ISOLATION

AFFECTING

AND

FEMALE

IDENTIFICATION

THE

MARINE

DEPARTMENT

WOODS

ACCESSORY

MICROBIAL

UNWERSITY

KATHRYN

CUTTLEFISH,

BIoLoGIcAL

HoLE,

OF

DivERsiTy

OF

L.

MASSACHUSETTS

MARINE

NIDAIVIENTAL

OF

PATrERSON

A

GEORGIA

PROBABLE

LABORATORY

SEPIA

SCWNCES

1998

OFFICJNALIS

GLAND BACTERIAL

OF PATHOGEN

conducted

room

necropsy

Materials

Diseased

female

nidamental

The

attributed

pigmented

accessory deaths

months

Introduction

accessory

temperature

The

cuttlefish

have

Most

Mass

at

and

cuttlefish

ISOLATION

TilE

at

to

and

nidamental

the

yellow,

accessory

gland

the

been

carotenoid

plated

female

mortalities

Marine

ACCESSORY

nidamental

Methods

MEL’s

at

for

(Van

attributed

die orange,

the

on

cephalopods

AND

three

mdamental

Biological

gland

shortly

marine

MRC

den

pigments

Microbial

of

IDENTIFICATION

gland

weeks

or

Branden

NIDAMENTAL

female

to

of

lose

red

blood after

a

the

probable

in

Laboratory’s

gland

found

at

in

possess

their

Diversity

female

cuttlefish

adult

onset

the

sexually

et

heart

al.,

characteristic

of

MRC

in

Sepia

of

bacterial

the

cuttlefish,

a

infusion

GLAND

symbiotic

1980).

OF

recently

infection.

laboratory.

(Sepia

mature

accessory

before

A

(MBL)

officinalis

1

PROBABLE

OF

The

pathogen.

agar

officinalis)

dead

manifests

females

a

bacteria

orange-red

FEMALE

Marine

secondary

accessory

nidamental

(MBHIA).

The

is

diseased

orange-red

BACTERIAL

secondary

(Van

that

The

Resources

shortly

CUTTLEFISH,

have

color

transfer nidamental

inhabit

affection,

den

cuttlefish

gland.

Cultures

been

and

after

Branden

in

transfer

PATHOGEN

Center

the

to

color,

occurring

turn

This

spawning.

MEHIA

which

was

were

glands

accessory

SEPIA

a

was

et

gland

and

(MRC).

mottled

swabbed

incubated

al.,

occurs

OFFICINALIS

of

incubated in

this

AFFECTING

was

is

1980).

diseased recent

color

brown.

The

in

at

at

the is

isolate

plates

Results

light

aerobically

(SWC)

program

database.

probable

analysis universal

to

techniques

metal

prepared

electron pathogen,

at

amplify

room

microscopy

within

was

stained

Small,

The

agar

DNA

of

(Bruce

temperature

microscopy

pathogen

for

reverse

Phylogenic

predominantly

isolated

incubated

16S

the

(Microbial

probable

medium

24

TEM

round,

was

with

16S

ribosomal

hours.

Paster,

and

(position

extracted

isolate

on

on

1%

rRNA

mucoid

pathogen

in

observed

(IBM). and

for

analysis

copper

MBHIA,

Diversity

After phosphotungstic

personal

order

four

examined

composed

RNA

were

gene

1492)

from

colonies

24

to

grids

days

Isolated

isolated

in

of

was

compared

was

determine

(rRNA)

hours

communication).

course

a

the bacterial

primers

at

dark

coated

after

conducted

examined

of

sequences

13°C,

with

incubation,

bacterial

on

small

guide,

room

acid

gene

24

were

to

MBF1IA

with

if

colonies

yellow

19°C,

hours

the

16S

(PTA;

rapidly

sequences.

in

1998).

utilizing

with

2

used

plastic

colonies,

was

isolate

order

rRNA

and

light

pigmentation

and

plates

purified

pH

PCR

conducted

for

moving

30°C.

Polymerase

to

48

and

microscopy

7).

was

light

gene

sequencing.

assess

products.

diluted

was

Universal

hours

carbon.

bioluminescent.

on

microscopy

Cultures

rods

sequences

plated

IvIBITIA

using

bioluminescent

of

were

in

with

chain

incubation.

