capsules when the eggs were enclosed in a tough leath-
of chemical ripening was observed in the egg masses of ery outer casing or embedded in a firm matrix. Several
Dicathais orbita, where the toxic compound, tyrindo- independent collections of the egg masses of some com-
leninone, is converted into tyriverdin, which is bacte- mon species were made to test for any variation in
riostatic but not cytolytic, and then, at the time of antimicrobial activity. A summary of all the species
hatching, tyriverdin is converted into Tyrian purple, tested, the type of egg mass, the number of collections,
which shows no antibiotic properties (Benkendorff et and the total amount collected is provided in Table 1.
al., 2000a). Furthermore, there is evidence that the In general, the egg masses were collected shortly
requirement for antimicrobial agents in some mollus- after they were laid and were used immediately or
kan egg masses diminishes as the embryos develop. placed in the freezer (⫺20°C) until used. Antimicrobial
The early stage, shell-less embryos of the muricids activity was also assessed at a later stage of develop-
Nucella emarginata and Nucella lapillus were both ment for the egg masses of seven species. The develop-
found to suffer high mortality outside their leathery mental stage was determined by changes in the colora-
egg capsules (Pechenik et al., 1984; Rawlings, 1995), tion or firmness of the egg masses and confirmed by
which was apparently due to bacterial infection (Lord, inspection of the embryos or veligers under a dissecting
1986). However, N. lapillus embryos can be reared microscope. An open aperture at the top of leathery egg
outside the capsule in nonsterile seawater after they capsules also indicated the commencement of hatch-
have developed shells (Pechenik et al., 1984), indicat- ing.
ing that they have acquired resistance to disease.
In this study, the benthic egg masses of 7 species of
Preparation of Egg Material
marine mollusks from a broad range of taxonomic
groups were screened for antimicrobial activity against The egg masses were prepared differently for two
a suite of marine pathogenic bacteria. To determine antimicrobial assays (see below). The assay used de-
whether antimicrobial activity is a general phenomena pended on the availability of egg material, and a sub-
in benthic invertebrate egg masses, a further 32 mol- sample of 15 mollusks and 2 polychaetes were tested in
lusks and 4 polychaetes were screened for activity both assays (Table 1). When there was sufficient ma-
against 3 human pathogenic bacteria (Escherichia coli, terial, organic solvent extracts were prepared and
Staphylococcus aureus, and Pseudomonas aeruginosa), tested for activity in the fluorescein diacetate (FD)
which are routinely used for the testing of disinfectants assay. Egg masses that were rare, difficult to find, or
(Olsen, 1979). These include the gelatinous egg masses difficult to collect were tested in the zone of inhibition
and leathery egg capsules from 23 families of mollusks. (ZI) assay.
The majority of species were marine, although 3 estu- For use in the ZI assay, gelatinous egg masses were
arine, 2 freshwater, and 1 terrestrial species were also cut into approximately 1-cm pieces and ground with a
 ANTIBACTERIAL ACTIVITY IN BENTHIC INVERTEBRATE EGG MASSES 111

TABLE 1
Species of Invertebrates, Arranged Phylogenetically, That Were Screened for Antibacterial Activity in Their Egg Masses
Natural Concentration
Egg Amount
Family Species mass collected Assay CHCl 3 MeOH

