The production of tetrodotoxin-like substances by nemertean worms in conjunction with bacteria

The production of tetrodotoxin-like substances by

nemertean worms in conjunction with bacteria

Stuart Carroll, Eric G. McEvoy, Ray Gibson*

School of Biological and Earth Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK

Received 9 September 2002; received in revised form 4 November 2002; accepted 5 December 2002

Abstract

Evidence is presented which strongly indicates a relationship between the presence of Vibrio

bacteria, probably Vibrio alginolyticus, and the synthesis of tetrodotoxin (TTX)-like chemicals in

seven species of British nemerteans. The occurrence of these substances and associated Vibrio

bacteria in these species was investigated by bacteriological, chromatographic, spectroscopic and

ultraviolet spectrometric techniques. It is suggested that these toxins are utilised by the nemerteans as

a chemical defence against potential predators.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Bacteria; Nemerteans; Tetrodotoxin; Vibrio

1. Introduction

Nemertean worms are soft-bodied invertebrates with no morphological or behavioural

means of protection against potential predators (Heine et al., 1991). They are known to

rely upon a variety of toxic or noxious chemicals for their defence (Kem, 1985). The

earliest report that nemerteans possessed toxins was the discovery of two neurotoxins,

from Amphiporus and Lineus, respectively, by Bacq (1936, 1937). He suggested that these

substances, which he called ‘amphiporine’ and ‘nemertine’, served in a defensive role,

rather than being offensive venoms such as are frequently associated with prey capture by

the nemertean proboscis. This suggestion is supported by Kem’s (1971, 1973) reports that

some 70% of the total anabaseine present in the hoplonemertean Paranemertes peregrina

is located in the integument. Species of Amphiporus, Cerebratulus, Lineus and Tetra-

0022-0981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0022-0981(02)00595-6

* Corresponding author. Tel.: +44-151-231-2175; fax: +44-151-298-1014.

E-mail address: [email protected] (R. Gibson).

www.elsevier.com/locate/jembe

Journal of Experimental Marine Biology and Ecology

288 (2003) 51–63

stemma have been found to contain other toxins, including neurotoxic polypeptides and

pyridyl alkaloids (Kem, 1973, 1988; Kem et al., 1976). More recently, tetrodotoxin (TTX),

long known to occur in the ovaries and livers of pufferfish (Tetraodontidae) (Goto et al.,

1965), has been reported from two species of Japanese nemerteans, the palaeonemertean

Tubulanus punctatus and the heteronemertean Lineus fuscoviridis (Miyazawa et al., 1988).

A third species, the palaeonemertean Cephalothrix linearis, was subsequently found to

contain TTX and related substances (Ali et al., 1990). Extremely high levels of these

toxins have also been recorded from a Cephalothrix species living amongst cultured

oysters in Hiroshima Bay (Asakawa et al., 2000).

The comparatively few records of identified nemertean species being used as prey

organisms provide further evidence for defence by toxins. Todd (1905) recorded a single

occasion where a specimen of the heteronemertean Lineus bilineatus had been found in a

cod stomach, the South African heteronemertean, Polybrachiorhynchus dayi, is regarded

as an excellent bait by local fishermen (Gibson, 1977), the heteronemertean Cerebratulus

lacteus has been found in the gizzard of a Black-bellied Plover (Hicklin and Smith, 1979),

Gibson et al. (1990) reported an incidence of a palaeonemertean, Procephalothrix

hermaphroditicus, having fed on a hoplonemertean, Amphiporus nelsoni, and Heine et

al. (1991) record two instances of the benthic Antarctic fish, Trematomus bernacchii,

feeding on pieces of the heteronemertean Parborlasia corrugatus. McDermott (2001), in

his review of marine nemerteans as prey organisms, adds a further eight identified

nemertean species as the prey of various animal groups. More frequently unidentified

nemerteans are reported as comprising a percentage (usually small) of the diet of various

fish species (e.g., see Jewett and Feder, 1980; Langton and Watling, 1990; Matlock and

Garcia, 1983; Miller, 1967; McDermott, 2001).

