- Email: [email protected]
Two symbiotic bacteria of the entomopathogenic nematode Heterorhabditis spp. against Galleria mellonella
Accepted Manuscript Two symbiotic bacteria of the entomopathogenic nematode Heterorhabditis spp. against Galleria mellonella Chunli Liao, Along Gao, Bingbing Li, Mengjun Wang, Linna Shan PII:
S0041-0101(16)30601-8
DOI:
10.1016/j.toxicon.2016.11.257
Reference:
TOXCON 5513
To appear in:
Toxicon
Received Date: 20 August 2016 Revised Date:
17 November 2016
Accepted Date: 23 November 2016
Please cite this article as: Liao, C., Gao, A., Li, B., Wang, M., Shan, L., Two symbiotic bacteria of the entomopathogenic nematode Heterorhabditis spp. against Galleria mellonella, Toxicon (2016), doi: 10.1016/j.toxicon.2016.11.257. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Short communication
2
Two symbiotic bacteria of the entomopathogenic nematode Heterorhabditis spp. against Galleria mellonella
3
Chunli Liao*, Along Gao, Bingbing Li*, Mengjun Wang, Linna Shan
4
College of Life Science and Engineering, Henan University of Urban Construction, Pingdingshan, China
5
*Corresponding author: Bingbing Li or Chunli Liao
6
Address: College of Life Science and Engineering, Henan University of Urban Construction, Pingdingshan 467036,
7
Henan, China
8
E-mail: [email protected] (B. Li), [email protected] (C. Liao)
9
Tel.: +86-0375-2089090;
10
Fax: +86-0375-2089090;
SC M AN U
11 12 13 14 15
20 21 22 23 24 25
EP
19
AC C
18
TE D
16 17
RI PT
1
26 27 28 29
1
ACCEPTED MANUSCRIPT Abstract: The entomopathogenic nematode Heterorhabditis spp. is considered a promising agent in the biocontrol of
31
injurious insects of agriculture. However, different symbiotic bacteria associated with the nematode usually have
32
different specificity and virulence toward their own host. In this study, two symbiotic bacteria, LY2W and NK, were
33
isolated from the intestinal canals of two entomopathogenic nematode Heterorhabditis megidis 90 (PDSj1 and PDSj2)
34
from Galleria mellonela, separately. To determine their species classification, we carried out some investigations on
35
morphology, culture, biochemistry, especially 16S rDNA sequence analyses. As a result, both of them belong to
36
Enterobacter spp., showing the closest relatedness with Enterobacter gergoviae (LY2W) and Enterobacter cloacae
37
(NK), respectively. Moreover, the toxicity to Galleria mellonella was examined using both the metabolites and washed
38
cells (primary and secondary) of these two strains. The results indicated both metabolites and cells of the primary-type
39
bacteria could cause high mortalities (up to 97%) to Galleria mellonella, while those of the primary-type bacteria only
40
killed 20%. These findings would provide new symbiotic bacteria and further references for biological control of the
41
agricultural pest.
42
Keywords: Entomopathogenic nematode, Symbiotic bacteria, Galleria mellonella, Toxicity, Biological control
M AN U
SC
RI PT
30
43 44
48 49 50 51 52 53 54
EP
47
AC C
46
TE D
45
55 56 57 58 59 2
ACCEPTED MANUSCRIPT 60
1.
Introduction Galleria mellonella (the greater wax moth or honeycomb moth) is a moth of the family Pyralidae, which is the only
62
member of the genus Galleria (Wand et al., 2015). It can be found in most of the world, including Europe and adjacent
63
Eurasia, its presumed native range, and as an introduced species on other continents, including North America and
64
Australia (Wand et al., 2015). This moth flies from May to October in the temperate parts of its range, such as Belgium
65
and the Netherlands. The caterpillar larvae, or wax worms, feed on the honeycomb inside bee nests and may become
66
pests of apiculture (Olsen et al., 2011). Less often, they are found in bumblebee and wasp nests or feeding on dried figs
67
(Wand et al., 2015). Current control recommendations for this injurious insect are focused on chemical pesticide usages
68
(Malik et al., 2012). Due to the difficulty in controlling this insect with chemical insecticides and the additional
69
environmental pollution associated with their use, the development of biological control alternatives is extremely urgent.
SC
RI PT
61
Entomopathogenic nematodes in the Steinernematidae and Heterorhabditidae families are lethal parasites of a large
71
number of insects (Rahimi et al., 1993) and have been used effectively to control many burrowing insects (Ali and
72
Wharton, 2015; Ansari and Butt, 2012; Ayaad et al., 2001). Entomopathogenic nematodes have a symbiotic association
73
with bacteria in the Enterobacter (Bisch et al., 2015), which can be used as a biocontrol alternative of Galleria
74
mellonella (Puza and Mracek, 2009). However, existing as the bacterial symbionts of nematode, these bacteria, therefore,
75
cannot be isolated directly from the natural environment. They are not described until the 1960s and therefore regarded
76
as a new special Enterobacter (Puza and Mracek, 2009).
TE D
M AN U
70
To date, the described mutualistic bacteria have been classified into two genera, Xenorhabdus spp. and
78
Photorhabdus spp., associated with the entomopathogenic nematodes Steinernema spp. and Heterorhabditis spp.,
79
respectively (Hsieh et al., 2009; Park and Kim, 2000). Steinernema spp. carry in Xenorhabdus spp., and Heterorhabditis
80
spp. carry throughout Photorhabdus spp. (Park and Kim, 2000; Rahimi et al., 1993). The infective nematode juveniles
81
actively search for hosts and penetrate through their natural openings (anus, mouth, and spiracles) (Park and Kim, 2000).
82
Once inside, these nematodes will release pathogenic bacteria which rapidly multiply and then cause the death of the
83
insect usually within 24-48 h (Fairbairn et al., 2000; Hebert et al., 2014). The bacteria also release other pathogenic
84
factors which will cause immune system depression of their host and will inhibit the growth of non-symbiotic
85
microorganisms (Yi et al., 2015). Therefore, nematodes have been widely used against larvae of different curculionid
86
species, such as Curculio elephas (Shapiro-Ilan et al., 2006), Otiorhynchus sulcatus (Westerman, 1998), Diaprepes spp.
87
(Shapiro-Ilan et al., 2010), Rhytidoderes plicatus (Rahimi et al., 1993), and Curculio caryae (Shapiro-Ilan et al., 2006).
88
Therefore, the full investigation and exploitation of entomopathogenic nematodes and their symbiotic bacteria are of
89
great significance for biological control of pest and environmental protection.
AC C
EP
77
3
ACCEPTED MANUSCRIPT 90
Therefore, our objectives were (i) to isolate several native symbiotic bacteria of the entomopathogenic nematode
91
against Galleria mellonella and (ii) to determine the virulence of the symbiotic bacteria to the larvae of this insect in
92
order to provide further references for the control of Galleria mellonella. We expect the knowledge obtained will enable
93
the bio-insecticide of Galleria mellonella to be improved and provide effective bioresource species for the biocontrol of
94
injurious agricultural insects.
95
2.
96
2.1 Isolation of bacteria, media and culture
RI PT
Materials and methods
Two entomopathogenic nematodes (Heterorhabditis spp. PDSj1 and Heterorhabditis spp. PDSj2) were originally
98
collected from a soil sample in a suburb field, Pingdingshan, China, and both were finally identified as Heterorhabditis
99
megidis 90 by the third party (Sangon Biotech, Shanghai, China). The obtained Heterorhabditis spp. PDSj1 and
100
Heterorhabditis spp. PDSj2 were surface sterilized using 10% (v/v) formaldehyde and then cleaned using sterilized water.