Phylum: Mollusca—Class Gastropoda

Neritopsidae Nerita atramentosa TC 25 capsules ZI (c) NA NA

Littorinidae Bembicium nanum G 161.5 g ZI (c), FD 1.3–5.8 NC
Naticidae Conuber sordidus G 150 g ZI (c), FD 0.7–0.8 4.4
Ranellidae Cabestana spengleri TC 15 g FD 20.5 38
Muricidae Dicathais orbita TC 292 g ZI, FD 14.2–18.6 24
Agnewia tritoniformis TC 10.5 g ZI, FD 8.6–11.3 NC
Morula marginalba TC 0.26 g FD NC NC
Bedeva hanleyi TC 1.56 g FD NC NC
Lepsiella reticularis TC 1.2 g FD NC NC
Trunculariopsis trunculus* TC 8.2 g FD 11.3 NA
Ceratostoma erinaceum* TC 1.2 g FD 23.9 NA
Mitridae Mitra carbonaria TC 10.4 g ZI, FD NC NC
Conidae Conus papilliferus TC 4.3 g ZI, FD NC NC
Amphibolidae Salinator fragilis G 25.4 g ZI (c), FD 5.6 12.8
Salinator solida G 52.9 g ZI (c), FD 3.2 NC
Siphonariidae Siphonaria denticulata G 24.3 g ZI, FD NC NC
Siphonaria zelandica G 5.6 g FD NC NC
Planorbidae Isidorella hainesi** G 28.8 g ZI, FD 6.3 19.9
Glyptophysa gibbosa** G 8 masses ZI NA NA
Camaenidae Meridolum gulosum** TC 6 capsules ZI NA NA
Aplysiidae Aplysia juliana G 170 g ZI, FD 5–8.3 10
Aplysia sydneyensis G 11.7 g ZI, FD 6.8 11.3
Aplysia parvula G 2g ZI NA NA
Aplysia dactylomela G 1 ribbon ZI NA NA
Stylocheilus longicauda G 30.6 g FD 12.4 14.5
Dolabrifera dolabrifera G 1/6 ribbon ZI NA NA
Philinidae Philine angasi G 178.4 g ZI, FD NC NC
Pleurobranchidae Pleurobranchus peroni** G 1 ribbon ZI NA NA
Pleurobranchea sp.** G 2 ribbons ZI NA NA
Bullinidae Bullina lineata** G 2 ribbons ZI (c) NA NA
Hydatinidae Hydatina physis** G 1 ribbon ZI (c) NA NA
Oxynoidae Oxynoe viridis G 1 ribbon ZI NA NA
Dendrodorididae Dendrodoris fumata** G 2 ribbons ZI NA NA
Dorididae Platyodoris galbannus** G 2 ribbons ZI NA NA
Jorunna pantherina** G 1 ribbon ZI NA NA
Goniodorididae Goniodoris sp. G 4 ribbons ZI NA NA
Polyceridae Plocampherus imperialis** G 1 ribbon ZI NA NA
Glaucidae Spurilla neopolitana* G 1.4 g FD 38.9 NA

Phylum: Mollusca—Class Cephalopoda

Loliginidae Sepioteuthis australis TC 67 g FD 8.2–12 NA

Phylum: Annelidia — Class Polychaeta

Eupolymnia sp.* G 1.8 g ZI, FD NC NC

Unidentified sp. 1 G 6 masses ZI NA NA
Unidentified sp. 2 G 2 masses ZI NA NA
Unidentified sp. 3 G 7.2 g ZI (c), FD NC NC

Note. All species are from the intertidal area on the Wollongong Coast, N.S.W, Australia, except for those marked *, which were
obtained from the Mediterranean Sea, Spain. The egg masses were collected from the field except those marked **, which were collected
from aquaria. The types of egg mass are TC, tough capsules and G, gelatinous egg mass. The total amount of material collected for
antimicrobial testing is recorded per egg mass for species that were tested only in the zone of inhibition assay but is provided in grams
for material that was solvent-extracted (wet weight except for the Mediterranean specimens *, which were freeze-dried). The
antimicrobial assays used are the zone of inhibition (ZI) and fluorescein diacetate (FD) assays. In the ZI assay some of the egg masses
were contaminated by foreign microbial growth when placed on agar plates, which has been indicated by (c). The natural concentration
refer to the concentration of cholorform (CHCl 3 ) and methanol/water (MeOH) extracts obtained from the egg masses (in mg extract per
ml of egg mass). NA refers to not applicable for egg masses that were not extracted and tested in the FD assay. NC indicates that the
natural concentration was not calculated.
112 BENKENDORFF, DAVIS, AND BREMNER

