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Characterized non-transient microbiota from stinkbug (Nezara viridula) midgut deactivates soybean chemical defenses

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The Southern green stinkbug (N. viridula) feeds on developing soybean seeds in spite of their strong defenses against herbivory, making this pest one of the most harmful to soybean crops. To test the hypothesis that midgut bacterial community allows stinkbugs to tolerate chemical defenses of soybean developing seeds, we identified and characterized midgut microbiota of stinkbugs collected from soybean crops, different secondary plant hosts or insects at diapause on Eucalyptus trees. Our study demonstrated that while more than 54% of N. viridula adults collected in the field had no detectable bacteria in the V1-V3 midgut ventricles, the guts of the rest of stinkbugs were colonized by non-transient microbiota (NTM) and transient microbiota not present in stinkbugs at diapause. While transient microbiota Bacillus sp., Micrococcus sp., Streptomyces sp., Staphylococcus sp. and others had low abundance, NTM microbiota was represented by Yokenella sp., Pantoea sp. and Enterococcus sp. isolates. We found some isolates that showed in vitro β-glucosidase and raffinase activities plus the ability to degrade isoflavonoids and deactivate soybean protease inhibitors. Our results suggest that the stinkbugs´ NTM microbiota may impact on nutrition, detoxification and deactivation of chemical defenses, and Enterococcus sp., Yokenella sp. and Pantoea sp. strains might help stinkbugs to feed on soybean developing seeds in spite of its chemical defenses.
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RESEARCH ARTICLE
Characterized non-transient microbiota from
stinkbug (Nezara viridula) midgut deactivates
soybean chemical defenses
Virginia Medina
1
, Pedro M. Sardoy
1
, Marcelo Soria
2
, Carlos A. Vay
3
, Gabriel O. Gutkind
3,4
,
Jorge A. Zavala
1,4
*
1Universidad de Buenos Aires, Facultad de Agronomı
´a, Ca
´tedra de Bioquı
´mica -Instituto de Investigaciones
en Biociencias Agrı
´colas y Ambientales (INBA-CONICET), Buenos Aires, Argentina, 2Universidad de
Buenos Aires, Facultad de Agronomı
´a, Ca
´tedra de Microbiologı
´a -Instituto de Investigaciones en Biociencias
Agrı
´colas y Ambientales (INBA-CONICET), Buenos Aires, Argentina, 3Universidad de Buenos Aires,
Facultad de Farmacia y Bioquı
´mica, Buenos Aires, Argentina, 4Consejo Nacional de Investigaciones
Cientı
´ficas y Te
´cnicas de Argentina, (CONICET), Buenos Aires, Argentina
*zavala@agro.uba.ar
Abstract
The Southern green stinkbug (N.viridula) feeds on developing soybean seeds in spite of
their strong defenses against herbivory, making this pest one of the most harmful to soy-
bean crops. To test the hypothesis that midgut bacterial community allows stinkbugs to tol-
erate chemical defenses of soybean developing seeds, we identified and characterized
midgut microbiota of stinkbugs collected from soybean crops, different secondary plant
hosts or insects at diapause on Eucalyptus trees. Our study demonstrated that while more
than 54% of N.viridula adults collected in the field had no detectable bacteria in the V1-V3
midgut ventricles, the guts of the rest of stinkbugs were colonized by non-transient micro-
biota (NTM) and transient microbiota not present in stinkbugs at diapause. While transient
microbiota Bacillus sp., Micrococcus sp., Streptomyces sp., Staphylococcus sp. and others
had low abundance, NTM microbiota was represented by Yokenella sp., Pantoea sp. and
Enterococcus sp. isolates. We found some isolates that showed in vitro β-glucosidase and
raffinase activities plus the ability to degrade isoflavonoids and deactivate soybean protease
inhibitors. Our results suggest that the stinkbugs´ NTM microbiota may impact on nutrition,
detoxification and deactivation of chemical defenses, and Enterococcus sp., Yokenella sp.
and Pantoea sp. strains might help stinkbugs to feed on soybean developing seeds in spite
of its chemical defenses.
Introduction
Even if developing soybean seeds respond to stinkbug damage up-regulating important
defenses against herbivore insects, as cysteine proteases inhibitors and isoflavonoids produc-
tion [1,2], these induced chemical defenses are not sufficient to stop attack by the southern
PLOS ONE | https://doi.org/10.1371/journal.pone.0200161 July 12, 2018 1 / 23
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OPEN ACCESS
Citation: Medina V, Sardoy PM, Soria M, Vay CA,
Gutkind GO, Zavala JA (2018) Characterized non-
transient microbiota from stinkbug (Nezara
viridula) midgut deactivates soybean chemical
defenses. PLoS ONE 13(7): e0200161. https://doi.
org/10.1371/journal.pone.0200161
Editor: Erjun Ling, Institute of Plant Physiology and
Ecology Shanghai Institutes for Biological
Sciences, CHINA
Received: March 2, 2018
Accepted: June 20, 2018
Published: July 12, 2018
Copyright: ©2018 Medina et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This study was supported by Consejo
Nacional de Investigaciones Cientı
´ficas y Te
´cnicas
(CONICET), PIP-2012-0136, and Ministerio de
Ciencia, Tecnologı
´a e Innovacio
´n Productiva
(MINCyT), PICT-2015-0684 to JAZ. The funders
had no role in study design, data collection and
green stinkbug (N.viridula) or the red banded stinkbug (Piezodorus guildinii), what makes
them the most harmful pests to soybean crops [3,4].
N.viridula has an annual life cycle that generally comprehends five generations [5]. During
the onset of bloom and podset in late summer, soybean becomes attractive to stinkbugs, and
the third generation of adults migrates into the crop. Subsequently, the fourth and fifth genera-
tions also develop within this crop, at a time when they can feed on their developing seeds [5].
However, when soybean (primary host) is not available stinkbugs specific preference for soy-
bean pods changes to many other plant species depending on its maturity and phenology
(secondary hosts: SH), and plants in stage of fruit and pod formation are more attractive [6].
Whereas in tropical or subtropical areas with mild winters N.viridula feeds on any secondary
hosts available [6], in colder areas the last generation to reach adult stage seeks shelter under
the bark of trees beginning diapause, which is a critical period for the population [7].
Diversification and evolutionary success of insects have depended in part on a number of
relationships with beneficial microorganisms that have been known to increase the nutritional
value of diets and allow the digestion of recalcitrant compounds [811]. Several studies have
discussed the potential effects of diets on gut microbial composition and insect host [1217]. A
study comprehending 62 insect species, including N.viridula, showed that diet certainly affects
intraspecific gut bacterial community profiles when the host and microbiota are intimately
associated, such as in lignocellulose digestion [18]. Moreover, gut bacterial diversity is signifi-
cantly higher in omnivorous than in stenophagous (carnivorous and herbivorous) insects
[19]. It has been suggested that technologies used in agricultural production systems, such as
changes in soil management by crop rotations or use of agrochemicals like pesticides and her-
bicides, may shape the microbial communities of soil and plants, inducing insects to adopt
these new microbial species, which may help them to adapt to these altered or changing envi-
ronments [20].
Symbiotic/aposymbiotc studies performed with two related stinkbug species (Megacopta
punctatissima and M.cribaria) suggest that these species success as soybean crops pests is
more related to their relationship with their obligate gut symbiont (Ishikawaella capsulate)
than to specific traits of the insect species [21]. Another interesting example is the variant of
the corn (Zea mays) pest western corn rootworm (WCR; Diabrotica virgifera virgifera Le
Conte) which in last years started feeding on soybean foliage and also acquired tolerance
against cysteine protease inhibitors, a specific defense against Coleopteran insects [22]. Gut
microbiota analysis of the new variant of WCR suggests that it is the bacterial community
what allows the insects to tolerate defenses and to feed on the new host (soybean) [23].