101
After that, they were suspended in 0.2% thimerosal (Sigma) for 30 min and then washed with sterilized water prior to
102
inoculation onto nutrient agar plates (214010/214030 Agar, Bacto, BD, USA), supplemented with 0.025% (w/v)
103
bromothymol blue and 0.004% (w/v) triphenyl tetrazolium chloride (referred to as NBTA). The plates were finally
104
maintained at 28 oC for 3-5 days in the dark. Colonies that faded bromothymol blue on the NBTA plates were regarded
105
as symbiotic bacteria associated with Heterorhabditis spp. (Hsieh et al., 2009). They were then collected and stored at
106
-20 oC in TSB containing 20% (v/v) glycerol for further identification.
107
2.2 Phenotypic characteristics of bacterial isolates
TE D
M AN U
SC
97
Symbiotic bacterial isolates were subcultured on NA and NBTA plates. After 24 h maintenance at 28 oC, the
109
characteristics of morphology and pigment absorption were recorded timely. Isolates from randomly selected colonies
110
were Gram-stained and their morphology was studied with the aid of a research microscope (optical and electronic
111
microscopes). Biochemical identification referred to the methods in Manual of Bacterium Identification.
112
2.3 Phylogenetic analyses of isolates
AC C
EP
108
113
Genomic DNA of the isolates was extracted using a ZR bacterial DNA kit (Biotechnology Research Corporation,
114
New York, USA). Polymerase chain reactions (PCR) were performed using TaKaRa Ex TaqTM, 10 Ex TaqTM buffer and
115
deoxynucleoside triphosphate (dNTP) mixture (Takara Bio Inc., Shiga, Japan). According to the 16S rDNA of symbiotic
116
bacteria
117
5′-GAGCGGATAACAATTTCACACAGG-3′ and 5′-CGCCAGGGTTTTCCCAGTCACGAC-3′, respective (Fischer-Le
118
Saux et al., 1999). The amplification volume was 50 µL and the PCR conditions were as follows: 30 cycles of
119
amplification, after an initial 5 min denaturation step at 94 oC; each cycle consisted of 1 min at 94 oC, 1 min at the
in
GenBank,
the
upstream
and
4
downstream
primers
were
designed
as
ACCEPTED MANUSCRIPT annealing temperature of 55 oC and 1.5 min at 72 oC. Amplified products were ligated into a PMD18-T vector (Promega
121
Corporation, Madison, Wisconsin, USA), cloned into E. coli DH5α and sequenced by the third party (DALIANBAO
122
Biotechnology Corporation, Dalian, China). Sequences were analyzed using BLAST (Basic Local Alignment Search
123
Tool, National Center for Biotechnology Information, National Library of Medicine, Bethesda, Maryland, USA) and
124
compared with known sequences in GenBank. Phylogenetic trees were established using MEGA (Molecular
125
Evolutionary Genetics Analysis) soft, version 6.06, according to the Neighbor-Joining method.
126
2.4 Virulence test design
RI PT
120
Symbiotic bacterial isolates were maintained in Luria-Bertani (LB) Broth at 28 oC for 24 h (shaking speed:
128
180r/min). The fermentation broth was centrifuged (1000 g) at 4 oC for 10 min and the supernatant was then filtered (pore
129
size 0.22 µm) under the sterile condition, which therefore contained active metabolites of cells. The larvae of Galleria
130
mellonella were cleaned using the sterile water and were injected separately with 10 µL filtered fermentation broth and
131
10 µL washed cells (106 CFU/mL) of which concentration was corresponding to that of fermentation broth. Each
132
treatment included 30 larvae and was set in triplicate. Larvae in control group were injected using 10 µL sterile LB broth
133
or saline water (0.85% m/v). All larvae were finally maintained in sterilized Petri dishes at 28 oC and their death was
134
recorded every 2 h. The mortality (M) and corrected mortality (CM) of larvae were calculated by the following formulas:
135
M (%) = n/N*100%
136
CM (%) = (M1-M0) / (1-M0) *100%
137
Where n=number of dead larvae, N=total number of larvae, M1=mortality of the treatment, and M0=mortality of the
138
control.
139
3.
140
3.1 Morphological characteristics of isolates
TE D
(1)
(2)
EP
Results
M AN U
SC
127
All colonies (about twenty) absorbed bromthymol blue on NBTA plate and were Gram-negative rods with most
142
isolates belonging to well-reported Photorhabdus spp. However, two isolates (LY2W and NK) shown differences in
143
other phenotypic properties. The morphological characteristics of LY2W and NK, when subcultured on NA and NBTA
144
plates, were shown in Table 1 and Fig. 1. After 2-day maintenance at 28 oC, both LY2W and NK colonies did not
145
generate bioluminescence whether on NA or on NBTA, which therefore indicated these two strains did not belong to
146
Photorhabdus spp. Moreover, their primary types had cloudily-spread colonies while their secondary types not. This also
147
indicated that the primary type of these strains was capable of motility while the secondary not.
148
3.2 Biochemical characteristics of LY2W and NK
AC C
141
5
ACCEPTED MANUSCRIPT The biochemical reactions in both primary and secondary type strains were shown in Table 2. As a result, both types
150
had positive reactions with catalase, maltose, cellobiose and nitratase. They were able to utilize citrate, but not able to
151
liquefy gelatin and produce esterase, oxidase as well as tryptophan reductase. The isolate NK not only hydrolysed
152
esculoside and starch but had a positive reaction with lecithinase and methyl red, while the isolate LY2W not. Isolate NK
153
could hydrolyze urea while only the primary type of isolate LY2W could. However, the primary type of isolate LY2W
154
generated phenylalanine reductase while both types of isolate NK not. These biochemical characteristics showed that
155
isolates LY2W and NK had the closest relationship to Enterobacter gergoviae and Enterobacter cloacae, respectively.
156
3.3 Phylogenetic relatedness of LY2W and NK
RI PT
149
The 16S rDNA sequences of the two strains were compared with sequences of representative strains of different
158
species in the Genbank. All sequences from both strains LY2W and NK showed a high percentage of homology (above
159
99%) to the sequences derived from three genera: Pantoea spp., Klebsiella spp., and Enterobacter spp. (Fig. 2).
160
However, based on the above biochemical identification both Pantoea spp. and Klebsiella spp. were excluded. Moreover,
161
the analyses of MEGA 6.06 and Neighbor-Joining indicated strains LY2W and NK had the closed relationships
162
(confidence coefficient: 100%) with Enterobacter spp. (Fig. 2). Therefore, combined with the results of biochemical
163
reaction, we concluded that strains LY2W and NK might be the same species to Enterobacter gergoviae and
164
Enterobacter cloacae, respectively.
165
3.4 Virulence of metabolites of LY2W and NK to G. mellonella
TE D
M AN U
SC
157
The mortalities of G. mellonella caused by the metabolites of strains LY2W and NK were shown in Fig. 3a. As a
167
result, the metabolites of primary type strains performed significantly higher mortalities (97-100%) than those of the
168
secondary type (about 20%). Moreover, virulence of metabolites of the NK primary type strain increased fastest within
169
6-8 hours after injection, and then reached the highest (100% mortality). However, mortalities caused by metabolites of
170
the primary type of strain LY2W increased slowly from 10% to 50% between 2 and 10 hours after injection and then
171
increased faster to about 97% within 16 hours. For the metabolites of the secondary type strains, their virulence to G.
172
mellonella was not significant or rapid, only starting to increase after 8-10 hours and finally remaining low (less than
173
20% mortality).
AC C
EP
166
174
In contrast, the mortalities caused by washed cells of both strains showed different patterns over the experiment (see
175
Fig. 3b). After a short lag phase, the primary type cells of LY2W and NK stated a dramatic increase of mortality to G.
