Akhurst, R.J.

Horizon / Pleins textes

A Xenorhabdus sp. (Eubacteriales : Enterobacteriaceae) 
symbiotically  associated  with Steinernema kraussei 
(Nematoda : Steinernernatidae) 
Raymond J. AICHURST 
CSIRO, Division of Entomology,  Stowell   Aue.,   Hobart,   Tasmania,   Australia,  7000. 
SUMMARY 
Examination of Stein.ernerna kraussei recently collected from  the field revealed that  the  bacterial  symbiont of 
these  nematodes  was a Xenorhabdus sp. and  not  the Flavobacterium sp.  reported  by MrBEelr (1977).  Like Neoaplec- 
f a n a  bibionis, S. kraussei infectives carry cells of the  bacterium  in a specialised vesicle in  the  foregut. 
RÉSUMÉ 
Xenorhabdus sp.  (Eubacteriales : Enterobacteriaceae)  associé  symbiotiquement à Steinernema  kraussei 
(Nemaioda : Steinernematidae) 
L’examen  de spécimens de Steinernema kraussei, fraîchsment récoltés au  champ, a montré  que  leur  symbionte 
bactérien  était  un Xenorhabdus sp.  et  non  un Flavobacterium sp., comme il  l’avait  été signalé par MraEek (1977). 
Gomme Neoaplectana bibionis, les S. kraussei infectés transportent les cellules bactériennes  dans  une vésicule di€- 
férenciée située à la partie  antérieure  de  l’intestin. 
Insect pathogenic nematodes of the families Stei- 
nernematidae and Heterorhabditidae are symbioti- 
cally  associated  with specific bacteria.  The  non- 
feeding, free-living infective stage nematode carries 
cells of its bacterial symbiont monoxenically in the 
intestine.  After  penetrating  an  insect  host,  he 
nematode  migrates  to  the  haemocoel  where it 
releases  its  symbiont.  The  bacteria  proliferate, 
killing the insect and providing suitable conditions 
for  growth  and  reproduction of the  nematode  (Poinar, 
1979). 
MrAEek (1977)  identified  nematodes  in  diseased 
Cephalia abietis  L. from ceskjr Rudolec, Czechoslo- 
valria as Steirzerrzema  kraussei (Steiner)  and  their 
bacterial symbiont as a Flavobacterium sp. MraCek 
sent a monoxenic  culture of S. kraussei and the 
Flavobacterium sp. to this laboratory where it was 
found that the bacterium was quite different from 
al1 other  bacteria  symbiotically  associated  with 
Steinernematidae  and  Heterorhabditidae  (Akhurst, 
unpubl.). Moreover, although Xenorhabdus  could be 
readily seen in smear preparations of and isolated 
from the intestine of the infective stage of other 
steinernematid  species,  the Flauobacteriurn could 
neither  be  seen  in  nor  isolated  from S. krausse i  
infectives. It was  concluded tha t   the  Flavobacterium 
sp. may  not  be  the  symbiont of S. kraussei.  
At  the  author’s request, Dr. MrACek lcindly col- 
lected  more of these  nematodes  from  ceskjr  Rudolec 
for  further  examination.  This  paper  eports  the 
results of this  examination. 
Materials and methods 
The S. kraussei used in this study were obtained 
in  1980  by Dr. Z. MrACelc, CSAV, ceské  Budzjovice,. 
Czechoslovalria. He collected  diseased C .  abietis from 
the field, harvested infective stage nematodes pro- 
duced within them and supplied these infectives to 
this  laboratory. 
The  intestine of infective  stage  nematodes  was 
examined for bacteria after extrusion and staining 
by  the  method of Poinar  (1966). 
Bacteria were isolated  from  surface-sterilised 
infective stage nematodes by homogenisation (Ak- 
Kevue   Némato l .  5 ( 2 )  : 277-280 (1982) 277 
R. J .  Akhurst 
hurst, 1980). The  homogenate  was  streaked  ont0 
nutrient agar + 0.0025%  (w/v)  bromothymol  blue + 
0.004~0  (w/v)  triphenyltetrazolium chloride (NBTA) 
and incubated a t  280. 