Although the cyclic annual feeding behavior and host multiplicity of N.viridula could have
some impact on gut microbial community composition, it seems clear that understanding the
impact of feeding on developing soybean seeds on microbial community may explain stink-
bugs behavior.
Previously, it has been shown that Klebsiella pneumoniae and Enterococcus faecalis are pres-
ent in the midgut of N.viridula specimens collected in Brazil [24]. Moreover, Klebsiella pneu-
moniae was isolated from N.viridula adults collected in soybean fields near College Station,
TX [25]. Other studies have focused on an obligate symbiont resident in the caeca of N.viri-
dula that may have relevance on nymph survival, which has been consistently identified in
Brazil, Hawaii, California and Japan [24,2628]. Egg-surface sterilization disrupts nymphal
infection with the symbiont, indicating vertical transmission of the gut symbiont via egg sur-
face contamination[26]. Eliminating the symbiont resulted in severe nymphal mortality and
emergence of few adult insects. These results contrasts with those studies on Hawaiian popula-
tions of N.viridula [27,29], wherein elimination of the gut symbiont caused few fitness defects
in the host. Although Streptomyces sp strains were also associated with the caeca of lab reared
Stinkbug’s non-transient microbiota
PLOS ONE | https://doi.org/10.1371/journal.pone.0200161 July 12, 2018 2 / 23
analysis, decision to publish, or preparation of the
manuscript.
Competing interests: The authors have declared
that no competing interests exist.
stinkbug adults, no further analysis was performed [30]. In addition, a Pantoea sp. strain has
been associated with the caeca of another stinkbug pest, Halyomorpha halys [31], and the lack
of this symbiont decreases survivorship of stinkbug subsequent generations [32]. However,
little is known about the biological function of the bacteria present in stinkbugs ventricles
where digestion is performed (V1-V3 of the midgut)[33]. Since N.viridula is polyphytopha-
gous and tolerate plant defenses, characterization of the bacterial community associated with
insects collected in field production areas will improve our understanding of stinkbug-soybean
interactions.
To study the mechanisms of midgut bacteria-insect symbiosis that might help stinkbugs
to overcome plant defenses, we characterized the midgut microbial community potentially
related with digestion and associated to different stinkbug’s hosts (diet) along the year. In addi-
tion, we determined diapause influence on stability and composition of resident microbiota.
Finally, we characterized isolated bacteria and identified potential functional activities related
to soybean digestion, and inactivation of chemical defenses, such as cysteine protease inhibi-
tors. Our results allowed us to draw conclusions about the possible functions of midgut bacte-
rial community in circumventing soybean defences by the southern green stinkbug in the
main production areas of central Argentina.
Materials and methods
Sample collection and treatments
To assess variations of the microbial community composition in guts of stinkbugs collected
across different geographical locations, 173 N.viridula (Hemiptera, Pentatomidae) adults were
collected in 26 collecting events: 8 collections from different species of plants with the excep-
tion of soybean, which were considered as secondary hosts (SH; total of 52 stinkbugs), 9 collec-
tions from soybean crops (53 stinkbugs) and 9 from Eucalyptus trees (diapause; 68 stinkbugs)
at different moments of the year with a random sampling design along three years (2012–
2014). Sample collections events were distributed in 15 different sites located in central east
Argentina: Rafaela (-31,269161–61,484985), Parana
´(-31.866785, -60.483346) and Oliveros
(-32,578063, -60,853958) (Santa Fe
´Province), Pincen (-34,834096, -63,923950) and Marcos
Juarez (-32,699489, -62,100220) (Co
´rdoba Province), La Plata (-35,014814, -58,069611),
Lujan (-34,569906, -59,118805), Pergamino (-33,897777, -60,571060), Rojas (-34,198173,
-60,731049), Carabelas (-34,037867, -60,871811), Pila (-35,980229, -57,994852), San Antonio
de Areco (-34,265161, -59,449768), Chacabuco (-34,642247, -60,852295) and General Villegas
(-35,056980, -63,006592) (Buenos Aires Province), and the experimental field of our Facultad
de Agronomı
´a, Universidad de Buenos Aires in Buenos Aires city (-34,590259, -58,457565)
(S1 Table and S1 Fig). All collecting events were carried out on private land with the exception
of one carried out on Facultad de Agronomı
´a experimental fields that belong to our place of
work. The owners of private lands gave permission to conduct de collecting of Nezara viridula
and also gave information about pesticides applications. Field studies did not involve endan-
gered or protected species. The funders had no role in study design, data collection and analy-
sis, decision to publish, or preparation of the manuscript.
Insects were collected from soybean crops at reproductive stage or plant species around the
crops, where pesticides were never applied before each collection event. Geographical location
(latitude and longitude) of each site was registered by GPS and plant species where N.viridula
adults were found, identified and annotated (S1 Table). Samples were composed by 4 to 10
adults of stinkbugs that were handpicked and dissected to analyze bacterial community. Gut
community of stinkbugs was analyzed by Automated Ribosomal Intergenic Spacer Analysis
(ARISA) supplemented with agar plate culturing techniques on Trypticase Soy Agar (TSA)
Stinkbug’s non-transient microbiota
PLOS ONE | https://doi.org/10.1371/journal.pone.0200161 July 12, 2018 3 / 23
media. Based on differences in colony morphology on TSA plates, 21 different isolates were
preliminarily identified by 16S rRNA sequencing. Frequency and abundance of each identified
bacteria were annotated with the aim of hierarchize its biological importance. For those iso-
lates that needed more accurate identification MALDI-TOF MS bacterial identification tech-
nique was performed. Based on the ability of each bacterium to remain in the gut of stinkbugs
during diapause, and their abundance and their frequency of appearance, we classified bacte-
rium as either Non-Transient Microbiota (NTM) (bacterial count 10
4
CFU/mg gut and
present in SH, soybean and Eucalyptus trees) or Transient microbiota (TM) (bacterial count
under 100 CFU/mg gut, present only in SH and soybean). Members of the NTM were charac-
terized by API 20E for enterobacteria, and 50 carbon sources fermentation API 50CH strips
(Biomerieux). Phylogenetic analysis was performed by comparing sequences obtained from
the analysis of isolated bacteria with those of reference strains indexed at the LPSN site (http://
www.bacterio.net/). Bacterial localization was analyzed through microdissection of midgut
ventricles and ARISA (S2 Fig). To determine potential functionality of NTM bacteria isolated
and identified from the gut of stinkbugs, in vitro metabolic assays, including lipolytic, proteo-
lytic and glycolytic activities were performed. To assess the ability of bacteria to decrease inhib-
itory activity of soybean cysteine proteases inhibitors, soybean meal was fermented with NTM
isolated bacteria and compared against cysteine protease papain as control.