mortar and pestle. Leathery egg capsules were cut bacterial lawn was allowed to dry for approximately 5
open with a pair of sterile scissors to release the intra- min. Duplicate samples of the egg material were then
capsular fluid and then ground with a mortar and placed onto the microbial lawn with sterile forceps. The
pestle to homogenize the sample. Prior to use with each plates were incubated overnight at 37°C before being
new sample, the mortar and pestle were washed, checked for apparent zones of inhibition in the bacte-
rinsed in 100% ethanol, and completely dried under a rial lawn surrounding the egg material. Zones of inhi-
fume hood. bition were recorded only if there was a clear and
For the FD assay, the egg masses were blended in consistent ring that exceeded 2 mm in width surround-
solvent with a Waring blender or cut open with a pair ing the egg material.
of scissors. Organic extracts were prepared by the Chloroform and polar (methanol/water) extracts
soaking of macerated egg masses in chloroform/meth- from the molluskan egg masses were tested for anti-
anol (1:1, v:v). The solvent was decanted and replaced microbial activity in the FD assay. The crude extracts
after 3 h and after a further 6 h. The sample was then were tested for activity at 1 and 10 mg/ml in acetone,
left to soak overnight and the extracts were combined which were in the range of natural concentrations of
and filtered. A small volume of water was added to the extract found in the fresh egg masses (Table 1). It
extract when necessary to induce the formation of two should be noted, however, that the extracts did not
layers. The chloroform layers were separated from the completely dissolve at the notional 10 mg/ml, and the
water/methanol layers in a separating funnel. The sol- actual test concentration was found to be in the range
vent was then removed by rotary evaporation under of 3–7 mg/ml for a subsample of species. To calculate
vacuum. The dried extract was transferred to a small the actual test concentrations, the extracts remaining
weighed vial by sequential resuspension in small vol- after the FD assay were dried under a stream of nitro-
umes (⬃0.5 ml) of methanol (MeOH) and dichlorometh- gen gas, followed by 1 h in vacuo (see above). The
ane. The bulk of the remaining solvent was removed remaining dried extract was reweighed to calculate the
under a stream of nitrogen gas and the last traces were amount used, which was then divided by the number of
removed by evaporation in vacuo (0.1 mm Hg, 30 min, replicates for which the sample was used during the
room temperature). The natural concentration of the FD assay. The water/methanol extracts were also
extracts was recorded as milligrams of extract per mil- tested at higher concentrations, which were within the
liliter of fresh egg mass. The natural concentration of range of natural concentrations for these polar extracts
extract was not calculated for every species due to (20 –50 mg/ml), depending on the availability of sam-
incomplete extraction and/or the insolubility of the ple.
dried extract. Seawater was used as a control in both antimicrobial
assays. In the ZI assay, 100 ␮l of seawater was pipetted
Antimicrobial Assays directly onto the microbial lawn. Organic extracts from
seawater and residues from large volumes (300 ml) of
Four marine pathogens, Enterococcus sericolicida, evaporated solvent were prepared in the same way as
Vibrio anguillarum, Vibrio alginolyticus, and Vibrio the egg extracts for the FD assay.
harveyi were provided by the Department of Primary
Industries and Fisheries, Launceston, Tasmania. Statistical Analysis
Stock cultures of E. coli ACM845, S. aureus ACM844,
and P. aeruginosa ACM846 were obtained from the The relative frequency of antimicrobial activity in
Culture Collection at the University of Queensland and tough egg capsules compared to gelatinous egg masses
maintained at ⫺78°C in 15% glycerol. The microbial was analyzed by the Fisher exact test (Zar, 1984) with
cultures were prepared according to Benkendorff et al. the JMP statistical package. Two-tailed tests were
(2000b). Activity against the marine pathogens was used, with ␣ set at 5%.
tested with the FD assay (Chand et al., 1994), accord-
ing to Benkendorff et al. (2000b). Activity against hu-
RESULTS
man pathogens was assessed with both the FD assay
and the ZI assay (Benkendorff et al., 2000b), which is a
Antimicrobial Activity against Marine Pathogens
modification of the traditional agar diffusion assay
(e.g., Spooner and Skyes, 1972). Organic extracts from the egg masses of seven ma-
Molluskan egg masses were tested directly for anti- rine mollusks from five orders were tested for bacteri-
microbial activity in the ZI assay by the placing of ostatic activity against four marine pathogenic bacte-
samples of the egg material onto a lawn of bacteria (see ria. The chloroform extracts from all of these species
Benkendorff et al., 2000b). Exponentially growing cul- were found to have some inhibitory activity toward all
tures (1 ml) of the bacteria were first pipetted onto the of the marine pathogens (Table 2). In general, activity
agar and evenly spread across the surface. The excess was observed at concentrations similar to the natural
culture liquid was removed with a pipette and the concentration of extract in the fresh egg mass (Table 2).
 ANTIBACTERIAL ACTIVITY IN BENTHIC INVERTEBRATE EGG MASSES 113

TABLE 2
Bacteriostatic Activity in the Egg Masses of Seven Marine Mollusks against Four Marine Pathogenic Bacteria, Vibrio
anguillarum (V. ang.), Vibrio alginolyticus (V. alg.), Vibrio harveyi (V. har.), and Enterococcus sericolicida (E. ser.) Tested in
the Fluorescein Diacetate (FD) Hydrolysis Assay
Minimum inhibitory concentration
Natural Concn.
Species Extract (mg/ml) V. ang. V. alg. V. har. E. ser.