Transfer of Xenorhabdus  by S. kraussei from one 
insect to  another  was  tested. Galleria mellonella (L.) 
larvae were buried in moist sterile sand in 100 ml 
specimen jars and 100 S. kraussei infectives  were 
pipetted  ont0  the surface.  After five days a t  200 
haemolymph  from  two  cadavers was streaked  on 
NA. When infective nematodes were released from 
the remaining cadavers, they were  examined  and 
sampled for bacteria as described above. 
Cells of the  bacterium  harvested  from  24  h  nutrient 
broth  cultures  and  iluted  with  sterile  Ringer’s 
solution (Cruickshank, Duguid & Swain, 1970) were 
injected into the haemocoel of G. mellonella larvae 
to assess pathogenicity.  Seven dosages of each  species 
and a Ringer’s solution control were injected into 
20  larvae/dosage. 
Bacteria isolated from infective nematodes, from 
the haemocoel of infected insect cadavers and from 
monoxenic  cultures  established  by  the  method of 
Bedding (1981) and bacteria isolated from MraCek’s 
original  monoxenic  cultures were  identified from  the 
results of a number of tests  conducted a t  280. Mean 
ce11 dimensions were estimated by measurement of 
50  cells in  wet  mounts of 24  h  culture  in  yeast-salt,s 
broth (YSB) (NH,H,PO,, 0.5 g ; K,HPO,, 0.5 g ; 
MgS04.7H,0,  0.2 g ; NaCl,  5.0 ; yeast  extract,  5.0  g ; 
water 1 1 ; Dye, 1968). Motiljty was  assessed by  
examination of hanging  drops of 24  h  YSB  cultures 
and flagella position  determined  by  transmission 
electron microscopy . after negatively staining cells 
from  24  h  YSB  cultures.  Air  dried films of 24 h  Y  SB 
cultures  were  stained  for  the  Gram  reaction. Colony 
and  cultural  characteristics  were  studied  on  nutrient 
agar, NBTA,  Simmons  cihate agar (Difco) and  triple 
sugar  iron  agar  (Difco). 
Examination for  bioluminescence  was  made in 
total  darkness  with  48 h nutrient  agar  cultures. 
Moeller’s (1955)  method  was used to  test for  arginine 
dihydrolase and lysine, ornithine and glutamic acid 
decarboxylases. Cytochrome oxidase was tested by 
Schaeffer’s (1961) method, phosphatase as described 
by Cowan and  Steel  (1974)  and  phenylalanine  deami- 
nase  by  the  method of the Difco Supplement  (1962). 
Gelatin  hydrolpsis  was  tested  in Oxoid nutrient 
gelatin and casein hydrolysis on Dye’s  (1968)  OY 
agar containing  10%  (w/v)  skim  milk.  Methods 
described by  Dye (1968)  were  used  for the  remaining 
tests. 
Antibiotic production was tested by the method 
of Poinar,  Hess  and  Thomas  (1980)  with  twenty 
species of bacteria. 
Dr. R.Bedding, CSIRO, examiged adult nematodes .-
278 
~ 
and confirmed that  these were of the  same species as 
those origihally sent in monoxenic culture with the 
Flauobacterium sp.  by  Dr. MrAEek. 
Results 
S. kraussei infectives contained a vesicle within 
the foregut (Fig. 1A) similar to that described for 
Neoplecfana  bibionis  by Bovien  (1937)  (Fig. 1 B). 
Bacteria were seen in the vesicle in 15/16 infectives 
from C .  abietis parasitised in the field, 20/20 from 
G. mellonella parasitised by  such  infectives  and  19/20 
from monoxenic cultures wjth the Xenorhabdus  sp. 
Bacteria  could  not be seen in  any of over  100 infec- 
tives  from  monoxenic  culture  with  the Flauobacteriunz 
sp. or isolated by homogenisation of surface  sterilised 
infectives  (100  from  each of three  such  cultures). 