176
mellonella until the mortality reached about 95%. However, the secondary type cells of the two strains performed a
177
long-time lag (8 hours) and subsequently caused only 20% mortality at the end of the experiment. Moreover, the
178
mortality curves also showed that washed cells of both strains need a longer period to reach the corresponding mortality 6
ACCEPTED MANUSCRIPT 179
of G. mellonella compared to their metabolites. Meanwhile, the final mortalities caused by both secondary cells and
180
metabolites are about the same (~20%), showing a low toxicity to G. mellonella.
181
4.
Discussion This study has managed to isolate two symbiotic bacteria (LY2W and NK) associated with entomopathogenic
183
nematode Heterorhabditis spp. Always, symbiotic bacteria of entomopathogenic nematode are mainly found in the
184
genera Photorhabdus and Xenorhabdus. Photorhabdus spp. can generate catalase and fluorescence while Xenorhabdus
185
spp. not (Hsieh et al., 2009; Rahimi et al., 1993). Neither LY2W nor NK can generate fluorescence but they both have
186
catalase, suggesting these two strains do not belong to Photorhabdus spp. or Xenorhabdus spp. Phylogenetic relatedness
187
showed the two strains have the closest relationship with Enterobacter spp., with the confidence coefficient of 100%.
188
Based on the phenotypic and genetic identification, isolates LY2W and NK were defined as Enterobacter gergoviae and
189
Enterobacter cloacae, respectively. Although Enterobacteriaceae are well defined, it is not clear between the genera of
190
Enterobacter because there are still many complex debates in the classification (Daubner, 1967; Hiroki, 1966). On the
191
basis of phenotypic and genetic identification, even if we initially defined strains LY2W and NK as Enterobacter
192
gergoviae and Enterobacter cloacae, respectively; some differences still exist between them. Therefore, further studies
193
on molecular biology and biological taxonomy are needed.
M AN U
SC
RI PT
182
The primary-type strain usually has motility on NBTA while the secondary-type strain does not have on NA
195
(Rasmussen et al., 2011). This may be because secondary-type strains cannot express the synthetic genes of flagellin, and
196
therefore they cannot form flagellum (Rasmussen et al., 2011). From Fig. 1, the primary-type strain has flagellum while
197
the secondary-type not, sharing the characteristics with Photorhabdus spp. Moreover, the differences in mobility between
198
the primary type and secondary type strains show differences of adaptability (Puza and Mracek, 2009). Using flagellum,
199
the primary type can move throughout the intestine of entomopathogenic nematode, and they, therefore, are easier to
200
enter the special positions of the intestine and kill their host (Shapiro-Ilan et al., 2010; Yi et al., 2015). Meanwhile, the
201
virulence of metabolites of strains NK and LY2W has also indicated that their primary type has caused significantly
202
higher mortalities (97-100%) to G. mellonella than their secondary type (18-20%) (Fig. 3a). The similar patterns are seen
203
in washed cells of both strains, except that a longer infection (24 hours) period is needed (Fig. 3b).
AC C
EP
TE D
194
204
Strains NK and LY2W were well deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen
205
(DSM 26 379) and the American Type Culture Collection (ATCC BAA-2478). These two strains, which are
206
representative of the bacteria associated with the entomopathogenic nematode Heterorhabditis spp., are two members of
207
Enterobacteriaceae. This may lead to the development of more efficacious use of entomopathogenic nematodes as
208
biocontrol agents of insect pests and may lead also to the discovery of antibiotics from the bacterial cultures. Moreover, 7
ACCEPTED MANUSCRIPT the virulence test showed that the primary type may have significantly higher lethality to G. mellonella than the
210
secondary type, which would help to clarify how symbiotic bacteria function in different growth forms when infecting
211
their host G. mellonella. Meanwhile, these findings provide further references on how to use symbiotic bacteria at the
212
optimal time for biological control of agriculture pests. These two strains NK and LY2W would also provide new species
213
resources and alternatives for further study of symbiotic bacteria of the entomopathogenic nematode Heterorhabditis spp.
214
against G. mellonella.
215
5.
216
In the present study, we obtained two symbiotic bacteria (LY2W and NK) from the intestinal canals of two
217
entomopathogenic nematode Heterorhabditis megidis 90 (PDSj1 and PDSj2) from Galleria mellonela, separately. Based
218
on the analyses of biochemistry and molecular biology, both of them were identified as members of Enterobacter spp.,
219
showing the closest relatedness with Enterobacter gergoviae (LY2W) and Enterobacter cloacae (NK), respectively.
220
Moreover, the virulence to Galleria mellonella was examined using both the metabolites and washed cells (primary and
221
secondary) of these two strains. The results indicated both metabolites and cells of the primary-type bacteria could cause
222
high mortalities (up to 97%) to Galleria mellonella, while those of the primary-type bacteria only killed 20%. These
223
findings would provide new symbiotic bacteria and further references for biological control of the agricultural pest.
224
Acknowledgements
225
We gratefully acknowledge the financial support by the Henan key science and technology research (Grant No.
226
092102310069 and Grant No. 142102210106), and The university young teachers project.
227
Conflict of interest
228
The authors declare that there are no conflicts of interest.
229
References
230
Ali, F., Wharton, D.A., 2015. Infective Juveniles of the Entomopathogenic Nematode, Steinernema feltiae Produce Cryoprotectants in
232 233
EP
TE D
M AN U
SC
Conclusion
AC C
231
RI PT
209
Response to Freezing and Cold Acclimation. PLoS One 10, e0141810.
Ansari, M.A., Butt, T.M., 2012. Evaluation of entomopathogenic fungi and a nematode against the soil-dwelling stages of the crane fly
Tipula paludosa. Pest Manag Sci 68, 1337-1344.
234
Ayaad, T.H., Dorrah, M.A., Shaurub el, S.H., el-Sadawy, H.A., 2001. Effects of the entomopathogenic nematode, Heterorhabditis
235
bacteriophora HP 88 and azadirachtin on the immune defence response and prophenoloxidase of Parasarcophaga surcoufi
236
larvae (Diptera: Sarcophagidae). J Egypt Soc Parasitol 31, 295-325. 8
ACCEPTED MANUSCRIPT 237
Bisch, G., Pages, S., McMullen, J.G., 2nd, Stock, S.P., Duvic, B., Givaudan, A., Gaudriault, S., 2015. Xenorhabdus bovienii CS03, the
238
bacterial symbiont of the entomopathogenic nematode Steinernema weiseri, is a non-virulent strain against lepidopteran
239
insects. J Invertebr Pathol 124, 15-22.
Daubner, I., 1967. [On the present classification of bacteria of the family Enterobacteriaceae]. Biologia (Bratisl) 22, 692-698.
241
Fairbairn, J.P., Fenton, A., Norman, R.A., Hudson, P.J., 2000. Re-assessing the infection strategies of the entomopathogenic nematode
242
RI PT
240
Steinernema feltiae (Rhabditidae; Steinernematidae). Parasitology 121 ( Pt 2), 211-216.
Fischer-Le Saux, M., Viallard, V., Brunel, B., Normand, P., Boemare, N.E., 1999. Polyphasic classification of the genus Photorhabdus
244
and proposal of new taxa: P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P.
245
luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov. and P.
246
asymbiotica sp. nov. Int J Syst Bacteriol 49 (Pt 4), 1645-1656.
M AN U
248
Hebert, L., Rince, I., Sanna, C., Laugier, C., Rince, A., Petry, S., 2014. The host model Galleria mellonella is resistant to taylorellae
infection. Lett Appl Microbiol 59, 438-442.