TTX, a low molecular weight neurotoxin which exists in many isomers (Goto et al.,

1965), has now been found in representatives from several different marine phyla,

including fish (Noguchi and Hashimoto, 1973; Sato et al., 1998), chaetognaths (Thuesen

et al., 1988), gastropod molluscs (Jeon et al., 1984; Narita et al., 1981, 1984; Noguchi et

al., 1981), octopus (Savage and Howden, 1977; Sheumack et al., 1978, 1984), echino-

derms (Maruyama et al., 1984, 1985; Noguchi et al., 1982) and horseshoe crabs (Tanu and

Noguchi, 1999).

Bacteria of several genera occur with TTX-like toxins in association with a diverse

range of animals (Cheng et al., 1995; Hormansdorfer et al., 2000; Lee et al., 2000; Matsui

et al., 2000; Matsumura, 1995; Simidu et al., 1990). This led to the suggestion that marine

bacteria were involved in toxin synthesis, and in particular a number of studies has shown

a direct link between the presence of Vibrio alginolyticus and TTX production (Noguchi et

al., 1987; Thuesen and Kogure, 1989). McEvoy et al. (1998) found evidence for V.

alginolyticus-like bacteria in 10 species of free-living predatory marine nemerteans, nine

of which showed bacterial growth in their epidermal mucus. One other species, which

gave negative results, was Gononemertes parasita, a commensal in ascidians; McEvoy et

al. (1998) suggested that this might be because Gononemertes, unlike the other species

studied, was not an active predator.

A possible precursor of TTX has also been identified by Noguchi et al. (1991) as an

unstable tetrodonic acid-like substance (TDA) which, at pH 3.0, rapidly converts into

TTX. More than 100 years ago McIntosh (1873–1874) and Wilson (1900) noted

S. Carroll et al. / J. Exp. Mar. Biol. Ecol. 288 (2003) 51–6352

respectively that the skin and surface mucus of L. bilineatus and C. lacteus was strongly

acidic. Heine et al. (1991) reported that the epidermal mucus of P. corrugatus had a pH of

3.5. McEvoy et al. (1998) suggested that in nemerteans TTX or its precursors came from

internal symbiotic or parasitic microorganisms. They further commented that the acidic,

Alcian blue positive, epidermal mucus of nemerteans (Gibson, 1972) could favour the

conversion of bacterially synthesised TDA into TTX.

Bacteriological, chromatographic, spectroscopic and ultraviolet spectrometric methods

have accordingly been used to investigate both the occurrence of TTX or TTX-like toxins

and the presence of associated Vibrio bacteria in seven species of British nemerteans.

2. Materials and methods

The species of nemerteans were collected intertidally, from either Llandudno, North

Wales (53j19VN, 3j49VW) or Rhosneigr, Anglesey (53j14VN, 4j30VW), in March and

April 2002. Listed taxonomically, the species were:

Class Anopla

Subclass Palaeonemertea

Cephalothrix rufifrons (Johnston, 1837)

Subclass Heteronemertea

Lineus longissimus (Gunnerus, 1770)

Lineus ruber (Muller, 1774)

Lineus viridis (Muller, 1774)

Ramphogordius sanguineus (Rathke, 1799)

Riseriellus occultus Rogers, Junoy, Gibson and Thorpe, 1993

Class Enopla

Subclass Hoplonemertea

Amphiporus lactifloreus (Johnston, 1828)

The nemerteans were maintained in the laboratory in clean, filtered seawater at room

temperature. Investigations were carried out on extracts made by homogenising 0.5 g of

fresh nemertean tissue in 3 ml 0.03M acetic acid. To three of the replicates for each species

0.5 ml sterile seawater was added to give a final volume of 1 ml. Cod tissue was used as a

control, 1.5 g of tissue being homogenised in 9 ml acetic acid to maintain the same tissue–

solvent ratio. Pufferfish (Fugu rubripes) liver extract, known to contain TTX, was used as

a positive control, 5 g liver tissue being homogenised in 30 ml acetic acid.