Bembicium nanum CHCl 3 5.8 1–10 ⬎10 1–10 1–10

Cabestana spengleri CHCl 3 20.5 5 5–10 5 5
Dicathais orbita CHCl 3 17.6 1–10 1–10 1–10 1–10
MeOH/water 24 — — — —
Salinator fragilis CHCl 3 7.8 1–10 1–10 1–10 1–10
Aplysia juliana CHCl 3 8.3 1–10 1–10 1–10 10
MeOH/water 10 ⬎10 — — —
Stylocheilus longicauda CHCl 3 12.4 5–10 5–10 5 5
Sepioteuthis australis CHCl 3 12 10 ⬎10 10 ⬎10

Note. The natural concentration of extract in the egg mass and the minimum inhibitory concentrations are recorded as mg/ml. The
minimum inhibitory concentration refers to the lowest concentration at which the hydrolysis of FD was completely suppressed and is
presented as falling within a range of test concentrations when partial inhibition was observed at the lower concentration and complete
inhibition was observed at the higher concentration, which was incompletely soluble in acetone; ⬎ indicates that the extracts partially
inhibited the hydrolysis of FD at the maximum test concentration; — indicates that there was no apparent inhibition of FD at the maximum
test concentration (10 mg/ml).

However, V. alginolyticus appears to be relatively re- around the egg masses. These have all been recorded
sistant to extracts from the gelatinous egg mass of as not active in Table 3 due to the ambiguity of the
Bembicium nanum and the tough egg capsules of Se- results. Foreign microbial growth was found to sur-
pioteuthis australis. E. sericolicida was also relatively round the egg masses of 1 polycheate and 7 mollusks
resistant to extracts from S. australis egg masses. The when these were placed on agar plates on the ZI assay
polar extracts from the egg masses of D. orbita and A. (Table 1).
juliana showed little activity against any of the marine In the FD assay, antimicrobial activity was detected
pathogens (Table 2). in the egg masses of 22 mollusks and 2 polychaetes.
The activity was apparent in all replicated collections
Antimicrobial Activity against Terrestrial Pathogens of the egg masses. However, there was some variation
The freshly laid egg masses from 39 molluscs and 4 in the minimum inhibitory concentration of samples
polychaetes were also tested for antimicrobial activity from some species that were collected from the same
against 3 human pathogenic bacteria, and 34 of these headland on different days and between samples from
species effectively inhibited the growth of at least 1 test different locations. Most extracts were active only at
microorganism in at least one assay (Table 3). Two their maximum solubility, although S. aureus and E.
polychaetes and species from 18 of the 23 families of coli were susceptible to some of the extracts at 1 mg/ml.
mollusk tested were found to have antimicrobial prop- The active concentrations were generally within the
erties in their egg masses. These included representa- range of natural concentrations. The main exception
tives of estuarine (Naticidae and Amphibolidae), fresh- was the egg mass of Conuber sordidus, which is com-
water (Planorbidae), and terrestrial families (Ca- posed primarily of water and produced a very small
maenidae), in addition to marine species from two amount of organic extract. The lipophilic (chloroform)
classes (Gastropoda and Cephalopoda). extracts from the molluskan egg masses showed more
The full details of the antimicrobial activity in the antimicrobial activity than the polar extracts, with
egg mass of each species in the two assays are available activity in 24 of 25 species. Strong antimicrobial activ-
in Benkendorff (1999). In the ZI assay, a total of 16 of ity was detected in the polar extracts from 3 species in
38 species were found to unambiguously inhibit bacte- the family Aplysiidae and 1 fresh water pulmonate,
rial growth (Table 3). Consistent results were observed Isidorella hainesi. No antimicrobial activity was ob-
with separate collections of egg masses from the same served with the seawater or solvent controls.
species. No zones of inhibition were produced by sea- Consistent results were found in the two assays for 8
water controls. The egg ribbons of the sea hares, Aply- of the 15 species that were tested in both (Table 3).
sia spp., consistently produced the largest zones of However, antimicrobial activity was detected in the
inhibition against all the test organisms. Two uniden- egg masses from 6 species in the FD assay but not with
tified polychaetes, 1 Dorid nudibranch, and Philine any certainty in the ZI assay. The egg masses from 5 of
angasi produced inconsistent zones of inhibition these species were contaminated by epibiotic microor-
114 BENKENDORFF, DAVIS, AND BREMNER