TE D
247
SC
243
Hiroki, H., 1966. [New classification of enterobacteriaceae]. Shigaku 54, 64-71.
250
Hsieh, F.C., Tzeng, C.Y., Tseng, J.T., Tsai, Y.S., Meng, M., Kao, S.S., 2009. Isolation and characterization of the native
251
entomopathogenic nematode, Heterorhabditis brevicaudis, and its symbiotic bacteria from Taiwan. Curr Microbiol 58,
252
564-570.
254 255 256 257 258
AC C
253
EP
249
Malik, A., Gade, G., Lange, A.B., 2012. Sequencing and biological effects of an adipokinetic/hypertrehalosemic peptide in the stick
insect, Baculum extradentatum. Peptides 34, 51-56.
Olsen, R.J., Watkins, M.E., Cantu, C.C., Beres, S.B., Musser, J.M., 2011. Virulence of serotype M3 Group A Streptococcus strains in
wax worms (Galleria mellonella larvae). Virulence 2, 111-119.
Park, Y., Kim, Y., 2000. Eicosanoids rescue Spodoptera exigua infected with Xenorhabdus nematophilus, the symbiotic bacteria to the
entomopathogenic nematode Steinernema carpocapsae. J Insect Physiol 46, 1469-1476.
9
ACCEPTED MANUSCRIPT
260 261 262
Puza, V., Mracek, Z., 2009. Mixed infection of Galleria mellonella with two entomopathogenic nematode (Nematoda: Rhabditida)
species: Steinernema affine benefits from the presence of Steinernema kraussei. J Invertebr Pathol 102, 40-43.
Rahimi, F.R., McGuire, T.R., Gaugler, R., 1993. Morphological mutant in the entomopathogenic nematode, Heterorhabditis
bacteriophora. J Hered 84, 475-478.
RI PT
259
Rasmussen, L., White, E.L., Pathak, A., Ayala, J.C., Wang, H., Wu, J.H., Benitez, J.A., Silva, A.J., 2011. A high-throughput screening
264
assay for inhibitors of bacterial motility identifies a novel inhibitor of the Na+-driven flagellar motor and virulence gene
265
expression in Vibrio cholerae. Antimicrob Agents Chemother 55, 4134-4143.
267
Shapiro-Ilan, D.I., Cottrell, T.E., Brown, I., Gardner, W.A., Hubbard, R.K., Wood, B.W., 2006. Effect of Soil Moisture and a
M AN U
266
SC
263
Surfactant on Entomopathogenic Nematode Suppression of the Pecan Weevil, Curculio caryae. J Nematol 38, 474-482.
Shapiro-Ilan, D.I., Morales-Ramos, J.A., Rojas, M.G., Tedders, W.L., 2010. Effects of a novel entomopathogenic nematode-infected
269
host formulation on cadaver integrity, nematode yield, and suppression of Diaprepes abbreviatus and Aethina tumida. J
270
Invertebr Pathol 103, 103-108.
TE D
268
Wand, M.E., Baker, K.S., Benthall, G., McGregor, H., McCowen, J.W., Deheer-Graham, A., Sutton, J.M., 2015. Characterization of
272
pre-antibiotic era Klebsiella pneumoniae isolates with respect to antibiotic/disinfectant susceptibility and virulence in
273
Galleria mellonella. Antimicrob Agents Chemother 59, 3966-3972.
275 276 277
Westerman, P.R., 1998. Penetration of the entomopathogenic nematode heterorhabditis spp. into host insects at 9 and 20 degreesC. J
AC C
274
EP
271
Invertebr Pathol 72, 197-205.
Yi, Y., Wu, G., Lv, J., Li, M., 2015. Eicosanoids mediate Galleria mellonella immune response to hemocoel injection of
entomopathogenic nematode cuticles. Parasitol Res.
278 279 280 281 282 10
ACCEPTED MANUSCRIPT Figure legends
284
Fig.1 Graph showing the morphology of strains (1000X amplification). a) the primary type of LY2W; b) the secondary
285
type of LY2W; c) the primary type of NK; d) the primary type of NK. Each sample observed was from a pure culture or
286
colony.
287
Fig.2 Phylogenetic trees of strains LY2W and NK. The maximum-likelihood trees were based on 16S rRNA gene
288
sequences.
289
Fig.3 Mortality of G. mellonella larvae caused by the metabolites (a) and cells (b) of strains LY2W and NK. Treatments
290
were conducted in triplicates. Data points are means ± SD (n = 3). All mortality data have been corrected based on
291
formula 2.
AC C
EP
TE D
M AN U
SC
RI PT
283
11
Table 1 Morphological characteristics of colony of strains NK and LY2W
ACCEPTED MANUSCRIPT
Strain NK
Strain LY2W
Media White, convex, NA
non-transparent, viscous
NBTA
Secondary type
Primary type
White, flat,
Grey, convex,
semi-transparent
viscous
Transparent,
Red, flat,
convex, red center
transparent circle
＋
Methyl red
＋
Urease
＋
Lecithinase
＋
Gelatin liquefaction
－
Tween 80
w
Nitratase
＋
Indol generation
－
H2S generation
－
Amylolysis
＋
reductase
1mm), convex,
mm), transparent
red center
SC
circle
Primary type
Secondary type
＋
＋
－
＋
－
＋
＋
－
＋
－
－
－
－
－
－
＋
＋
＋
＋
＋
－
－
－
－
＋
－
＋
－
－
－
－
＋
－
－
－
－
－
TE D
＋
AC C
Tryptophan reductase
Secondary type
EP
Phenylalanine
semi-transparent Red, flat, big (2-3
M AN U
Catalase
convex,
Strain LY2W
Characteristic Primary type
Grey, slightly
Little (less than
Table 2 Main biochemical characters of strains NK and LY2W
Strain NK
Secondary type
RI PT
Primary type
Citrate
＋
－
＋
－
Aesculin hydrolysis
＋
＋
＋
＋
Oxidase
－
－
－
－
Esterase
－
－
－
－
Maltose
＋
＋
＋
＋
Cellobiose
＋
＋
＋
＋
Note: + represents positive reaction; - represents negative reaction; w represents a little positive reaction.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights
2
1.
Two symbiotic bacteria (LY2W and NK) of entomopathogenic nematode were obtained.
3
2.
Strains LY2W and NK were identified as Enterobacter gergoviae and Enterobacter cloacae, respectively.
4
3.
These two strains showed high virulence to Galleria mellonella.
5
4.
The primary type strains caused significantly higher mortalities to G. mellonella.
AC C
EP
TE D
M AN U
SC
RI PT
1
1
ACCEPTED MANUSCRIPT
Conflicts of interest The authors declare that they have no conflict of interest. And we all reported the following statements:
RI PT
1. All third-party financial support for the work in the submitted manuscript. 2. All financial relationships with any entities that could be viewed as relevant to the general area of the submitted manuscript.
SC
3. All sources of revenue with relevance to the submitted work who made payments to you,
M AN U
or to your institution on your behalf, in the 36 months prior to submission. 4. Any other interactions with the sponsor of outside of the submitted work should also be reported.
5. Any relevant patents or copyrights (planned, pending, or issued).
TE D
6. Any other relationships or affiliations that may be perceived by readers to have influenced,
AC C
EP
or give the appearance of potentially influencing, what you wrote in the submitted work.
ACCEPTED MANUSCRIPT
Ethics in publishing The authors reported the following ethics in publishing: We have prepared our manuscript according to the author guidance, and the paper length is well
RI PT
controlled as journal’s requirements. This work has not been published before and it is also not being considered by another journal. All authors have read the submitted version of the manuscript
AC C
EP
TE D
M AN U
manuscript, and it is approved by all authors for publication.