Insect dissection, bacteria isolation and DNA extraction
Stinkbugs were dissected under aseptic conditions no later than 8 h after collection. Guts were
entirely removed from insects and pooled on 1000 μL sterile buffer phosphate pH 7, and dis-
rupted by homogenization with a plastic pestle. For bacterial count and isolation, 100 μl ali-
quot were serially diluted and plated on Trypticase Soy Agar and cultured at 37 ˚C for 18 h
under aerobic conditions, Colonies with morphological differences of 17 different agar plates
(bacterial gut communities of individual insects) were chosen for further isolation, identifica-
tion, ARISA chromatograms performance, and in vitro activities (S2 Table). Remaining
homogenates of individual guts from each sample were pooled and total gut DNA was purified
with PowerFecal DNA Isolation kit (MOBIO) to perform gut bacterial community analysis
with ARISA (S2 Table).
Frequency and abundance of bacteria
Frequency of each identified bacterium was defined as positive results on plate counts (isolated
and identified) and/or ARISA detection. Relative abundance was the ratio between colony
forming units in 1 mg of intestine (CFU/mg of gut) of each species of bacteria identified and
the total number of colonies counted on the agar plate.
Isolated bacteria identification
For initial characterization, Gram staining and oxidase test [34] were used. DNA extraction of
each isolate was performed with UltraClean Microbial DNA Isolation Kit (MOBIO) and two
independent 16S rRNA fragments were PCR-amplified and sequenced. Firstly, the variable
region V4 was amplified with universal primers 530f (5´-GTGCCAGCMGCCGCGG-´3) and
1392r (5´-ACGGGCGGTGTGTRC-3´)according to Geib, SM et al (2009)[35]. For those iso-
lates that needed more accurate identification, a near-full length 1,450 bp fragment of 16S
rRNA was amplified according to Lehman, RM et al. (2009) [36]. PCR products were ligated
into pGEM-T Easy vector (Promega), and Escherichia coli DH5a were transformed. Plasmids
were extracted with a QIAprep
1
Spin Miniprep kit (QIAGEN, Valencia, CA). Briefly, the
inserts were amplified using the vector flanking sequences as primers (T7 and sp6 promoters).
Stinkbug’s non-transient microbiota
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A second pair of internal primers Sp6L01 (5`-AGTTTAT CACTGGCAGTCTCC-3´) and
T7H01 (5´-GTACTTTCAG CGAGGAGAAG-3´)were used for sequencing the central 700
bp region. The BioEdit Sequence Alignment Editor software was used to build the entire
sequence. Sequencing was done at Leloir Institute Facility, Buenos Aires, Argentina. The com-
plete 16S rRNA gene sequences of strains isolated in this study are sequences of known species
previously isolated by other groups, and these sequences are available for electronic retrieval
from the EMBL, GenBank.
All isolates were deposited in the National Bank of Microorganisms of the Institute of
Investigations in Agricultural and Environmental Biosciences (INBA-CONICET) of the
Agronomy School at University of Buenos Aires, Argentina.
MALDI–TOF MS for bacterial identification
To identify NTM isolates, we used MALDI-TOF (Matrix Assisted Laser Desorption Ioniza-
tion-Time of Flight) mass spectrometry [37]. Cultures were grown in TSA medium (Trypticase
Soy Agar, Laboratorios Britania S.A), incubated at 37 ˚ C for 18h. Samples were processed on a
Microflex MALDI-TOF MS spectrometer (Bruker Daltonics, Bremen, Germany) and analyzed
using the coupled software FlexControl v3.0 (Bruker Daltonics). Protein # 1 standard (BTS,
Bruker Daltonics) was included for calibration. All samples were analyzed in duplicate. The
analysis was performed by direct extraction methodology "on spot". A colony was deposited
without prior extraction step using a wooden stick and allowed to be air dried at room temper-
ature on a metal plate in MALDI-TOF. The samples were fixed with 1 μl of formic acid and
then with 1 μl of α-cyano-4-hydroxycinnamic acid to allow co-crystallization of the matrix
solution with the sample at room temperature. The positrons were extruded linearly at an
acceleration of 20 kV. The obtained spectra represent the sum of the ions obtained after the
impact of 350 automatic shots of the laser. The spectra were analyzed in a range of m/z (mass/
ionic charge ratio) of 3,500 to 20,000. Identification was performed using the MALDI Bioty-
perTM v3.1 program (Bruker Daltonics) by comparison of the mass spectra obtained for the
microorganisms under study with those included in their database. A possible error of a +10
variation of the peak value of m/z was considered. The results were interpreted from the score
assigned by the software to each sample (in the context of the analysis performed on the micro-
organisms under study). According to the criteria proposed by the manufacturer, a result was
considered valid (accurate identification to the species level) whenever the score value was
2.0 [38].
Phylogenetic analysis
Phylogenetic trees were constructed for all the identified bacteria, adding previously reported
Yokenella,Pantoea and Enteroccocus 16S rRNA 1450 bp sequences. To evaluate the phyloge-
netic proximity of Yokenella sp isolates to those bacteria identified as Klebsiella pneumoniae
in N.viridula guts collected in Brazil [24], we included sequences available from the type strain
of Yokenella regensurgei ATCC 49455, other strain of Yokenella sp., the type strains Klebsiella
oxytoca ATCC 13182, Klebsiella michiganensis ATCC BAA-2403 and Klebsiella pneumoniae
ATCC 13885.
In a second tree, NvP01 sequence was compared with 23 type strains of Pantoea species. A
third phylogenetic tree was constructed to analyze the proximity our enterococci to other
Enterococcus sp type strains (Enterococcus faecalis JCM 5803 and Enterococcus moriaviensis
ATCC BAA-383), and those enterococci identified in guts of N.viridula collected in Brazil
[24].
Stinkbug’s non-transient microbiota
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All phylogenetic analyses were conducted in MEGA software version 6.06 [39]. Sequences
corresponding to the 16S rRNA gene were aligned using the Muscle algorithm. A survey of
genetic distances based on the alignments was performed and the Kimura 2-parameter [40]
substitution model with Gamma corrections for variations of the mutation rate across sites
was chosen. For gap treatment, complete deletions were considered. The neighbor joining
algorithm [41] was used to generate the phylogenetic trees and they were validated with a boot-
strap of 1000 replicates.
Bacterial localization and cysteine protease activity in individual midgut
ventricles of reared N.viridula adults
Stinkbug adults were kept under rearing conditions in plastic cages at 23˚C; 10:12h dark/light;
60% humidity, and they were fed with mature rehydrated soybean seeds, dehulled sunflower
seeds and unsalted peanuts seeds. Ten reared adults were dissected under sterile conditions
and guts were microdissected and pooled to obtain samples of individual midgut ventricles.
ARISA detection and plate count was performed as it is reported elsewhere. Cystein protease
activity was measured according to Zavala et al (2008)[42] Here, cysteine proteinase activity
was estimated by using the chromogenic substrate p-Glu-Phe-Leu-pNA. Then 10 μl of the 18×
diluted enzyme was added to 20 μl of 0.38 mM p-Glu-Phe-Leu-pNA [in 0.1 M NaPhosphate,
0.3 M KCl, 0.1 mM EDTA, and 3 mM dithioerythreitol (pH 6.0)] and incubated at 37˚C.
Absorbance at 410 nm from wells on the microtiter plate was measured at 20-s intervals for
20 min with N.viridula guts enzymes. Initial rates of hydrolysis were estimated from the slopes
of the resulting absorbance versus time graphs. One cysteine activity unit was defined as the
amount of enzyme required to produce 1 mM 4-nitroaniline per minute at 37˚C using p-Glu-
Phe-Leu-pNA as a substrate under given assay conditions. Here, cysteine protease activity
against specific substrate (p-Glu-Phe-Leu-NA; Sigma) is normalized with total protein content
in the gut (Bradford-BioRad). Enzymatic kinetics curves were performed on a microplate spec-
trophotometer BIOTEK 808xl, with 402 nm filter. Enzymatic kinetics curves were performed
on a microplate spectrophotometer BIOTEK 808xl, with 402 nm filter. The assay was per-
formed in triplicate.