TABLE 3
Proportion of Invertebrate Egg Masses Found to Have Antimicrobial Activity against at Least One Microorganism
(E. coli, S. aureus, P. aeuriginosa) in the Zone of Inhibition (ZI) and Fluorescein Diacetate (FD) Assays
Proportion of species active

Superorder Order/infraorder Family ZI FD Total

Phylum: Mollusca
Neritopsina Neritodea Neritidae 0/1 0/1
Caenogastropoda Littorinimorpha Littorinidae 0/1 1/1 1/1
Naticidae 0/1 1/1 1/1
Ranellidae 1/1 1/1
Neogastropoda Muricidae 3/4 7/7 7/7
Mitridae 1/1 0/1 1/1
Conidae 1/1 1/1 1/1
Heterobranchia Basommatophora Amphibolidae 0/2 2/2 2/2
Siphonariidae 1/1 2/2 2/2
Planorbidae 2/2 1/1 2/2
Eupulmonata Camaenidae 1/1 1/1
Anaspidea Aplysiidae 5/6 3/3 6/6
Cephalaspidea Bullinidae 0/1 0/1
Hydatinidae 0/1 0/1
Philinidae 0/1 1/1 1/1
Notaspidea Pleurobranchidae 1/2 1/2
Saccoglossa Oxynoidae 1/1 1/1
Nudibranchia Dendrodorididae 1/1 1/1
Dorididae 1/2 1/2
Gonidorididae 0/1 0/1
Polyceridae 0/1 0/1
Glaucidae 0/1 1/1 1/1
Debrachia Teuthoidea Loliginidae 0/1 1/1 1/1
Phylum: Annelidia—Polychaeta 1/4 2/2 2/4
Total egg masses 16/38 24/25 34/43

ganisms on the agar plates. The remaining species (Table 4). A Fisher exact test showed that there was no
produced inconsistent areas of inhibition around the significant difference in the overall frequency of anti-
tissue. The egg mass from 1 species of Mitridae clearly microbial activity for the two types of egg mass (P ⫽
inhibited microbial growth in the ZI assay but extracts
from this egg mass did not inhibit the hydrolysis of
fluorescein diacetate (Table 3). TABLE 4
Of the human pathogens, the Gram-positive bacte- Antibacterial Activity in Tough Egg Capsules Compared to
rium S. aureus was the most susceptible to components Gelatinous Egg Masses of Mollusks
of the molluskan egg masses. The growth of S. aureus
Origin of Tough Gelatinous All egg
was inhibited by 79% of the egg masses, whereas E. coli pathogen Species capsules masses masses
was inhibited by only 69% and P. aeruginosa was in-
hibited by 72% of species (Table 4). Furthermore, S. Marine V. anguillarum 3/3 4/4 7/7
aureus was inhibited at lower concentrations of extract V. alginolyticus 2/3 3/4 5/7
V. harveyi 3/3 4/4 7/7
for several species in the FD assay and the largest E. sericolicida 2/3 4/4 7/7
zones of inhibition were produced against this bacte- Combined 10/12 15/16 25/28
rium in the ZI assay. At least one 3/3 4/4 7/7
Terrestrial S. aureus 12/13 18/25 30/38
Gelatinous Egg Masses vs Tough Egg Capsules E. coli 10/13 17/26 27/39
P. aeuriginosa 6/9 15/20 21/29
Antimicrobial activity against at least one ecologi- Combined 28/35 50/71 75/103
cally relevant (marine) pathogen was observed in both At least one 12/13 20/26 32/39
gelatinous egg masses and tough capsules of mollusks Note. The frequency of antibacterial activity is recorded as the
(Table 4). A Fisher exact test found no significant dif- proportion of egg masses that effectively inhibited the marine and
ference in the overall frequency of antimicrobial activ- terrestrial pathogens in at least one antibacterial assay. The propor-
ity against the marine pathogens in these two types of tion of egg masses with activity has been combined for the marine
and terrestrial pathogens to give the overall incidence of antimicro-
egg mass (P ⫽ 0.5604 ; df ⫽ 27). Antimicrobial activ- bial activity against these two groups of pathogens. The proportion of
ity was also observed in both tough egg capsules and species showing activity against at least one pathogen has also been
gelatinous egg masses against the human pathogens recorded for the bacteria of marine and terrestrial origin.
 ANTIBACTERIAL ACTIVITY IN BENTHIC INVERTEBRATE EGG MASSES 115