SC
and agreed to submit this work to our journal. No conflict of interest exits in the submission of this
S0041-0101(16)30601-8
DOI:
10.1016/j.toxicon.2016.11.257
Reference:
TOXCON 5513
To appear in:
Toxicon
Received Date: 20 August 2016 Revised Date:
17 November 2016
Accepted Date: 23 November 2016
Please cite this article as: Liao, C., Gao, A., Li, B., Wang, M., Shan, L., Two symbiotic bacteria of the entomopathogenic nematode Heterorhabditis spp. against Galleria mellonella, Toxicon (2016), doi: 10.1016/j.toxicon.2016.11.257. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Short communication
2
Two symbiotic bacteria of the entomopathogenic nematode Heterorhabditis spp. against Galleria mellonella
3
Chunli Liao*, Along Gao, Bingbing Li*, Mengjun Wang, Linna Shan
4
College of Life Science and Engineering, Henan University of Urban Construction, Pingdingshan, China
5
*Corresponding author: Bingbing Li or Chunli Liao
6
Address: College of Life Science and Engineering, Henan University of Urban Construction, Pingdingshan 467036,
7
Henan, China
8
E-mail: [email protected] (B. Li), [email protected] (C. Liao)
9
Tel.: +86-0375-2089090;
10
Fax: +86-0375-2089090;
SC M AN U
11 12 13 14 15
20 21 22 23 24 25
EP
19
AC C
18
TE D
16 17
RI PT
1
26 27 28 29
1
ACCEPTED MANUSCRIPT Abstract: The entomopathogenic nematode Heterorhabditis spp. is considered a promising agent in the biocontrol of
31
injurious insects of agriculture. However, different symbiotic bacteria associated with the nematode usually have
32
different specificity and virulence toward their own host. In this study, two symbiotic bacteria, LY2W and NK, were
33
isolated from the intestinal canals of two entomopathogenic nematode Heterorhabditis megidis 90 (PDSj1 and PDSj2)
34
from Galleria mellonela, separately. To determine their species classification, we carried out some investigations on
35
morphology, culture, biochemistry, especially 16S rDNA sequence analyses. As a result, both of them belong to
36
Enterobacter spp., showing the closest relatedness with Enterobacter gergoviae (LY2W) and Enterobacter cloacae
37
(NK), respectively. Moreover, the toxicity to Galleria mellonella was examined using both the metabolites and washed
38
cells (primary and secondary) of these two strains. The results indicated both metabolites and cells of the primary-type
39
bacteria could cause high mortalities (up to 97%) to Galleria mellonella, while those of the primary-type bacteria only
40
killed 20%. These findings would provide new symbiotic bacteria and further references for biological control of the
41
agricultural pest.
42
Keywords: Entomopathogenic nematode, Symbiotic bacteria, Galleria mellonella, Toxicity, Biological control
M AN U
SC
RI PT
30
43 44
48 49 50 51 52 53 54
EP
47
AC C
46
TE D
45
55 56 57 58 59 2
ACCEPTED MANUSCRIPT 60
1.
Introduction Galleria mellonella (the greater wax moth or honeycomb moth) is a moth of the family Pyralidae, which is the only
62
member of the genus Galleria (Wand et al., 2015). It can be found in most of the world, including Europe and adjacent
63
Eurasia, its presumed native range, and as an introduced species on other continents, including North America and
64
Australia (Wand et al., 2015). This moth flies from May to October in the temperate parts of its range, such as Belgium
65
and the Netherlands. The caterpillar larvae, or wax worms, feed on the honeycomb inside bee nests and may become
66
pests of apiculture (Olsen et al., 2011). Less often, they are found in bumblebee and wasp nests or feeding on dried figs
67
(Wand et al., 2015). Current control recommendations for this injurious insect are focused on chemical pesticide usages
68
(Malik et al., 2012). Due to the difficulty in controlling this insect with chemical insecticides and the additional
69
environmental pollution associated with their use, the development of biological control alternatives is extremely urgent.
SC
RI PT
61
Entomopathogenic nematodes in the Steinernematidae and Heterorhabditidae families are lethal parasites of a large
71
number of insects (Rahimi et al., 1993) and have been used effectively to control many burrowing insects (Ali and
72
Wharton, 2015; Ansari and Butt, 2012; Ayaad et al., 2001). Entomopathogenic nematodes have a symbiotic association
73
with bacteria in the Enterobacter (Bisch et al., 2015), which can be used as a biocontrol alternative of Galleria
74
mellonella (Puza and Mracek, 2009). However, existing as the bacterial symbionts of nematode, these bacteria, therefore,
75
cannot be isolated directly from the natural environment. They are not described until the 1960s and therefore regarded
76
as a new special Enterobacter (Puza and Mracek, 2009).
TE D
M AN U
70
To date, the described mutualistic bacteria have been classified into two genera, Xenorhabdus spp. and
78
Photorhabdus spp., associated with the entomopathogenic nematodes Steinernema spp. and Heterorhabditis spp.,
79
respectively (Hsieh et al., 2009; Park and Kim, 2000). Steinernema spp. carry in Xenorhabdus spp., and Heterorhabditis
80
spp. carry throughout Photorhabdus spp. (Park and Kim, 2000; Rahimi et al., 1993). The infective nematode juveniles
81
actively search for hosts and penetrate through their natural openings (anus, mouth, and spiracles) (Park and Kim, 2000).
82
Once inside, these nematodes will release pathogenic bacteria which rapidly multiply and then cause the death of the
83
insect usually within 24-48 h (Fairbairn et al., 2000; Hebert et al., 2014). The bacteria also release other pathogenic
84
factors which will cause immune system depression of their host and will inhibit the growth of non-symbiotic
85
microorganisms (Yi et al., 2015). Therefore, nematodes have been widely used against larvae of different curculionid
86
species, such as Curculio elephas (Shapiro-Ilan et al., 2006), Otiorhynchus sulcatus (Westerman, 1998), Diaprepes spp.
87
(Shapiro-Ilan et al., 2010), Rhytidoderes plicatus (Rahimi et al., 1993), and Curculio caryae (Shapiro-Ilan et al., 2006).
88
Therefore, the full investigation and exploitation of entomopathogenic nematodes and their symbiotic bacteria are of
89
great significance for biological control of pest and environmental protection.
AC C
EP
77
3
ACCEPTED MANUSCRIPT 90
Therefore, our objectives were (i) to isolate several native symbiotic bacteria of the entomopathogenic nematode
91
against Galleria mellonella and (ii) to determine the virulence of the symbiotic bacteria to the larvae of this insect in
92
order to provide further references for the control of Galleria mellonella. We expect the knowledge obtained will enable
93
the bio-insecticide of Galleria mellonella to be improved and provide effective bioresource species for the biocontrol of
94
injurious agricultural insects.
95
2.
96
2.1 Isolation of bacteria, media and culture
RI PT
Materials and methods
Two entomopathogenic nematodes (Heterorhabditis spp. PDSj1 and Heterorhabditis spp. PDSj2) were originally
98
collected from a soil sample in a suburb field, Pingdingshan, China, and both were finally identified as Heterorhabditis
99
megidis 90 by the third party (Sangon Biotech, Shanghai, China). The obtained Heterorhabditis spp. PDSj1 and
100
Heterorhabditis spp. PDSj2 were surface sterilized using 10% (v/v) formaldehyde and then cleaned using sterilized water.