Identification of V4 midgut symbiont in N.viridula
A consistent peak of 745 pb was detected in all field collected samples and in reared stinkbugs
when bacterial community ARISA was performed (S2 Fig). Ventricle microdisecction allow us
to locate this peak on V4 midgut ventricle and purified this ITS fragment from agarose gel
using Agarose gel PCR purification kit. This fragment was cloned and sequence as it is
reported in previous sections. After sequencing, BLAST data base was used to confirm bacte-
rial origin and identify at family level.
Bacterial gut community Automated Ribosomal Intergenic Spacer Analysis
(ARISA)
To characterize stink bug gut bacterial community, ARISA electropherograms of total gut and
isolated bacteria were compared. ARISA specifically amplifies 16S and 23S rRNA intergenic
spacer and allows identifying the presence of uncultivable bacteria. The polymerase chain reac-
tion (PCR) step was performed according to the method described by Kent and Bayne (2010)
[43]. Intergenic spacers (ITS) were amplified with primers 23Sr (5´-GGGTTBCCCCATTCRG-
) and 1406f (5´-TGYACACACCG CCCGT-3´) marked at the 5´end with 6-FAM fluores-
cent dye. Denaturing capillary electrophoresis was carried out for each PCR reaction using an
Stinkbug’s non-transient microbiota
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ABI 3130 Genetic Analyzer (Applied Biosystems) at the Biotechnology Institute, INTA Caste-
lar, Argentina. Estimation of DNA fragment sizes was accomplished by using synthetic molec-
ular weight size standard ABI GeneScan1200 LIZ. 748bp and 756bp peaks were used as
positive control as they were consistently found in all samples (field collected and reared
adults). As ARISA and bacterial count on agar plates were conducted in parallel, we were able
to determine that sensibility of ARISA technique was at least of 10
3
CFU/mg gut, as it was seen
for Collecting event N˚ 25 (S3 Table).
Functional digestive activities of NTM isolates
Glycolytic activity of enterobacteria and enterococci was followed by using the 50 carbon
source fermentation strip API 50CHE (Biomerieux) accordingly to the manufacturer’s recom-
mendations. Fermentation of soybean seeds main components, sucrose, manose, cellulose and
raffinose was recorded at 24 h and 48 h. Proteolytic activity was detected by plating (as a spot)
5μl of overnight pure cultures adjusted to 0.1 McFarland onto Skim Milk Agar, incubated 96
h at 23-30-37 ˚C. Any transparent halo around the site of inoculation was considered as a posi-
tive proteolytic result. Lipolytic activity was evaluated by Rodhamine B-olive oil agar, incu-
bated as described above, and any pink/orange fluorescent halo was considered as a positive
result using a florescent lamp with an excitation wavelength of 350 nm [44].
Soybean whole meal fermentation and cysteine protease inhibitory activity
determination
Whole mature seeds of soybean cv. Williams were grounded with a coffee grinder to obtain
fine flour. The flour was sieved through a 0.5 mm pore metal mesh and suspended 20% in ster-
ile distilled water. Ten ml of the suspension were transferred into each of several 15 ml Falcon
tubes. An aliquot was immediately stored at -20˚C (non-pasteurized control). Remaining ali-
quots were pasteurized in a water bath at 50˚C for 1 h to reduce protein denaturation, includ-
ing cysteine protease inhibitors. 100 μl of a 10
8
CFU/ml suspension of overnight cultures were
inoculated in the pasteurized suspensions, and cultures were maintained for 24 h at 37 ˚C.
One tube was left without inoculation of bacteria (non-inoculated control). After this period,
soybean ferments and control were homogenized with vortex for 30 seconds and 1 ml aliquots
of each ferment and control were centrifuged at 12000 g for 20 min to obtain cysteine protease
inhibitors extracts. Cysteine proteases inhibitory activity of fermented and non-fermented
(control) soybean whole meal extracts was measured against papain by following the release of
p-nitroaniline (pNA; 37˚C for up to 20 min at 410 nm) after adding the synthetic substrate p-
Glu-Phe-Leu-pNA [42]. Briefly, 30 μl of 28 μg/ml papain was incubated in a 96-microplate
with 0–10 μl of supernatant of soybean fermented extracts at 37˚C for 10 min before addition
of the substrate. Cysteine protease inhibitors concentration was normalized with total protein
content in the gut (Bradford-BioRad)[42]. Enzymatic kinetics curves were performed on a
microplate spectrophotometer BIOTEK 808xl, with 402 nm filter. The assay was performed in
triplicate.
Statistical analysis
To evaluate the relationship between the presence of bacteria in the gut of N.viridula and the
host where stinkbugs were feeding on, Chi square (and Fisher´s exact) test was performed. To
evaluate the efficiency of isolated bacteria to inactivate cysteine protease inhibitor of soybean
whole meal, ANOVA with Dunnet posttest was performed, and non-inoculated pasteurized
soybean meal was used as control. To evaluate the effects of incubation of bacteria with
Stinkbug’s non-transient microbiota
PLOS ONE | https://doi.org/10.1371/journal.pone.0200161 July 12, 2018 7 / 23
soybean meal on cysteine proteases inhibitory activity, a t-test was performed. All analysis
were made with Prism 5.01 2007 (GraphPad Software Inc).
Results
Insect host survey
Before we started the study of bacterial community in the gut of N.viridula, we performed a
survey to determine the main hosts where these stinkbugs naturally feed on. During the south-
ern hemisphere winter (June to August), N.viridula adults were found on diapause, sheltering
under the bark of Eucalyptus trees present at the edges and in internal patches of soybean
crops (Fig 1). In spring, as result of longer photoperiod and increase of average temperature,
N.viridula begins to colonize different plant species as hosts (secondary hosts: SH) around the
trees. From mid-September to late January, adults were found feeding on mulberry (Morus
nigra L.), passion flower (Passiflora sp), honey locust (Acacia megaloxylon) and burdock (Arcti-
cum lappa) (Fig 1). Stinkbugs were also collected from maize (Zea mays), rapeseed (Brassica
napus), wheat (Triticum aestivum) and pecan (Carya illinoinensis) (Fig 1). We were not able to
find representative number of stinkbugs between November and December, probably because
of the low number of adults and the dispersion of stinkbugs among many plant species. How-
ever, from the beginning of February until the end of April stinkbugs moved from secondary
hosts to soybean crop (primary host), where they were collected (Fig 1). N.viridula colonized
soybean crops from the edges of the field during pods elongation (R4 according to Fehr and
Caviness, [45] and the population started to grow inside the field. The last generation of stink-
bugs turned to adult feeding on senescing soybean pods, thus they sought shelter under Euca-
lyptus bark to survive through winter (diapause) (Fig 1).