TABLE 5 approximate the natural concentrations of extract in

Antibacterial Activity of Seven Molluskan Egg Masses the egg masses, particularly when the incomplete sol-
Tested at Different Stages of Embryonic Development in the ubility of the extracts at 10 mg/ml is taken into con-
Zone of Inhibition (ZI) and Fluorescein Diacetate (FD) Assays sideration. Consequently, the antimicrobial compo-
Developmental stage nents in these egg masses could realistically play an
ecological role in the prevention of microbial infection.
Well The idea that egg masses of marine invertebrates pro-
Freshly laid developed vide protection against microbial infection was first
Species ZI FD ZI FD suggested by Thorson (1950) but until now has not
been rigorously tested.
Agnewia tritoniformis ⫹ ⫹ — — Antimicrobial properties in benthic egg masses ap-
Dicathais orbita ⫹ ⫹ — —
Mitra carbonaria ⫹ — — —
pear to have evolved in two different invertebrate
Conus papilliferus ⫹ ⫹ — — phyla and at least twice within the Mollusca. The dep-
Siphonaria denticulata ⫹ ⫹ — — osition of benthic egg masses has evolved in two sepa-
Isidorella hainesi ⫹ ⫹ — — rate classes of mollusk, Gastropoda and Cephalopoda
Aplysia juliana ⫹ ⫹ — — (Smith et al., 1989), and species from both of these
Note. ⫹ Indicates that the specimen unambiguously inhibited the classes were found to have antimicrobial activity in
growth of at least one terrestrial microorganism in the given assay; their egg masses (Table 1). Within the class Gas-
— indicates that the specimen showed no clear antibacterial prop- tropoda, egg masses have evolved in one subclass, the
erties.
Orthogastropoda, but could have evolved indepen-
dently in two clades, the apogastropods (including cae-
nogastropods and heterobranchs) and the neritopsines
0.3539, df ⫽ 105) or the proportion of species that
(including Neritoidea) (Ponder and Linberg, 1997).
were active against at least one human pathogen (P ⫽
Most of the species tested in this study are apogastro-
0.3884, df ⫽ 38).
pods. The egg capsules from one species of Neritoidea
(Nerita atramentosa) were tested for antimicrobial ac-
Antimicrobial Properties at Different Stages of
tivity in the ZI assay but did not show clear antimicro-
Development
bial activity due to contamination by foreign microor-
The egg masses from seven species of mollusk were ganisms surrounding the eggs (Table 2). Future re-
tested for antimicrobial activity at two different stages search should include the testing of a greater range of
of development (freshly laid and hatching). The freshly species from the gastropod superorder Neritopsina and
laid egg masses from all of these species inhibited the the class Cephalopoda against marine pathogens.
growth of at least one test microorganism, in at least It remains possible that antimicrobial activity in
one antimicrobial assay (Table 5). However, none of the benthic invertebrate egg masses is even more wide-
hatching egg masses were found to completely inhibit spread than indicated by this study. All of the species
the growth of any test microorganisms in either assay that did not exhibit clear activity were tested only in
(Table 5). the zone of inhibition assay, which has a number of
associated limitations (Chand et al., 1994; Benkendorff
DISCUSSION et al., 2000b). Several species tested negative in the ZI
assay but showed clear activity in the FD assay (Table
The benthic egg masses of invertebrates are poten- 3). Interestingly, most of these egg masses were con-
tially vulnerable to microbial infection, which may taminated by the growth of unidentified microorgan-
have led to the evolution of chemical defence mecha- isms when placed on agar in the ZI assay. Further
nisms. In support of this hypothesis, antimicrobial ac- experiments are required to determine whether these
tivity was recorded in 79% of the invertebrate egg microorganisms are epibiotic. However, it is possible
masses tested in this study (Table 3). The egg masses that there is a commensal relationship between the
of mollusks from 18 families, in addition to 2 species of benthic egg masses of some invertebrates and the
polychaetes, were found to inhibit the growth of human epibiotic microbes. Many organisms are protected from
pathogenic bacteria. The antibacterial activity in mol- pathogenic microorganisms as long as they retain their
luskan egg masses appears to extend across marine normal associated microflora (Austin, 1988). Further-
and terrestrial environments, with 26 marine, 3 estu- more, symbiotic microorganisms on the surface of eggs
arine, 2 freshwater, and 1 terrestrial species testing from some crustaceans have been shown to produce
positive. Extracts from the egg masses of 7 marine antimicrobial compounds (Gil-Turnes and Fenical,
mollusks were also found to inhibit the growth of 4 1992; Gil-Turnes et al., 1989).
marine pathogenic bacteria (Table 2), 3 of which are It is also significant that the egg masses from several
known to infect mollusks (Austin, 1988; Sutton and species that tested negative in this study were collected
Garrick, 1993; Koh, 1997). The active concentrations from aquaria (Table 1). The deposition of egg masses by
116 BENKENDORFF, DAVIS, AND BREMNER