101
After that, they were suspended in 0.2% thimerosal (Sigma) for 30 min and then washed with sterilized water prior to
102
inoculation onto nutrient agar plates (214010/214030 Agar, Bacto, BD, USA), supplemented with 0.025% (w/v)
103
bromothymol blue and 0.004% (w/v) triphenyl tetrazolium chloride (referred to as NBTA). The plates were finally
104
maintained at 28 oC for 3-5 days in the dark. Colonies that faded bromothymol blue on the NBTA plates were regarded
105
as symbiotic bacteria associated with Heterorhabditis spp. (Hsieh et al., 2009). They were then collected and stored at
106
-20 oC in TSB containing 20% (v/v) glycerol for further identification.
107
2.2 Phenotypic characteristics of bacterial isolates
TE D
M AN U
SC
97
Symbiotic bacterial isolates were subcultured on NA and NBTA plates. After 24 h maintenance at 28 oC, the
109
characteristics of morphology and pigment absorption were recorded timely. Isolates from randomly selected colonies
110
were Gram-stained and their morphology was studied with the aid of a research microscope (optical and electronic
111
microscopes). Biochemical identification referred to the methods in Manual of Bacterium Identification.
112
2.3 Phylogenetic analyses of isolates
AC C
EP
108
113
Genomic DNA of the isolates was extracted using a ZR bacterial DNA kit (Biotechnology Research Corporation,
114
New York, USA). Polymerase chain reactions (PCR) were performed using TaKaRa Ex TaqTM, 10 Ex TaqTM buffer and
115
deoxynucleoside triphosphate (dNTP) mixture (Takara Bio Inc., Shiga, Japan). According to the 16S rDNA of symbiotic
116
bacteria
117
5′-GAGCGGATAACAATTTCACACAGG-3′ and 5′-CGCCAGGGTTTTCCCAGTCACGAC-3′, respective (Fischer-Le
118
Saux et al., 1999). The amplification volume was 50 µL and the PCR conditions were as follows: 30 cycles of
119
amplification, after an initial 5 min denaturation step at 94 oC; each cycle consisted of 1 min at 94 oC, 1 min at the
in
GenBank,
the
upstream
and
4
downstream
primers
were
designed
as
ACCEPTED MANUSCRIPT annealing temperature of 55 oC and 1.5 min at 72 oC. Amplified products were ligated into a PMD18-T vector (Promega
121
Corporation, Madison, Wisconsin, USA), cloned into E. coli DH5α and sequenced by the third party (DALIANBAO
122
Biotechnology Corporation, Dalian, China). Sequences were analyzed using BLAST (Basic Local Alignment Search
123
Tool, National Center for Biotechnology Information, National Library of Medicine, Bethesda, Maryland, USA) and
124
compared with known sequences in GenBank. Phylogenetic trees were established using MEGA (Molecular
125
Evolutionary Genetics Analysis) soft, version 6.06, according to the Neighbor-Joining method.
126
2.4 Virulence test design
RI PT
120
Symbiotic bacterial isolates were maintained in Luria-Bertani (LB) Broth at 28 oC for 24 h (shaking speed:
128
180r/min). The fermentation broth was centrifuged (1000 g) at 4 oC for 10 min and the supernatant was then filtered (pore
129
size 0.22 µm) under the sterile condition, which therefore contained active metabolites of cells. The larvae of Galleria
130
mellonella were cleaned using the sterile water and were injected separately with 10 µL filtered fermentation broth and
131
10 µL washed cells (106 CFU/mL) of which concentration was corresponding to that of fermentation broth. Each
132
treatment included 30 larvae and was set in triplicate. Larvae in control group were injected using 10 µL sterile LB broth
133
or saline water (0.85% m/v). All larvae were finally maintained in sterilized Petri dishes at 28 oC and their death was
134
recorded every 2 h. The mortality (M) and corrected mortality (CM) of larvae were calculated by the following formulas:
135
M (%) = n/N*100%
136
CM (%) = (M1-M0) / (1-M0) *100%
137
Where n=number of dead larvae, N=total number of larvae, M1=mortality of the treatment, and M0=mortality of the
138
control.
139
3.
140
3.1 Morphological characteristics of isolates
TE D
(1)
(2)
EP
Results
M AN U
SC
127
All colonies (about twenty) absorbed bromthymol blue on NBTA plate and were Gram-negative rods with most
142
isolates belonging to well-reported Photorhabdus spp. However, two isolates (LY2W and NK) shown differences in
143
other phenotypic properties. The morphological characteristics of LY2W and NK, when subcultured on NA and NBTA
144
plates, were shown in Table 1 and Fig. 1. After 2-day maintenance at 28 oC, both LY2W and NK colonies did not
145
generate bioluminescence whether on NA or on NBTA, which therefore indicated these two strains did not belong to
146
Photorhabdus spp. Moreover, their primary types had cloudily-spread colonies while their secondary types not. This also
147
indicated that the primary type of these strains was capable of motility while the secondary not.
148
3.2 Biochemical characteristics of LY2W and NK
AC C
141
5
ACCEPTED MANUSCRIPT The biochemical reactions in both primary and secondary type strains were shown in Table 2. As a result, both types
150
had positive reactions with catalase, maltose, cellobiose and nitratase. They were able to utilize citrate, but not able to
151
liquefy gelatin and produce esterase, oxidase as well as tryptophan reductase. The isolate NK not only hydrolysed
152
esculoside and starch but had a positive reaction with lecithinase and methyl red, while the isolate LY2W not. Isolate NK
153
could hydrolyze urea while only the primary type of isolate LY2W could. However, the primary type of isolate LY2W
154
generated phenylalanine reductase while both types of isolate NK not. These biochemical characteristics showed that
155
isolates LY2W and NK had the closest relationship to Enterobacter gergoviae and Enterobacter cloacae, respectively.
156
3.3 Phylogenetic relatedness of LY2W and NK
RI PT
149
The 16S rDNA sequences of the two strains were compared with sequences of representative strains of different
158
species in the Genbank. All sequences from both strains LY2W and NK showed a high percentage of homology (above
159
99%) to the sequences derived from three genera: Pantoea spp., Klebsiella spp., and Enterobacter spp. (Fig. 2).
160
However, based on the above biochemical identification both Pantoea spp. and Klebsiella spp. were excluded. Moreover,
161
the analyses of MEGA 6.06 and Neighbor-Joining indicated strains LY2W and NK had the closed relationships
162
(confidence coefficient: 100%) with Enterobacter spp. (Fig. 2). Therefore, combined with the results of biochemical
163
reaction, we concluded that strains LY2W and NK might be the same species to Enterobacter gergoviae and
164
Enterobacter cloacae, respectively.
165
3.4 Virulence of metabolites of LY2W and NK to G. mellonella
TE D
M AN U
SC
157
The mortalities of G. mellonella caused by the metabolites of strains LY2W and NK were shown in Fig. 3a. As a
167
result, the metabolites of primary type strains performed significantly higher mortalities (97-100%) than those of the
168
secondary type (about 20%). Moreover, virulence of metabolites of the NK primary type strain increased fastest within
169
6-8 hours after injection, and then reached the highest (100% mortality). However, mortalities caused by metabolites of
170
the primary type of strain LY2W increased slowly from 10% to 50% between 2 and 10 hours after injection and then
171
increased faster to about 97% within 16 hours. For the metabolites of the secondary type strains, their virulence to G.
172
mellonella was not significant or rapid, only starting to increase after 8-10 hours and finally remaining low (less than
173
20% mortality).
AC C
EP
166
174
In contrast, the mortalities caused by washed cells of both strains showed different patterns over the experiment (see
175
Fig. 3b). After a short lag phase, the primary type cells of LY2W and NK stated a dramatic increase of mortality to G.