TEM

analysis

sterile

Sequences

forward

on

Nucleotide

the

detected

were

characteristic

present

seawater

using

reaction

RNA1

and

samples

seawater

examined

revealed

(position

SWC

transmission

standard

on

obtained

The

activity

in

computer

sequence

(PCR)

complete

the

were

the

plates

probable

(80%),

gaps

that

MBHIA

utilizing

Genbank

8)

heavy

for

was

and

the

were

in

were

the

used the

cultures

were

morphologies

incubator.

grew

was

only

other

that

were

2).

not

and

included

bacterial

cytoplasm

Curved

change

observed

the

rods

white

95%

well

present

species

The

Nucleotide

TEM

grown

cuttlefish

that

non-motile

morphologies

and

at

Cultures

on

(Figures

probable

after

rod-shaped

room

in

sequenced

showed

observed at

exhibited

SWC

96%

on

fewer

19°C.

24

isolate

SWC

temperature,

sequence

similarity

grown

hours

agar

la

pathogen

rods

the

numbers.

The

and

on

bacteria

spiral-like

plates

(Figures

(Figure

medium is

predominance

lacking

and

MBBIA

at

more

lack

ib).

analysis

13°C

to

incubated

48

isolate

and

lacking

V. 4).

of

Microscopy

similar

ic

hours

and

gaps

motility.

logeii

were

growth

incubated

may

and

However,

of

grew

were

in

of

the

flagella

of

id).

pigmented

to

be

at

and

the

incubation.

rod-shaped

at

Vibrio

13°C

cocci,

on

attributed

16S

The

The

conducted

cytoplasm,

19°C

V.

at

SWC

the

3

room

salmonicida,

rRNA

(Figure

appearance

and

additional

some

salmonicida

sequence

was

yellow

agar

30°C

to

temperature.

bacteria

gene

unexpected,

of

after

3)

inaccurate

long

medium

which

did

were

and

for

(500

morphological

of

48

respectively.

thin

not

and

with

resembled

colony

the

hours

also

were

base

at

exhibit

“string-like”

temperature

Vibrio

probable

Cultures

considering

single

13°C

observed

pair

incubation

cultures

in

chains.

bioluminescence.

the

logeii

and

poiar

sequence)

types

pathogen

grown

mixed

with

30°C.

on

non-motile

control

that

than

flagella

observed

Bacterial

showed

MBIHA

TEM

at

bacterial

the

to

showed

No

30°C

showed

by

any

isolate

(Figure

growth

but

mixed

the

rods, did

the

with

be

from

recently

However,

or

V.

4).

to

sequence

harveyii,

fish

isolated

V.

they

isolated

Vibrio

Discussion

light

Hitra

salmonicida

eachother

diseased

salmonicida

tufts

and

the

are

Vibrio

organ

Vibrio

spp.

Disease

dead

Nucleotide

cuttlefish

from

shellfish.

known

from

of

V.

analysis,

pleomorphism

cuttlefish

two

are

ordali,

than

fish

symbionts

logeii

skin

salmonicida

the

are

common

and

to

pathogens

(Egidius

to

(Egidius

diseased

appears

five

motile

lesions

The

V.

and

sequence

are

V.

the

is

salmonicida,

logeii

polar

other

bioluminescent

similar

other

V.

of

has

inhabitants

et

curved

and

to vulnificus

et

causes

cuttlefish

affecting

Sepiolid

al.,

been

flagella

be

are

pathogens

at.,

analysis

Vibrio

kidneys

to

1986;

pleomorphic.

two

rods

1986).

V. a

observed

V.

serious

squid

marine

spp.

of

logeii

(Benediktsdottir

is

of

(Bang

Enger logeii,

with

of

of

of

bacteria

marine

eight

include

Like

the

known

fish

the

(Fidopiastis

in

at

disease

in

et

organisms.

16S

et

and

species

that least

genus

V.

(Egidius

al.,

strains

4

environments

al.,

(Bang V.

salmonicida,

to

rRNA

the

both

1986).

alginolyticus,

nine

1989;

be

of

Vibrio

cuttlefish

of

of

et

salmonid

pathogenic

et

et

Vibrio

et

polar

gene

al., Vibrio

Pathogenic

V.

al.,

Fanner

al.,

al.,

The

salmonicida

and

1998).