L. longissimus, when handled, produces copious amounts of a thick, acrid-smelling,

mucus. Samples of this mucus were examined separately. For scanning spectroscopy and

ultraviolet spectrometry, the mucus had to be diluted in acetic acid to 25% of its original

concentration; undiluted mucus gave results that exceeded the scanning range of the

apparatus.

Bacterial cultures from the nemerteans were obtained by a modification of the method

used by McEvoy et al. (1998). Individual nemerteans were homogenised in a sterile

stainless steel blender with 10 ml sterile filtered seawater. Aliquots of 500 Al were then

S. Carroll et al. / J. Exp. Mar. Biol. Ecol. 288 (2003) 51–63 53

transferred to 10 ml of full strength (88 g l� 1) TCBS (thiosulphate, citrate, bile, salt

sucrose sloppy agar) and plated on to set TCBS, a selective medium known to inhibit the

growth on non-Vibrio bacteria but suitable for culturing most Vibrio species (Furniss et al.,

1978). Plates were maintained at 20jC for 24 h to permit bacterial growth through the

semi-liquid matrix. The gross cell morphology of the resulting colonies was examined

microscopically, a gram stain procedure was performed to facilitate their identification and

a hanging drop method was used to determine their motility. Sub-cultures were set up by

transferring bacterial colonies to new TCBS plates and allowing them to grow at 20jC for

2–3 days. Samples of these second generation bacteria were analysed in the same manner

as the parental colonies.

High performance liquid chromatography was performed on 0.01 ml of each extract

sample at a flow rate of 1 ml min� 1 using heptanesulfonic acid and methanol in 0.05M

potassium phosphate buffer. Pufferfish extract was run first in order to establish a reference

point, cod, nemertean and bacterial extracts being analysed afterwards.

An overlay scan at a rate of 20 nmcm-1 with a range of 350–550 nm was carried out in a

Shimadzu scanning spectrometer, commencing at k = 550 nm. Distilled water was used as

a blank. Extracts of all seven nemertean species and the mucus of L. longissimus, together

with those obtained from the pufferfish, cod and both bacterial generations, were

examined. Filtered seawater and acetic acid were also tested.

Ultraviolet spectrometry was carried out on a Perkin Elmer UV/VIS Spectrophotometer

Lambda 40 at wavelengths between 450 and 280 nm, starting at k= 450 nm. Pufferfish

extract, first and second generation bacteria and extracts of cod and all the nemertean

species were analysed in the same way. All samples were calibrated against acetic acid

used as a reference blank.

3. Results

3.1. Bacteriology

All the plates with nemertean extracts added to them developed significant growth

patterns. The bacteria were of a uniform appearance, indicative of only a single species

being represented. Their gross external morphology was of small yellow colonies, 3–4

mm in diameter, which were mucoid in consistency, gram-negative and motile. In order to

avoid confusion with Brownian movement, bacterial motility was confirmed by high

power microscopy. These findings are consistent with the results obtained by Lee et al.

(2000). Further microscopic examination of the bacteria showed that they possessed single

rod-shaped and slightly curved cells. Bacterial growth for all the nemertean species

extracts was identical. No growth was achieved on plates treated with sterile seawater,

acetic acid or cod extract.

3.2. Scanning spectroscopy

There was a distinct curve between wavelengths of approximately 350–400 nm. This

curve was very obvious in the pufferfish extract (Fig. 1A), but significant curves were also


Fig. 1. Scanning spectroscopy absorbance curves for extracts of the pufferfish control (A), C. rufifrons (B), L.

longissimus tissue (C), L. ruber (D), L. viridis (E), R. sanguineus (F), R. occultus (G), A. lactifloreus (H) and L.

longissimusmucus (I). Curves for both the tissue and mucus of L. longissimus were obtained from extracts diluted

to 25% of the original concentration.


found for all seven of the extracts from nemertean species (Fig. 1B–H) and for the mucus

of L. longissimus (Fig. 1I). Acetic acid and sterile seawater yielded no curves between 350

and 550 nm (Fig. 2A), whereas cod extract showed a slight increase in absorbance (Fig.