176
mellonella until the mortality reached about 95%. However, the secondary type cells of the two strains performed a
177
long-time lag (8 hours) and subsequently caused only 20% mortality at the end of the experiment. Moreover, the
178
mortality curves also showed that washed cells of both strains need a longer period to reach the corresponding mortality 6
ACCEPTED MANUSCRIPT 179
of G. mellonella compared to their metabolites. Meanwhile, the final mortalities caused by both secondary cells and
180
metabolites are about the same (~20%), showing a low toxicity to G. mellonella.
181
4.
Discussion This study has managed to isolate two symbiotic bacteria (LY2W and NK) associated with entomopathogenic
183
nematode Heterorhabditis spp. Always, symbiotic bacteria of entomopathogenic nematode are mainly found in the
184
genera Photorhabdus and Xenorhabdus. Photorhabdus spp. can generate catalase and fluorescence while Xenorhabdus
185
spp. not (Hsieh et al., 2009; Rahimi et al., 1993). Neither LY2W nor NK can generate fluorescence but they both have
186
catalase, suggesting these two strains do not belong to Photorhabdus spp. or Xenorhabdus spp. Phylogenetic relatedness
187
showed the two strains have the closest relationship with Enterobacter spp., with the confidence coefficient of 100%.
188
Based on the phenotypic and genetic identification, isolates LY2W and NK were defined as Enterobacter gergoviae and
189
Enterobacter cloacae, respectively. Although Enterobacteriaceae are well defined, it is not clear between the genera of
190
Enterobacter because there are still many complex debates in the classification (Daubner, 1967; Hiroki, 1966). On the
191
basis of phenotypic and genetic identification, even if we initially defined strains LY2W and NK as Enterobacter
192
gergoviae and Enterobacter cloacae, respectively; some differences still exist between them. Therefore, further studies
193
on molecular biology and biological taxonomy are needed.
M AN U
SC
RI PT
182
The primary-type strain usually has motility on NBTA while the secondary-type strain does not have on NA
195
(Rasmussen et al., 2011). This may be because secondary-type strains cannot express the synthetic genes of flagellin, and
196
therefore they cannot form flagellum (Rasmussen et al., 2011). From Fig. 1, the primary-type strain has flagellum while
197
the secondary-type not, sharing the characteristics with Photorhabdus spp. Moreover, the differences in mobility between
198
the primary type and secondary type strains show differences of adaptability (Puza and Mracek, 2009). Using flagellum,
199
the primary type can move throughout the intestine of entomopathogenic nematode, and they, therefore, are easier to
200
enter the special positions of the intestine and kill their host (Shapiro-Ilan et al., 2010; Yi et al., 2015). Meanwhile, the
201
virulence of metabolites of strains NK and LY2W has also indicated that their primary type has caused significantly
202
higher mortalities (97-100%) to G. mellonella than their secondary type (18-20%) (Fig. 3a). The similar patterns are seen
203
in washed cells of both strains, except that a longer infection (24 hours) period is needed (Fig. 3b).
AC C
EP
TE D
194
204
Strains NK and LY2W were well deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen
205
(DSM 26 379) and the American Type Culture Collection (ATCC BAA-2478). These two strains, which are
206
representative of the bacteria associated with the entomopathogenic nematode Heterorhabditis spp., are two members of
207
Enterobacteriaceae. This may lead to the development of more efficacious use of entomopathogenic nematodes as
208
biocontrol agents of insect pests and may lead also to the discovery of antibiotics from the bacterial cultures. Moreover, 7
ACCEPTED MANUSCRIPT the virulence test showed that the primary type may have significantly higher lethality to G. mellonella than the
210
secondary type, which would help to clarify how symbiotic bacteria function in different growth forms when infecting
211
their host G. mellonella. Meanwhile, these findings provide further references on how to use symbiotic bacteria at the
212
optimal time for biological control of agriculture pests. These two strains NK and LY2W would also provide new species
213
resources and alternatives for further study of symbiotic bacteria of the entomopathogenic nematode Heterorhabditis spp.
214
against G. mellonella.
215
5.
216
In the present study, we obtained two symbiotic bacteria (LY2W and NK) from the intestinal canals of two
217
entomopathogenic nematode Heterorhabditis megidis 90 (PDSj1 and PDSj2) from Galleria mellonela, separately. Based
218
on the analyses of biochemistry and molecular biology, both of them were identified as members of Enterobacter spp.,
219
showing the closest relatedness with Enterobacter gergoviae (LY2W) and Enterobacter cloacae (NK), respectively.
220
Moreover, the virulence to Galleria mellonella was examined using both the metabolites and washed cells (primary and
221
secondary) of these two strains. The results indicated both metabolites and cells of the primary-type bacteria could cause
222
high mortalities (up to 97%) to Galleria mellonella, while those of the primary-type bacteria only killed 20%. These
223
findings would provide new symbiotic bacteria and further references for biological control of the agricultural pest.
224
Acknowledgements
225
We gratefully acknowledge the financial support by the Henan key science and technology research (Grant No.
226
092102310069 and Grant No. 142102210106), and The university young teachers project.
227
Conflict of interest
228
The authors declare that there are no conflicts of interest.
229
References
230
Ali, F., Wharton, D.A., 2015. Infective Juveniles of the Entomopathogenic Nematode, Steinernema feltiae Produce Cryoprotectants in
232 233
EP
TE D
M AN U
SC
Conclusion
AC C
231
RI PT
209
Response to Freezing and Cold Acclimation. PLoS One 10, e0141810.
Ansari, M.A., Butt, T.M., 2012. Evaluation of entomopathogenic fungi and a nematode against the soil-dwelling stages of the crane fly
Tipula paludosa. Pest Manag Sci 68, 1337-1344.
234
Ayaad, T.H., Dorrah, M.A., Shaurub el, S.H., el-Sadawy, H.A., 2001. Effects of the entomopathogenic nematode, Heterorhabditis
235
bacteriophora HP 88 and azadirachtin on the immune defence response and prophenoloxidase of Parasarcophaga surcoufi
236
larvae (Diptera: Sarcophagidae). J Egypt Soc Parasitol 31, 295-325. 8
ACCEPTED MANUSCRIPT 237
Bisch, G., Pages, S., McMullen, J.G., 2nd, Stock, S.P., Duvic, B., Givaudan, A., Gaudriault, S., 2015. Xenorhabdus bovienii CS03, the
238
bacterial symbiont of the entomopathogenic nematode Steinernema weiseri, is a non-virulent strain against lepidopteran
239
insects. J Invertebr Pathol 124, 15-22.
Daubner, I., 1967. [On the present classification of bacteria of the family Enterobacteriaceae]. Biologia (Bratisl) 22, 692-698.
241
Fairbairn, J.P., Fenton, A., Norman, R.A., Hudson, P.J., 2000. Re-assessing the infection strategies of the entomopathogenic nematode
242
RI PT
240
Steinernema feltiae (Rhabditidae; Steinernematidae). Parasitology 121 ( Pt 2), 211-216.
Fischer-Le Saux, M., Viallard, V., Brunel, B., Normand, P., Boemare, N.E., 1999. Polyphasic classification of the genus Photorhabdus
244
and proposal of new taxa: P. luminescens subsp. luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P.
245
luminescens subsp. laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov. and P.
246
asymbiotica sp. nov. Int J Syst Bacteriol 49 (Pt 4), 1645-1656.
M AN U
248
Hebert, L., Rince, I., Sanna, C., Laugier, C., Rince, A., Petry, S., 2014. The host model Galleria mellonella is resistant to taylorellae
infection. Lett Appl Microbiol 59, 438-442.
TE D
247
SC
243
Hiroki, H., 1966. [New classification of enterobacteriaceae]. Shigaku 54, 64-71.