1998).

suggests

1978)

(Benediktsdottir

flagella

1986;

spp. the

probable

pathogen

shown

most

fishes

V.

and

probable

to

exhibit

and

According

anguillarum,

Benediktsdottir

Vibrio

marine

V.

probably

Hickman-Brenner,

(Egidius

that

to

called

logeii

pathogen

were

isolated

are

cause

yellow

the

spp.

pathogen

organisms

more

cold-water

recently

are

et

probable

is

et

to

disease

have

from

al.,

a

al.,

motile

isolated

V.

16S

pigmentation

closely

new

et

damsela,

1998),

isolated

been

1986).

kidneys

found

rRNA

al.,

in

(Figure

species.

pathogen

rods vibriosis

1992).

marine

related

from

1998).

and

to

V. of

nidamental

probe

bacteria

(FISH).

should

pleomorphic,

represent

types

needed

diseased

sequence

Suggestions

et

also

exoskeleton

bioluminescent

(Bang

al.,

been

in

of

be

1989;

The

to

Further

et

isolated

order

bacteria V.

Vibrio

cuttlefish.

different

compared

analysis

al.,

confirm

isolated

gland

logeii

bacterial

Farmer

(Farmer

for

like

to

1978).

spp.

studies

from

determine

organism.

observed

of

Future

has

its

of

that

forms

from

Sepia

are

to

Biochemical

contents

closest

and

the

been

However,

an

and

isolates

the

should

gamma-proteobacteria,

scallops,

apparently

probable

Hickman-Brenner,

Research

of

officinalis.

Hickman-Brenner,

from

shown

isolate

if

the

relative,

the

of

be

from

same

unlike

the

accessory

probable

conducted

analysis

fish

to

is

pathogen

MBHIA

healthy

indeed

be

diseased

Vibrio

species

intestines,

V.

pathogenic

logeii,

of

pathogen

nidamental

a

cuttlefish

salmonicida.

isolated

to

1992).

plates

the

new

and

cuttlefish.

1992;

confirm

5

and

the

isolate

and

hence

species

in

should

FISH

Benediktsdottir

is probable

from

marine

tanner

utilizing

glands

a

the

and

indicate

normal

A

could

of

the

be

useful

results

further

crabs,

sediments

Vibrio.

studied

pathogen

accessory

of

fluorescent

be

inhabitant

that

apparently

analysis

obtained

conducted causing

microscopic

et

the

The

to

al.,

(Bang

appears

determine

nidamental

probable

different

1998).

would

of

in

necrotic

by

healthy

situ

the

utilizing

et

16S

not

examination

al.,

accessory

be

hybridization

This

pathogen

if

morphological

rRNA

to

cuttlefish

gland

lesions

to

1978;

they

be

a genus

examine

gamma

a

of

gene

Enger

of

is

the

has

is the

Koch’s

agent cause

activity. bioluminescence, isolate

of

of

is

Finally,

Further

postulates

the

the

bioluminescent.

disease

accessory

studies

the

this

should

isolate

causing

does

nidamental

should

be

obtained

Although

not

the

employed

be

prove

mass

carried

gland

from

the

mortalities

that

to

isolates

infection

out

the

definitively

this

to

diseased

new

6

definitively

incubated

of

and

Vibrio

cuttlefish

prove

cuttlefish

not

sp.

the

on

show

that

is

at

result

SWC

incapable

the

must

the

whether

MRC.

isolate of

did

be

a

not

shown

secondary

of

the

is

demonstrate

bioluminescent

indeed

probable

to

be

infection.

the

the

pathogen

causal primary n 0 0

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Photobacterium

Vibrio

Vibrio

Vibrio

Vibrio

Vibrio

Cuttlefish

Vibrio

damsela

logei

alginolyticus

ordalil

harvey/i

mar/n

sa/monicida

fisher/i

metschniko

capsulatus

vulnificus

anguillarum

Roseobacter

us

(Sepia

angustum

Vibrio

leiognathi

phosphoreum

vii

officina/is)

cholera

denitrificans

pathogen WH51

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