Opisthobranchs in aquaria is thought to be a stress from the dog whelk N. lapillus. The loss of antimicro-
response (W. Rudman, pers. com.) and therefore these bial activity during larval development could also be an
species may not have accumulated the compounds that important component of the hatching process in gelat-
are normally produced to protect the egg mass. Signif- inous egg masses. Microbial degradation of the jelly
icantly, soft corals have been shown to accumulate the matrix around the time of hatching may facilitate the
defensive compounds found in the eggs only a few release of the larvae or juveniles into the water column
weeks prior to spawning (Coll et al., 1985). It is likely or onto the substratum (Harris, 1975; Eyster, 1986).
that antimicrobial compounds in some species are diet Antimicrobial activity in the egg masses of most
derived, as appears to be the case for the biologically marine mollusks tested in this study appears to be
active compounds that have been found in the egg attributable to components of low to moderate polarity.
masses of the Opisthobranchs Hexabranchus san- The activity in the egg masses of the mollusks was
guineus (Faulkner, 1992) and Elysia halimedae (Paul primarily restricted to the lipophilic extracts in the FD
and van Alstyne, 1988). Nevertheless, this is clearly assay. This would effectively reduce the loss of bioac-
not the case with the antibacterial components from D. tive components into the water column. However, the
orbita egg masses, which are derived from a precursor water/methanol-soluble components from extracts of
in the hypobranchial gland of the adults (Benkendorff four species showed clear antimicrobial activity (Table
et al., 2000a). 4). Three of these species are from the family Aplysi-
It appears that a physical barrier may not be suffi- idae and several cytolytic glycoproteins have been pre-
cient to protect reproductive material from microbial viously isolated from eggs and adult sea hares in saline
infection in the marine environment. Antimicrobial solutions (Yamazaki et al., 1984, 1985, 1990; Kisugi et
properties against both marine and terrestrial patho- al., 1987; Kamiya et al., 1984, 1988). Nevertheless, the
gens were found to occur at similar frequencies in both lipophilic extracts of Aplysia eggs were also found to
gelatinous egg masses and tough egg capsules of mol- have antimicrobial properties in this study and, signif-
lusks (Table 4). All of the egg masses showed some icantly, bioactive compounds have been isolated from
activity against at least one marine pathogen and the chloroform extracts of adult sea hares (e.g., Hollen-
there was no significant difference in the proportion of beak et al., 1979; Miyamoto et al., 1995).
the two types of egg mass that were active against at The egg masses of marine invertebrates appear to
least one human pathogen. Lord (1986) demonstrated have broad-spectrum antimicrobial activity. The egg
that the contents of N. lapillus egg capsules were free masses of the seven mollusks found to inhibit the
from bacterial contamination and suggested that the growth of marine pathogens were also found to have
capsule walls were impermeable to bacteria. However, some activity against human pathogens of consider-
reproductive tracts free of bacteria would also be re- able medical importance. Both Gram-negative and
quired to produce egg capsules with axenic contents, Gram-positive marine and human pathogens were sus-
where physical defense would be more problematic. ceptible to extracts from the egg masses. Overall, how-
Consequently, a bacteria-free environment may be dif- ever, the Gram-positive bacterium S. aureus was more
ficult to achieve by physical defence mechanisms alone. susceptible to the toxic compounds in the eggs than the
This study indicates that antibacterial compounds are other test organisms (Table 4). S. aureus is an impor-
equally common in tough egg capsules and gelatinous tant human pathogen and has evolved resistance to a
egg masses, suggesting that chemical defence is the large number of conventional antibiotics in the last
primary mechanism used to maintain an axenic envi- decade (Stinson, 1996). The greater resistance of the
ronment for embryonic development. Gram-negative bacteria to the molluskan egg masses is
The requirement for antimicrobial protection ap- likely due to the greater reinforcement in their cell
pears to be greatest at the early stages of molluskan walls (e.g., Martin and Beveridge, 1986).
embryonic development. An assessment of antimicro- Overall, our data indicate that benthic invertebrate
bial activity in the egg masses of seven mollusks dur- egg masses provide a novel source of potential antimi-
ing two stages of development indicates that egg crobial agents. We have recently isolated a potent non-
masses have significantly greater antimicrobial activ- cytolytic antibiotic from the egg mass of D. orbita (Ben-
ity when first laid and then lose activity as the larvae kendorff et al., 2000a), which is bacteriostatic at the
develop and hatch (Table 5). These findings are consis- therapeutically significant concentration of 0.5 ␮g/ml
tent with previous studies on molluskan egg masses and has a chemical structure different from that of all
that have reported the loss of antimicrobial activity antibiotics in current use. Previous studies on mollus-
during larval development (Kamiya et al., 1988; Ben- kan egg masses have reported agglutinating activity
kendorff et al., 2000a). The lack of antimicrobial activ- (Kamiya and Shimizu, 1981), antineoplastic and cyto-
ity at late stages of development suggests that mollus- lytic activity (Yamazaki et al., 1984, 1985; Roesner and
kan embryos lose their susceptibility to bacteria during Scheuer, 1986; Kisugi et al., 1987; Kamiya et al., 1988),
development. This is supported by the observations of antifeedant activity (Pennings, 1994), and tranquiliz-
Pechenik et al. (1984) on the survivorship of embryos ing effects (Marthy, 1976). These studies all demon-
 ANTIBACTERIAL ACTIVITY IN BENTHIC INVERTEBRATE EGG MASSES 117