250
Hsieh, F.C., Tzeng, C.Y., Tseng, J.T., Tsai, Y.S., Meng, M., Kao, S.S., 2009. Isolation and characterization of the native
251
entomopathogenic nematode, Heterorhabditis brevicaudis, and its symbiotic bacteria from Taiwan. Curr Microbiol 58,
252
564-570.
254 255 256 257 258
AC C
253
EP
249
Malik, A., Gade, G., Lange, A.B., 2012. Sequencing and biological effects of an adipokinetic/hypertrehalosemic peptide in the stick
insect, Baculum extradentatum. Peptides 34, 51-56.
Olsen, R.J., Watkins, M.E., Cantu, C.C., Beres, S.B., Musser, J.M., 2011. Virulence of serotype M3 Group A Streptococcus strains in
wax worms (Galleria mellonella larvae). Virulence 2, 111-119.
Park, Y., Kim, Y., 2000. Eicosanoids rescue Spodoptera exigua infected with Xenorhabdus nematophilus, the symbiotic bacteria to the
entomopathogenic nematode Steinernema carpocapsae. J Insect Physiol 46, 1469-1476.
9
ACCEPTED MANUSCRIPT
260 261 262
Puza, V., Mracek, Z., 2009. Mixed infection of Galleria mellonella with two entomopathogenic nematode (Nematoda: Rhabditida)
species: Steinernema affine benefits from the presence of Steinernema kraussei. J Invertebr Pathol 102, 40-43.
Rahimi, F.R., McGuire, T.R., Gaugler, R., 1993. Morphological mutant in the entomopathogenic nematode, Heterorhabditis
bacteriophora. J Hered 84, 475-478.
RI PT
259
Rasmussen, L., White, E.L., Pathak, A., Ayala, J.C., Wang, H., Wu, J.H., Benitez, J.A., Silva, A.J., 2011. A high-throughput screening
264
assay for inhibitors of bacterial motility identifies a novel inhibitor of the Na+-driven flagellar motor and virulence gene
265
expression in Vibrio cholerae. Antimicrob Agents Chemother 55, 4134-4143.
267
Shapiro-Ilan, D.I., Cottrell, T.E., Brown, I., Gardner, W.A., Hubbard, R.K., Wood, B.W., 2006. Effect of Soil Moisture and a
M AN U
266
SC
263
Surfactant on Entomopathogenic Nematode Suppression of the Pecan Weevil, Curculio caryae. J Nematol 38, 474-482.
Shapiro-Ilan, D.I., Morales-Ramos, J.A., Rojas, M.G., Tedders, W.L., 2010. Effects of a novel entomopathogenic nematode-infected
269
host formulation on cadaver integrity, nematode yield, and suppression of Diaprepes abbreviatus and Aethina tumida. J
270
Invertebr Pathol 103, 103-108.
TE D
268
Wand, M.E., Baker, K.S., Benthall, G., McGregor, H., McCowen, J.W., Deheer-Graham, A., Sutton, J.M., 2015. Characterization of
272
pre-antibiotic era Klebsiella pneumoniae isolates with respect to antibiotic/disinfectant susceptibility and virulence in
273
Galleria mellonella. Antimicrob Agents Chemother 59, 3966-3972.
275 276 277
Westerman, P.R., 1998. Penetration of the entomopathogenic nematode heterorhabditis spp. into host insects at 9 and 20 degreesC. J
AC C
274
EP
271
Invertebr Pathol 72, 197-205.
Yi, Y., Wu, G., Lv, J., Li, M., 2015. Eicosanoids mediate Galleria mellonella immune response to hemocoel injection of
entomopathogenic nematode cuticles. Parasitol Res.
278 279 280 281 282 10
ACCEPTED MANUSCRIPT Figure legends
284
Fig.1 Graph showing the morphology of strains (1000X amplification). a) the primary type of LY2W; b) the secondary
285
type of LY2W; c) the primary type of NK; d) the primary type of NK. Each sample observed was from a pure culture or
286
colony.
287
Fig.2 Phylogenetic trees of strains LY2W and NK. The maximum-likelihood trees were based on 16S rRNA gene
288
sequences.
289
Fig.3 Mortality of G. mellonella larvae caused by the metabolites (a) and cells (b) of strains LY2W and NK. Treatments
290
were conducted in triplicates. Data points are means ± SD (n = 3). All mortality data have been corrected based on
291
formula 2.
AC C
EP
TE D
M AN U
SC
RI PT
283
11
Table 1 Morphological characteristics of colony of strains NK and LY2W
ACCEPTED MANUSCRIPT
Strain NK
Strain LY2W
Media White, convex, NA
non-transparent, viscous
NBTA
Secondary type
Primary type
White, flat,
Grey, convex,
semi-transparent
viscous
Transparent,
Red, flat,
convex, red center
transparent circle
＋
Methyl red
＋
Urease
＋
Lecithinase
＋
Gelatin liquefaction
－
Tween 80
w
Nitratase
＋
Indol generation
－
H2S generation
－
Amylolysis
＋
reductase
1mm), convex,
mm), transparent
red center
SC
circle
Primary type
Secondary type
＋
＋
－
＋
－
＋
＋
－
＋
－
－
－
－
－
－
＋
＋
＋
＋
＋
－
－
－
－
＋
－
＋
－
－
－
－
＋
－
－
－
－
－
TE D
＋
AC C
Tryptophan reductase
Secondary type
EP
Phenylalanine
semi-transparent Red, flat, big (2-3
M AN U
Catalase
convex,
Strain LY2W
Characteristic Primary type
Grey, slightly
Little (less than
Table 2 Main biochemical characters of strains NK and LY2W
Strain NK
Secondary type
RI PT
Primary type
Citrate
＋
－
＋
－
Aesculin hydrolysis
＋
＋
＋
＋
Oxidase
－
－
－
－
Esterase
－
－
－
－
Maltose
＋
＋
＋
＋
Cellobiose
＋
＋
＋
＋
Note: + represents positive reaction; - represents negative reaction; w represents a little positive reaction.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights
2
1.
Two symbiotic bacteria (LY2W and NK) of entomopathogenic nematode were obtained.
3
2.
Strains LY2W and NK were identified as Enterobacter gergoviae and Enterobacter cloacae, respectively.
4
3.
These two strains showed high virulence to Galleria mellonella.
5
4.
The primary type strains caused significantly higher mortalities to G. mellonella.
AC C
EP
TE D
M AN U
SC
RI PT
1
1
ACCEPTED MANUSCRIPT
Conflicts of interest The authors declare that they have no conflict of interest. And we all reported the following statements:
RI PT
1. All third-party financial support for the work in the submitted manuscript. 2. All financial relationships with any entities that could be viewed as relevant to the general area of the submitted manuscript.
SC
3. All sources of revenue with relevance to the submitted work who made payments to you,
M AN U
or to your institution on your behalf, in the 36 months prior to submission. 4. Any other interactions with the sponsor of outside of the submitted work should also be reported.
5. Any relevant patents or copyrights (planned, pending, or issued).
TE D
6. Any other relationships or affiliations that may be perceived by readers to have influenced,
AC C
EP
or give the appearance of potentially influencing, what you wrote in the submitted work.
ACCEPTED MANUSCRIPT
Ethics in publishing The authors reported the following ethics in publishing: We have prepared our manuscript according to the author guidance, and the paper length is well
RI PT
controlled as journal’s requirements. This work has not been published before and it is also not being considered by another journal. All authors have read the submitted version of the manuscript
AC C
EP
TE D
M AN U
manuscript, and it is approved by all authors for publication.
SC
and agreed to submit this work to our journal. No conflict of interest exits in the submission of this