Identification and characterization of midgut microbiota and its
relationship with the insect host
Midgut microbial community analysis through ARISA and bacterial plate count of 173 stink-
bugs from 26 collecting events revealed that N.viridula is associated with few species of bacte-
ria (Table 1). Based on colony morphology, 21 gut bacteria were isolated and divided in two
groups (Table 1). The first group, with a bacterial count over 10
4
CFU/mg of gut, was identi-
fied as members of the Enterobacteriaceae and Enterococcaceae families (Non-Transient micro-
biota; NTM). These isolated bacteria were also detected by the culture independent technique
ARISA. Sequence analysis of the genes coding for the 16S rRNA showed that seven isolates of
Enterobacteriaceae were close to Yokenella (NvH01, NvO01, NvP02, NvR01, NvU01, NvU02,
NvW01) with a 99% similarity (Table 1). These isolates were further confirmed as related to
the Yokenella genus by MALDI-TOF MS bacterial identification. Isolates NvH01 and NvO01,
were identified as Y.regensburgei with scores 2.04 and 2.07 respectively, while NvP02, NvW01,
NvU01, NvU02 and NvR01, were identified as Yokenella sp. as their scores were <2 (1.73,
1.96, 1.94, 1.85 and 1.96, respectively) (S3 Table). In addition, we were able to identify one iso-
late as Pantoea sp (NvP01), and one as Cedecea sp (NvMJ01). Conversely, we found 4 gram-
positive isolates to be Enterococcus sp (NvH02, NvM04, NvS01 and NvW02).
Among isolates with sporadic presence and counts lower than 100 CFU/mg of gut, that
were not detected by ARISA (Transient microbiota; TM), other different bacteria were identi-
fied, such as Bacillus sp. (NvJ01, NvM02 and NvO02), Streptomyces sp. (NvI01), Micrococcus
sp. (NvI02 and NvS02) and Staphylococcus sp. (NvJ01) (Table 1). Since strict aseptic conditions
were used during dissection and plating, external contamination was preliminarily discarded.
ARISA profiles of identified bacteria allowed evaluating gut insect samples without further
Stinkbug’s non-transient microbiota
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specific isolation. For example, Yokenella sp. consistently showed ARISA peaks of 626, 710,
780 bp; Pantoea sp. peaks of 651, 667 and 858 bp; Cedecea peaks of 659 and 815 bp and Entero-
coccus sp. peaks of 502 and 602 bp (S2 Table).
We found a strong association between gut bacterial community and plant host of N.viri-
dula (Fisher´s test—X
2
: 23.0; df: 4, p = 0.001; Fig 2). NTM was found in the guts of 17% of
stinkbug adults feeding on SH and in 26% of those feeding on soybean, and in 26.5% of stink-
bugs in diapause under the bark of Eucalyptus trees (Fig 2). While transient microbiota was
present in 29% of stinkbugs guts that were feeding on SH and in 12% of those feeding on soy-
bean, this microbiota was absent in stinkbugs during diapause (Fig 2). Also, 54%, 62% and
73.5% of stinkbugs guts were found to be free of cultivable bacteria on TSA or ARISA detect-
able bacteria, when feeding on secondary hosts, soybean or under the bark of Eucalyptus trees,
respectively.
Gut bacterial community richness of collected samples (αdiversity) ranged between cero
and five, and was restricted to three main phyla: Enterobacteriaceae,Enterococcaceae,Bacilla-
ceae (S2 Table). Regarding enterobacterial isolates, Yokenella sp. was detected in ten collecting
events (two from SH; three from soybean, and five from Eucalyptus trees) (S2 Table), while
Pantoea sp. was detected in two collecting events (sampled in soybean and in Eucalyptus trees)
and Cedecea only in one (sampled in soybean) (S2 Table). Enterococci were isolated from five
collecting events (two sampled in SH, one in soybean and two in Eucalyptus trees). Enterococ-
cus sp. and Yokenella sp. were found cohabiting Nezara´s gut (collecting events N˚ 1, 9 and
23), where Enterococcus bacterial count was always one logaritmic order over Yokenella (S2
Table). Yokenella was more abundant (10
6
CFU/mg gut) when inhabiting the midgut without
Enterococcus competition (collecting events N˚ 2, 10, 14, 17, 19; S2 Table).
Fig 1. Nezara viridula hosts survey around the year cycle. Secondary hosts (SH) from late September to late January; soybean (primary host) from
February to late May and under the bark of Eucalyptus trees (diapause) from June to late September.
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Yokenella was the most frequent NTM in collected stinkbugs. We found Yokenella in eight
stinkbugs feeding on SH and in six feeding on soybean, and in 13 of those found in Eucalyptus
trees (Fig 3a). Conversely, Bacillus sp. was the most frequent among bacteria of the transient
microbiota group. We found Bacillus sp. in six stinkbugs feeding on SH and in three feeding
on soybean crop (Fig 3a). Relative abundance analysis of NTM showed that Enterobacteriaceae
represented 47% of bacterial communities in stinkbugs feeding on SH, and 80% in stinkbugs
feeding on soybean, and in those found under the bark of Eucalyptus trees (Fig 3b). Moreover,
Enterococcus sp. was 47% of the bacterial community in stinkbugs feeding on SH and 19% in
those feeding on soybean, and 20% in the stinkbugs collected from Eucalyptus trees (Fig 3b).
Finally, transient microbiota did not exceed 5% of bacterial population in the gut of stinkbug
feeding on SH and reached 1% in stinkbug feeding on soybean, and was not detected in those
in diapause (Fig 3b).
Bacterial localization in the midgut of N.viridula
Adults of N.viridula reared in laboratory were microdissected to evaluate digestive activity
and bacterial communities of individual ventricles (V). ARISA of midgut dissected ventricles
V1-V4 of N.viridula revealed the presence of enterococci and enterobacteria in ventricles
V1-V3, while caeca (V4) harbored a non-cultivable bacterium with 748 and 756 bp ITS frag-
ments (S2 Fig). Sequencing of the 748 bp fragment confirmed bacterial origin related to the
Enterobacteriaceae family. Cysteine protease activity was higher in ventricles V2-V3 (9.0 ±1.5
Table 1. Microscopic and molecular identification of 21 isolates from the midgut of field collected N.viridula adults.
Group Strain
1
ARISA detection log CFU/mg guton TSA
2
Gram stainig 16S ARNr V4 or 1492 bp
3
Selected for characterization
4
Non -Transient microbiota NvH01 Yes 6Gram—bacilli Yokenella sp. Yes
NvO01 Yes 6Gram—bacilli Yokenella sp. Yes
NvP01 Yes 4Gram—bacilli Pantoea sp. Yes
NvP02 Yes 6Gram—bacilli Yokenella sp. Yes
NvR01 Yes 4Gram—bacilli Yokenella sp. Yes
NvU01 Yes 6Gram—bacilli Yokenella sp. Yes
NvU02 Yes 6Gram—bacilli Yokenella sp. Yes
NvW01 Yes 4Gram—bacilli Yokenella sp. Yes
NvMJ01 Yes 5Gram—bacilli Cedecea sp. NO
NvH02 Yes 7Gram+ rods Enterococcus faecalis Yes
NvS01 Yes 5Gram—bacilli Enterococcus sp. Yes
NvW02 Yes 5Gram+ rods Enterococcus sp. Yes
NvM04 Yes 7Gram+ rods Enterococcus sp. Yes
Transient microbiota NvI01 NO 1 Gram+ bacilli Streptomyces sp. NO
NvI02 NO 1 Gram + rods MIcrococcus sp. NO
NvJ01 NO 1 Gram+ rods Staphylococcus sp. NO
NvJ02 NO 1 Gram + bacilli Bacillus sp. NO
NvM02 NO <1Gram + bacilli Bacillus sp. NO
NvM01 NO <1Gram—bacilli Bacillus sp. NO
NvO02 NO 1 Gram + bacilli Bacillus sp. NO
NvS02 NO <1Gram+ rods Micrococcus sp. NO
1
: Isolate identification code: NvXN˚; Nv (N.viridula) X (collecting event) N˚ (isolate number).