strate that invertebrate egg masses are suitable tar- Harris, L. G. 1975. Studies on the life history of two coral-eating
gets for further chemical, ecological, and pharmacolog- nudibranchs of the genus Phestilla. Biol. Bull. 149, 539 –550.
ical studies. Harvell, C. D. 1984. Why nudibranchs are partial predators: Intra-
colonial variability in bryozoan palatability. Ecology 65, 716 –724.
ACKNOWLEDGMENTS Havenhand, J. N. 1993. Egg to juvenile period, generation time, and
the evolution of larval type in marine invertebrates. Mar. Ecol.
The assistance of Bill Rudman and Ian Loch from the Australian Prog. Ser. 97, 247–260.
Museum in the species identification is greatly appreciated. We are Hollenbeak, K. H., Schmitz, F. J., Hossain, M. B., and Van Der Helm,
grateful to Dr. Manual Ballesteros (University of Barcelona) for D. 1979. Deodactol, antineoplastic sequiterpenoid from the sea
assistance in the collection and identification of the egg masses from hare Aplysia dactylomela. Tetrahedron 35, 541–545.
three Mediterranean mollusks and one polychaete. Dr. Jeremy Car-
Hunt, S. 1966. Carbohydrate and amino acid composition of the egg
son from the Fish Health Unit, Department of Primary Industry and
capsule of the whelk, Buccinum undatum. Nature 210, 436 – 437.
Fisheries, Tasmania kindly provided the strains of marine bacteria.
We also thank the Australian Flora and Fauna Research Centre, the Kamiya, H., Muramoto, K., Goto, R., and Yamazaki, M. 1988. Char-
Biomolecular Research Centre, and the Environmental Research acterisation of the antibacterial and antineoplastic glycoproteins
Institute, University of Wollongong for a supplementary postgradu- in a sea hare Aplysia juliana. Nippon Suisan Gakkaishi 54, 773–
ate scholarship to K.B. This is contribution No. from the Ecology and 777.
Genetics Group at the University of Wollongong. Kamiya, H., Muramoto, K., and Ogata, K. 1984. Antibacterial activ-
ity in the egg mass of a sea hare. Experientia 40, 947.
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