2B) but this did not correspond with either the nemertean or pufferfish extracts (Fig. 1). An

absorbance curve similar to the pufferfish control was elicited by extracts of the first

generation bacteria (Fig. 2C), but this curve was greatly reduced in extracts from second

generation bacteria (Fig. 2D).

Absorbance curves obtained from the pufferfish, nemerteans and L. longissimus

mucus are essentially similar in showing an increase within the range of about 350–

450 nm.

Fig. 2. Scanning spectroscopy absorbance curves for sterile seawater (A), and extracts of cod (B), first generation

bacteria (C) and second generation bacteria (D). The absorbance curve for acetic acid was essentially the same as

that for sterile seawater.


Fig. 3. Ultraviolet spectrometry traces obtained from extracts of the pufferfish control (A), C. rufifrons (B), L.

longissimus tissue (C), L. ruber (D), L. viridis (E), R. sanguineus (F), R. occultus (G), A. lactifloreus (H) and L.

longissimus mucus (I). Traces for both the tissue and mucus of L. longissimus were obtained from extracts diluted

to 25% of the original concentration.


Fig. 4. Ultraviolet spectrometry traces obtained from first generation bacteria (A) and the 0.03 M acetic acid

control (B).

Fig. 5. HPLC traces, showing time in minutes against percentage absorbance, obtained from extracts of the

pufferfish control (A), L. ruber (B), A. lactifloreus (C), the mucus of L. longissimus (D) and first generation

bacteria (E).


3.3. Ultraviolet spectrometry

The pufferfish control showed a significant peak, very similar to that obtained from the

scanning spectroscopy (Fig. 3A). Peaks for the extracts from all the nemertean species

tested, as well as the mucus of L. longissimus, correspond to that of the pufferfish control

(Fig. 3B–I).

First (Fig. 4A), but not second, generation bacteria yielded similar results, but the

absorbance curve yielded by the acetic acid control (Fig. 4B) was very different.

3.4. High performance liquid chromatography

Three very obvious peaks were recorded for the pufferfish control (Fig. 5A), at time

indices of 4.16, 4.58 and 4.72. A minor peak, at 14.50–15.00 time index, was not apparent

on the original trace but could be discerned when this was magnified five-fold. L. ruber

extracts also yielded three peaks (Fig. 5B) but only the first of these, at time index 3.42,

was major, the other peaks at time indices 4.79 and 5.06 being much lower. Two other

groups of peaks are apparent for this species, one set occurring between 12.41 and 12.83,

the other at The extracts from A. lactifloreus (Fig. 5C) showed three peaks at 3.42, 4.59

and 4.65, followed later by three further peaks at 12.16, 13.55 and The mucus of L.

longissimus (Fig. 5D) showed a major peak at a time index of 4.03, but also showed

several other peaks between about 11.0 and 11.5. First generation bacteria (Fig. 5E)

yielded a major peak at 3.61 time index, with a second smaller peak at 5.34 being followed

by several other peaks at time indices 12.84, 13.16, 14.40, 14.95 and 15.22. All these

HPLC traces thus show similar peaks occurring at roughly the same time indices. Though

there is some small degree of variation between the time indices, three main peaks can be

recognised at approximately 4.00, 12.00 and 15.00.