2
: bacterial count was performed on Trypticase Soy Agar, overnight, 37˚C.
3
: V4 sequences were compared against GenBank database. Complete sequence was compared against SILVA databe.
4
: ARISA detected isolates were chosen for further characterization.
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activity units and 16.8 ±2.2 activity units, respectively) than in V4 (0.5 ±0.05 activity units),
indicating that ventricles V2-V3 are specialized for diet degradation and absorption (S2 Fig).
Phylogenetic analysis of Enterobacteriaceae and Enterococcus sp.
16S rRNA sequences of gut isolates from adults of N.viridula matched with Yokenella sp.
sequences, which is a species phylogenetically close to Klebsiella (Fig 4). The phylogenetic
analysis of the 16S rRNA gene sequences revealed two different clusters (89% bootstrap confi-
dence); one of the (NvR01, NvO01 and NvU01) match showed a high similarity to the type
strain Yokenella regensburgei ATCC 49455, while the other (NvH01, NvP02, NvU02 y NvW01)
matched closer to a different strain deposited as Yokenella (S3 Table). The first group of Yoke-
nella sp. (NvR01, NvO01 and NvU01) also matches closer to Klebsiella pneumoniae strains
reported by Hirose (24), which are clearly separated from other Klebsiella species, suggesting
that the bacteria isolated in Brazil are more likely to be Yokenella than Klebsiella.
Fig 2. Presence/absence of gut microbiota in N.viridulaadults associated with the insect host. Presence of non-transient (black) and transient (light
grey) microbiota or absence of bacteria (Not infected; dark grey) in N.viridula adult’s V1-V3 midgut ventricles; and its distribution related to insect
host: secondary hosts (SH), soybean and under the bark of Eucalyptus trees. Numbers correspond to insect gut dissected.
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Fig 3. Gut bacterial communities associated with the insect host. Bacteria species inhabiting Nezara viridula infected V1-V3
midgut ventricles and its distribution among insect hosts (a) and relative abundance (CFU/mg gut x n˚ insects positive
-1
) of
bacterial groups of infected N.viridula V1-V3 midgut ventricles and its distribution among hosts (b). Enterobacteriaceae groups
Yokenella,Cedecea and Pantoea species. Numbers correspond to insect gut dissected.
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Another enterobacteria was identified as Pantoea sp., (NvP01) with a closer match to P.con-
spicua. Phylogenetic analysis included 22 species of Pantoea and one subspecies of P.stewartii
(Fig 5). Pantoea sp. was also detected with ARISA from stinkbugs collected on Eucalyptus trees
as it presented the same peak pattern as Pantoea sp. NvP01.
We also isolated and identified an Enterococcus sp. strain (NvH02), very closely related
(overall genetic distance 0.002 and similarity greater than 99%) to the Enterococcus faecalis
type strain (Fig 6 and S4 Table). In a separated branch, our other Enterococcus sp. isolates
(NvM04, NvS01 and NvW02) grouped together with those identified in N.viridula in Brazil
[24] (Fig 6 and S4 Table). These bacteria had also a 99% similarity to E.faecalis, and the search
in the RefSeq database of NCBI confirmed that the closest member was indeed E.faecalis.
In vitro enzymatic activities of bacteria in ventricles V1-V3 of N.viridula
To test whether gut isolated bacteria can help stinkbugs to digest soybean, in vitro analysis of
enzymatic activities using a specific culture media were performed. None of the Yokenella iso-
lates were able to utilize sucrose, the main sugar in soybean, and had no proteolytic activity on
casein under aerobic or fermentative conditions (Table 2). Yokenella sp. NvH01 obtained from
Fig 4. Phylogenetic placements of Yokenella strains (bold letters). We constructed a tree that included our isolates, those described as K.pneumoniae by Hirose
et al. (2006), the species reference strains K.pneumoniae ATCC 13885, K.michiganensis ATCC BAA-2403, K.oxytoca ATCC 13182, Y.regensburgei ATCC 49455
and one additional strain of Y.regensburgei. Gene bank accession numbers and insect host of bacteria isolated in this study appear in parentheses Escherichia coli
ATCC 11775 was included as an outgroup to root the tree. K.michiganensis and K.oxytoca were included because a blast search in the RefSeq database indicates
that both these species and Y.regensburgei lie at closer genetic distance to the Hirose´s strains than K.pneumoniae. Only the bootstrap values greater than 60% are
shown. Scale bar states for the phylogenetic distance with a common ancestor.
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stinkbugs feeding on Morus nigra and Yokenella sp NvU01 and NvW01 isolated from those
collected on Eucalyptus trees, had cellulase, maltase, esculinase and rafinase activity, evidenc-
ing α1–2, β1–4, α1–4, β1–6, α1–6 glycosidase activity, respectively (Table 2). In addition,
Yokenella sp. NvH01, Yokenella sp. NvO01 isolated from insects feeding on soybean and Yoke-
nella sp. NvR01 and NvU02 isolated from insects in diapause, were positive for lipase activity,
as they showed pink fluorescent colony and degradation halos on Rhodamine B/Olive oil
Agar plates (Table 2). Pantoea sp. NvP01 isolated from insects feeding on soybean was able to
degrade sucrose, cellulose and maltose, but was not able to perform any proteolytic or lipolytic
activity (Table 2).
All of the enterococci strains isolated from insect feeding on Morus nigra (NvH02), rape-
seed (NvS01), soybean (NvM04) or from stinkbugs in Eucalyptus trees (NvW02), were positive
for utilization of sucrose, cellulose and esculine, and negative for utilization of raffinose
(Table 2). E.faecalis NvH02 was also positive for utilization of maltose. No isolated enterococci
strain showed proteolytic or lipolytic activities under the conditions of the assay.
Fig 5. Phylogenetic positioning of Pantoea NvP01 (bold letters). We constructed a tree that included our isolate and type strains of 22 species of Pantoea
and a subspecie of Pantoea stewartii. Gene bank accession numbers and insect host of bacteria isolated in this study appear in parentheses. The sequence of
the type strain Klebsiella pneumoniae ATCC 13883 was included as an outgroup to root the tree. Only the bootstrap values greater than 65% are shown.
Scale bar states for the phylogenetic distance with a common ancestor.
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Enterobacteria and enterococci isolated strains decrease protease inhibitor
activity of soybean flour during fermentation
To determine whether soybean protease inhibitors can be deactivated through fermentative
process by bacteria from the midgut of stinkbugs, soybean flour was inoculated with represen-
tatives of the different isolated microorganisms and incubated. Incubation of the control (non-
Fig 6. Phylogenetic positioning of Enterococcus isolated strains (bold letters). We constructed a tree that included our isolates, those described as
Enterococcus faecalis by Hirose et al. (2006), and type strain Enterococcus faecalis JCM 5803. Gene bank accession numbers and insect host of bacteria
isolated in this study appear in parentheses. The sequence of the type strain Enterococcus moraviensis ATCC BAA-383 was included as an outgroup to
root the tree. Only the bootstrap values greater than 65% are shown. Scale bar states for the phylogenetic distance with a common ancestor.
https://doi.org/10.1371/journal.pone.0200161.g006
Table 2. Enzymatic activities of isolated strains with significance in soybean digestion.