The  bacterium  isolated  from  infectives  sent  by 
Dr. MraCek was  found to  be the  same as tha t  which 
overwhelmingly  dominated the flora of the G. mello- 
nella cadavers  and as tha t  recovered  from the infec- 
tives  produced  from  these  cadavers. 
Characteristics of the  bact4erium  isolated  from  the 
intestine of infective stage S. kraussei and of the 
Flauobacterium sp.  isolated  from ,MrBEek’s mono- 
xenic  cultures  are  listed  in  Tab.  1. On nutrient agar, 
the Flavobacterium sp. formed two colony types ; 
one  was  convex,  circular  with a smooth  margin,  and 
1 mm diameter at four days while the other was 
similar but surrounded by a flat, irregular apron. 
Colonies  formed by  the  bacterium  isolated  from 
infective  stage S. kraussei were  convex,  circular  with 
slightly  irregular  margin, 1.5-2 mm  diameter  at 
4d and slightly granular. Two colony types were . 
obtained  from  monoxenic  cultures of S. krartssei and 
the  bacterium isolated  from the  infective  stage ; one 
colony  type  was  as  described  for  the  bacterium 
isolated  from  the  infective  stage  while  the  other  was 
flatter, wider  (2.5-3.5 mm  at  four  days)  and less 
intensely  pigmented  on NA. The  latter  type  did  not 
absorb bromothymol blue from NBTA. 
While  intrahaemocoelic  injection  with  100 cells 
of the  bacterium  isolated  from  infective  stage 
S. kraussei was  inevitably  fatal  for G.  mellonella 
larvae  (LD50 = 16.5),  injection of 25 O00 cells of the 
Flauobacterium sp.  did not kill any G. mellonella 
larvae. 
Discussion 
The  bacterium  isolated  from  the  intestine of 
infective  stage S. kraussei conforms substantially  to 
the description of the genus Xenorhabdus  given by 
Thomas  and  Poinar -. (1979). . - It . also ~~ occurs  in  two 
Revue  Nèmatol. 5 ( 2 )  : 277-280 (1982) 
- --. - - -  
Table 1 
Xenorhabdus sp. symbiotically  associated  with Steinernema  kraussei 
.,... . ,,.,. . " " ,  '. ,.. ".. " ."., .,i 
Comparison of bacteria  isolated  from  intestinal  lumen 
of infective  stage S.  kraussei and of a Flavobacterium 
sp.  previously reported  as  being  symbiotically 
associated  with S. kraussei 
Characteristic  Bacterium Flavobacterium 
isolated f r o m  sp. isolated from 
intestinal  Mracek's 
l umen  of 
infective  stage 
monoxenic 
cultures 
S. kraussei 
No. isolates  6 
Ce11 morphology ' Rod 
Mean ce11 length 
Mean ce11 width 
(range) 4.8 (1.6-12.1) pm 
(range) 
Motilitv 
1.1 (0.8- 1.6) pm + 
Flagella Peritrichous 
Pigment  (nutrient 
Gram  stain 
Anaerobic growth -k 
Catalase - 
Oxidase ' 
Cytochrome oxidase 
' Hugh & Leifson-open -k 
Hugh & Leifson- 
Nitrate  reduction 
Indole 
MethyJ red 
Acetoin 
KCN, inhibition  by + 
Simmons citrate 
TSI  (slope/butt/H,S)  alkaline/acid/- 
Arginine dihydrolase - 
Lysine decarboxylase - 
Ornithine 
decarboxylase 
Glutamic acid 
decarboxylase - 
Phenylalanine 
Gelatine liquefaction 
deaminase - + 
Bioluminescence 
Absorbtion of BTB .r (l) 
Urease - 
Phosphatase 
Casein hydrolysis + 
Aesculin hydrolysis + 
Antibiotic  production + 
Carried within 
intestine of + 
Infective  stage 
S. kraussei 
agar) yellow 
- 
- 
- 
closed + .  