4. Discussion

Round, yellow bacterial colonies 3–4 mm in diameter grown on TCBS, with individual

cells being motile, possessing a curved rod shape and negative gram stain reaction, are

characteristic of Vibrio species (Lee et al., 2000). The bacteria are thus identified as a

Vibrio species, with the possibility that they are in fact V. alginolyticus according to results

obtained by previous studies (Cheng et al., 1995; Hashimoto and Kamiya, 1970;

Hormansdorfer et al., 2000; Lee et al., 2000; Matsumura, 1995; McEvoy et al., 1998;

Noguchi, 1993; Simidu et al., 1987).

TTX in marine organisms is known to exist in many different isomers, many of which

have now been isolated (e.g., see Asakawa et al., 2000; Endo et al., 1988; Goto et al.,

1965; Hanifin et al., 1999; Wu et al., 1996; Yotsu-Yamashita et al., 1995, 1999a,b). The

term ‘tetrodotoxin’ is frequently used in an umbrella sense to cover not only TTX itself but

also its various isomers and analogues.

The evidence from the spectroscopic, spectrometric and high performance liquid

chromatographic studies all strikingly support the interpretation that there are strong

similarities between the results obtained from the pufferfish, nemertean and bacterial


extracts. The liver of the pufferfish F. rubripes is known to contain high levels of TTX,

and peaks in the spectroscopic and spectrometric nemertean and bacterial extracts similar

to those obtained from the pufferfish are accordingly interpreted as TTX or TTX-like

substances.

This conclusion is supported by the HPLC studies. In an investigation carried out by

Lee et al. (2000), three significant HPLC peaks were obtained which corresponded to time

indices of 8, 16 and 19 min. These peaks represented tetrodoic acid, TTX and 4-epi-

tetrodotoxin, respectively. Different HPLC machines utilise different timescales; some use

the standard 60 s/min, others rely on a system based on 100 s to a minute. Whilst we are

not suggesting that the time index peaks of 4, 12 and 15 min in the present studies enable

us to identify the component substances which gave these peaks, possible differences in

the HPLC equipment used by Lee et al. (2000) and the present studies do allow our

findings to be interpreted as representing TTX-like isomers or analogues.

The question remains as to whether it is the bacteria or the nemerteans which are

biosynthesising these substances. That second generation bacteria show reduced activity in

their extracts compared with those of first generation growths may indicate that bacteria in

isolation tend to lose their ability to synthesise whatever substances are involved. A

reduction in toxicity has been recorded for wild-type toxic pufferfish maintained in

captivity which reverted to a non-toxic condition after approximately 24 weeks in captivity

(Kobayashi et al., 1999). To synthesise TTX, the bacteria may thus require some substrate

either from the nemertean epidermal mucus or from within their body tissues; perhaps

Vibrio species cannot synthesise TTX without their host species. The high degree of

activity evidenced by the mucus of L. longissimus may indicate that it is in this epidermal

secretion that the bulk of the substrate is found.

5. Conclusions

The evidence presented from spectroscopic, spectrometric and chromatographic inves-

tigations strongly suggest that there is some relationship between the presence of Vibrio

bacteria, possibly V. alginolyticus, and the synthesis of tetrodotoxin-like substances in

seven species of British marine nemerteans. The results further suggest that all traces from

both nemertean and bacterial extracts are of the same or closely similar compounds but

that discernible differences in the heights of each curve may indicate either their presence

in different concentrations in each species or the occurrence of different isomers. It is

suggested that whatever the chemical nature of these TTX-like toxins may be, they are

utilised by the nemerteans as a chemical defence against potential predation.

Acknowledgements

We are most grateful to Dr. Greg Elgar of the Fugu genomics project, Cambridge, for

generously providing us with the pufferfish used as control. Thanks are also offered to Dr.

Richard Sands, Liverpool John Moores University, for bacteriological advice and the

identification of the Vibrio species, to Stan Lambert, Colin Armstrong and Martin Wood


for considerable technical assistance, and Drs. Janet Moore and Richard Sands for

providing helpful criticisms and comments about an early draft of the manuscript. [SS]

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The production of tetrodotoxin-like substances by nemertean worms in conjunction with bacteria