Enzymatic
activity Glycolytic
1
Proteolytic
2
Lipolytic
3
α1–2
glucoside
β1–4
glucoside
α1–4
glucoside
β1–6
glucoside
α1–6
galactoside Total
Proteases Cystein proteases lipases
Substrate Sacarose Cellulose Maltose Esculine Rafinose Casein P Glu Phe Leu
NA
Olive oil
Yokenella sp.NvH01 - + + + + - - +
NvU01 - + + + + - - -
NvW01 - + + + + - - -
NvO01 - - - - - - - +
NvU02 - - - - - - - +
NvR01 - - - - - - - +
NvP02 - - - - - - - -
Pantoea sp.NvP01 + + + - - - - -
Enterococcus
sp.
NvH02 + + + + - - - -
NvS01 + + - + - - - -
NvM04 + + - + - - - -
NvW02 + + - + - - - -
1
. Bacterial glycolytic activities were evidence trough API 50CH strips (Biomerieux).
2
: Bacterial proteolytic activity were evidence on Skim Milk Agar and broth, and against specific cysteine protease substrate GluPheLeu—pNA (Sigma).
3
: Bacterial Lipolytic activity was evidence on Rhodamine B-Olive oil- Agar.
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inoculated soybean flour) increased the inhibitory capacity of papain activity by 21.3%, proba-
bly due to hydration of soybean meal and activation of cysteine protease inhibitors (p = 0.04).
However, the fermentative activities of Yokenella sp. NvH01, NvO01, NvR01 and NvU02, and
Enterococcus faecalis NvH02 and NvM04 reduced the inhibitory capacity of soybean cysteine
protease inhibitors from 35% to 75% (p<0.001; Fig 7). There were no significant differences
among the different tested microorganisms (ANOVA; p = 0.07; Fig 7). Finally, bacterial cyste-
ine proteases activity on fermented soybean meal was evaluated to identify potential activity
that could mask papain inhibition. There was no cysteine proteases activity detected for any
isolated strain or control.
Discussion
Changing gut environment by bacterial communities allow insects to rapidly adapt to new
hosts and to tolerate plant chemical defenses [10,16,20,23,4649]. To test the hypothesis that
midgut bacterial community of stinkbugs (N.viridula) deactivates chemical defenses of soy-
bean developing seeds, we identified and characterized midgut microbiota of stinkbugs col-
lected from soybean crops, different secondary plant hosts (SH) or Eucalyptus trees (Fig 1).
Our study demonstrated that while more than 54% of N.viridula adults collected in the field
Fig 7. Inhibition capacity of fermented soybean cv. Williams whole meal extracts against cysteine protease papain. Whole meal was inoculated
with non-transient microbiota (NTM) isolated bacteria and fermented during 24h at 37˚C. Yokenella sp. NvH01, NvO01, NvR01, NvU01, NvU02 and
NvW01; Pantoea sp. NvP01; Enterococcus faecalis NvH02 and Enterococcus p: NvM04. Control extracts correspond to a pasteurized non-inoculated and
incubated soybean whole meal suspension.
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had no detectable bacteria in V1-V3 midgut ventricles, the guts of the rest of stinkbugs were
colonized by non-transient microbiota (NTM) and transient microbiota not present in stink-
bugs at diapause (Fig 2).
Whereas transient microbiota had low abundance and was represented by a variable num-
ber of genera, such as Bacillus and Micrococcus, NTM microbiota was composed by enterobac-
teria and enterococci, which were represented by Yokenella sp., Pantoea sp., Cedecea sp. and
Enterococcus sp. isolates (Table 1,Fig 3 and S2 Table). ARISA together with MALDI-TOF and
16S rRNA sequencing techniques permitted us identify the NTM (S3 Table), and phylogenetic
trees allowed us positioning Enterococcus sp. Yokenella sp. and Pantoea sp. among similar spe-
cies, and even suggesting that some microorganisms previously isolated were perhaps errone-
ously identified (Figs 46). Our in vitro results suggests that stinkbugs NTM may impact on
nutrition, detoxification and deactivation of chemical defenses (Fig 7 and Table 2), indicating
that Enterococcus sp. Yokenella sp. and Pantoea sp. isolated strains might help stinkbugs to feed
on soybean developing seeds in spite of its chemical defenses. To our knowledge no study
before has characterized the biological functions of N.viridula microbiota.
Stinkbugs collected from different areas showed that NTM was present in midgut ventri-
cles V1-V3 where the highest enzymatic activity levels were measured, and suggested that
Yokenella sp., Pantoea sp. and Enterococcus sp. may play an important role on insect nutri-
tion and deactivation of chemical defenses (Fig 3). Midgut isolates of NTM were able to
degrade galactosyl derivatives of sucrose, such as raffinose (Table 2), which is the second
more abundant sugar in soybean seeds and is considered responsible of reducing digestibility
because it has some activity as protease inhibitor [50]. Moreover, digestion of raffinose
results on lower pH and prebiotic short chain fatty acids [51]. In addition, gut bacteria
may deactivate protease inhibitors (Fig 7), which are the main defense of soybean against
insect herbivores that can decrease the activity of digestive cysteine proteases of stinkbugs,
reducing insect performance [1,52]. Although biochemical characterization of Enterococus
sp. Yokenella sp. and Pantoea sp. isolated strains showed no extracellular proteolytic activity
(Table 2), the NTM reduced the inhibitory capacity of cysteine protease activity of soybean
whole meal after 24h of in vitro fermentation (Fig 7), and might help stinkbugs to feed on
soybean developing seeds. Furthermore, midgut isolates of NTM showed β-glycosidase and
α-galactosidase activities, and might hydrolyze the glyosidic bond of the isoflavonoids, genis-
tin and daizin in the insect gut (Table 2). Isoflavonoids participate in the defense against
insect attack, and damage produced by N.viridula increases these phenolic compounds pro-
duction in attacked seeds [1,2,53,54]. It is not clear yet whether the isoflavonoids glycosides
or aglycones are toxic to stinkbugs. Although NTM may play a role in helping stinkbugs to
feed on soybean, we did not detect any bacteria in V1-V3 ventricles in more than 54% of col-
lected insect (Fig 2).
Microorganisms of plant ecto- and endophytic communities are ingested during insect
feeding and some of them can become part of the gut microbiota, depending on pH, enzymatic
activity, redox potential and other intestinal conditions [8,9,55]. Midguts of N.viridula adults
are acidic with a pH of 4.5–6.5, with sectored compartments and high enzymatic activity
[24,33]. The lack of bacteria or low diversity of NTM found in V1-V3 of many stinkbugs (Figs
2and 3), may be explained by feeding behavior, moulting, gut biochemical and physiological
characteristics, non-gregarious behavior, among others [8,9,55,56]. Non gregarious behavior
of N.viridula adults limit oral-fecal transference of bacteria and conformation of complex gut
bacterial communities, as can be seen in social insects, which allows the stability of symbiotic
bacteria in the population [8,9,55,56]. In addition, piercing sucking feeding behavior of N.viri-
dula could limit horizontal transfer of bacteria by ingestion of endophytes, leaving behind phy-
loplane bacteria [10,57].
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Host also can exert control over their microbiota in midguts by monitoring and targeting
the species to either promote or hinder their proliferation [58]. We found that only few species
of bacteria (NTM) can reside in the midgut of N.viridula (Table 1). Detecting harmful and
beneficial traits of bacteria is a robust way for a host to monitor microbiota. There is a signifi-
cant variability in antimicrobial peptides in host species, suggesting that the secretion of theses
peptides from the host epithelium helps to determine which microbial genotypes prosper [59].