- - 
- - 
- 
- 
- 
2 
Rod 
2.7 (1.6-3.2) pm 
0.5 (0.3-0.5) pm 
none 
yellow 
- 
- 
+ + 
-t + 
- 
- 
- 
- 
- 
- 
- 
nc (,))lm+ 
- 
- - 
- 
- 
4- - 
- 
- + + 
- 
- - 
- 
(l) By one colony only. 
(z) No change. 
Revue Ndmatol ,  5 ( 2 )  : 277-280 (1982) 
Fig. 1. Ventricular  portion of the  intestine of the 
infective  stage  after  extrusion  by  the  method of 
Poinar  (1966)  and  staining  with  safranin ( x 100). 
A : Steinernema kraussei ; B : Neoaplectana bibionis ; 
b : bacteria ; O : basal  bulb of oesophagus ; v : vesicle. 
forms  as  described  by  Alrhurst  (1980)  for Xerzorhabdus 
spp.  and  the  primary  form,  like  the  primary  form of 
Xenorhabdus  isolated  from  infectives of seven  species 
of steinernematid  and  heterorhabditid  nematodes 
(Akhurst,  unpubl.),  produces  an  antibiotic  factor. 
This  bacterium is clearly  a Xerzorhabdus sp.  and  very 
different  from  the Flavobacteriurn sp.  originally 
thought by Mr5Eek (1977) to be the symbiont of 
S. kraussei  (Tab. 1). The  discovery of a Xerzorhabdus 
sp. symbiotic  with S. kraussei means  that  infectives 
of each species of nematode of the families Steiner- 
nematidae, and Heterorhabditidae so far examined 
(Thomas & Poinar, 1979 ; Akhurst, unpubl.) carry 
a  bacterium of this  genus  monoxenically  within  their 
intestines. 
279 
R.J. Akhurs t  
The Flar1obacterium sp.  originally  considered  sym- 
biotic  with S. kraussei does not  have  the  same  rela- 
tionship  with  the  nematode  as does the Xenorhabdus .  
This latter bacterium is transported from cadaver 
t o  fresh host, in a specialised organ of the infective’s 
intestine ; the Flavobacterium sp.  cannot  be so carried. 
The  ability of the  nematode  to  grow  and  reproduce 
in  monoxenic  culture  with  the Flavobacterium sp. is 
of little significance since in  such  cultures  fecundity 
is  much lower than  in  cultures  with  the X’erzorhabdus 
sp.  and  several Neoaplectana spp.  have  also  been 
cultured  monoxenically  with  this Flavobacferium 
sp. (Akhurst, unpubl.). Moreover, the Xenorhabdus  
sp. was highly pathogenic for insects and able to 
inhibit  the  growth of other  microorganisms while the 
Flavobacterium sp.  was  not. 
The bacterial symbiont was obviously lost. from 
the S. krausse i  described by MraCek (1977). This is 
not  the  only  instance of an  unwitting  separation of a 
steinernematid  nematode  and  its  bacterial  symbiont ; 
the  symbiont of Neoaplectana  glaseri  and of an  
undescribed Neoapleclana sp. (Akhurst,  unpubl.)  have 
also been accidentally lost. The potential of these 
nematodes as control agents cannot be adequately 
tested  in  the  absence of their  symbionts. Conse- 
’ quently it is most important that the symbiont be 
isolated as soon as possible after  detection of a new 
steinernematid population. Since the infective stage 
of steinernematids  contains  only  the  symbiotic 
bacterium  and  otker  stages may also contain a 
number of bacterial species from  t,he  insect  cadaver 
(Poinar, 1966 ; Akhurst,  unpubl.),  the  symbiont 
should be isolated from surface sterilised infectives. 
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- - - .- - -  - ~- . 
Revue   Némato l .  5 (2)  : 277-280 (1982) 


A Xenorhabdus sp. (Eubacteriales : Enterobacteriaceae) symbiotically associated with Steinernema kraussei (Nematoda : Steinernematidae)

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