In addition, host diet has a major impact on the available resources within insects, which
results in the microbial species dominating in the midgut. The diversity and frequency of
NTM were higher in stinkbugs feeding on soybean than in those feeding on secondary hosts
(Figs 2and 3). This may be due to diet quality as soybean is a better substrate for enterobacte-
ria [60]. Also, these bacteria had the ability to remain in the intestine during diapause (Euca-
lyptus trees) probably by colonization of midgut epithelium or the lumen (Fig 3). During
isolation of NTM microbiota, enterobacteria showed fast in vitro growing (data not shown),
suggesting a fast lumen colonization.
To compare the phylogenetic relations of the NTM species isolated from V1-V3 of stink-
bugs collected from the field, we used type strains of the related species to perform 16S rRNA
alignment trees, which has been the primary reference for bacterial phylogeny [61]. We found
close phylogenetic relationship between bacteria identified in this study (Figs 4and 5) with
those previously isolated from midguts of N.viridula in Brazil [24]. Some of our isolates are
closely related to others previously identified as K.pneumoniae, [24] our identification (includ-
ing MALDI-TOF identification) suggest that the isolated bacteria are Yokenella sp. rather than
K.pneumoniae (Fig 4). Some biochemical tests also would discard them as klebsiellas, but we
do not have access to the microorganisms previously isolated. Different Enterococcus sp identi-
fied in our study are also closely related to those identified in Brazil (Fig 6). However, our dis-
crimination among the different enterococci may not be sensitive enough. It is possible that
Yokenella sp. and Enterococcus sp. might have been associated to N.viridula probably during
migration from equatorial regions of Brazil to southern fields in Argentina or through a close
interaction with soybean, explaining the presence of the bacterium species in the gut of stink
bugs. Yokenella sp. has also been associated to the firebug (Pyrrhocoris apterus; Hemiptera,
Pyrrhocoridae), a common cotton pest in Europe [62]. In addition, we isolated and identified
Pantoea sp. from guts of stinkbugs collected from two different sites (Fig 5 and Table 1), which
to our knowledge has never been identified in stinkbugs before. This bacteria has been previ-
ously isolated from different environmental samples (e.g., water, soil, plant material) and
insects, including mosquitoes (Diptera), thrips (Thysanoptera), bees (Hymenoptera), and
hemipterans [55], suggesting that this gram negative rod has a wide ecological distribution.
Our study showed that N.viridula adults feed on many different hosts, and suggested a
strong association between gut bacterial community and plant hosts (Fig 2). Field surveys
showed that adults of N.viridula feed on soybean crops, in winter move to Eucalyptus trees to
spend diapause, and then in spring start to feed on different SH such as, mulberry (Morus
nigra L.), passion flower (Passiflora sp.), honey locust (Acacia megaloxylon) and some crops
like, maize (Zea mays), rapeseed (Brassica napus), wheat (Triticum aestivum) (Fig 1). Eucalyp-
tus trees unable the insect to adapt to non-tropical regions, as they are used for sheltering
during cold winters [6,63,64]. Although secondary hosts are not suitable for correct nymph
development, they allow adults emerging from diapause to obtain resources to begin a new
cycle [6567]. Since between 17% and 26% of stinkbugs collected in the field from all hosts
contain NTM in the midgut (Fig 2), NTM may help N.viridula to tolerate plant defenses and
feed on different hosts. Enterobacteria and enterococci that infected the midgut of N.viridula
may change the biochemical environment of guts, improving digestibility trough inactivation
of protease inhibitors and other antinutrients. However, the small number of stinkbugs
Stinkbug’s non-transient microbiota
PLOS ONE | https://doi.org/10.1371/journal.pone.0200161 July 12, 2018 18 / 23
infected with NTM could indicate possible detrimental effects of these bacteria over the insect.
Future work will focus on the effects of Enterobacteriaceae and Enterococcus midgut coloniza-
tion on N.viridula.
Supporting information
S1 Table. Geographical placement of collecting sites.
(PDF)
S2 Table. Bacterial communities in the midgut of Nezara viridula associated to the insects
hosts.
(PDF)
S3 Table. Bacteria isolated in this work and those used to build phylogenetic trees of Yoke-
nella.
(PDF)
S4 Table. Bacteria isolated in this work and those used to build phylogenetic trees of
Enterococcus sp.
(PDF)
S1 Fig. Map of Argentina (a) and a zoom of central east Argentina (b) were 26 collecting
events were performed during 2012–2014. Nezara viridula adults were handpicked from sec-
ondary hosts (light grey spots), Soybean (dark grey spots) or from under de bark of Eucalyptus
trees (black spots).
(PDF)
S2 Fig. (a) Cysteine protease activity of N.viridula V1-V4 midgut ventricles. Statistical
differences are denoted by different letters. (b) Distribution of ARISA detected bacteria
among N.viridula V1-V4 midgut ventricles. Bacterial ITS fragments appear as blue peaks
and LIZ 1200 weight standard fragments appear as yellow peaks. On a black square are 748 y
756bp cloacae symbiont ITS fragments. Numbers are reference for weight standard.
(PDF)
Author Contributions
Conceptualization: Gabriel O. Gutkind, Jorge A. Zavala.
Data curation: Marcelo Soria, Gabriel O. Gutkind.
Formal analysis: Virginia Medina, Marcelo Soria, Carlos A. Vay, Jorge A. Zavala.
Funding acquisition: Jorge A. Zavala.
Investigation: Virginia Medina.
Methodology: Pedro M. Sardoy, Marcelo Soria, Carlos A. Vay, Gabriel O. Gutkind.
Project administration: Jorge A. Zavala.
Supervision: Gabriel O. Gutkind.
Validation: Jorge A. Zavala.
Writing – original draft: Virginia Medina.
Writing – review & editing: Jorge A. Zavala.
Stinkbug’s non-transient microbiota
PLOS ONE | https://doi.org/10.1371/journal.pone.0200161 July 12, 2018 19 / 23
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... Whereas some insects can avoid plant chemical defenses by different behavioral and biochemical mechanisms, [5][6][7] recently laboratory in vitro experiments have suggested that soybean proteases inhibitors can be deactivated by N. viridula's gut microbiota, such as the enterobacteria Yokenella sp. 8 Some specific gut bacteria enable insects to feed on poor diets or digest recalcitrant compounds and increase insect performance. 9,10 Moreover, shifts on bacterial gut communities have allowed contemporary adaptation of insects to overcome new technologies of pest control. ...
... No bacteria were detected in the V1-V3 midgut sections of 60% of field collected N. viridula adults, and the rest were naturally infected with cultivable non-transient bacteria, such as Yokenella sp. 8 This stink bug species houses an obligate symbiont in the caeca (V4) not directly involved in food digestion, which is present in N. viridula from a wide variety of places, including Hawaii, California, Japan, and Brazil. [39][40][41] However, little is known about the biological function of the bacteria present in stink bugs ventricles where digestion is performed (V1-V3 of the midgut). ...
... 38 In field conditions adults of N. viridula have low diverse gut microbiota, and over 60% of individuals lack cultivable bacteria over 10 3 CFU mg −1 gut. 8 However, stink bugs relay upon a single non-cultivable strict symbiont located in a special gut section, and have low diverse and transient microbiota associated to the digestive midgut. 38,45 Therefore, laboratory overcrowding may compromise the immune response of non-social insects as N. viridula, which promote the infection or transfection of bacteria that will change substantially the gut microbiota. ...
Article
Background: The southern green stinkbug (Nezara viridula) is a mayor pest of soybean. However, the mechanism underlying stinkbug resistance to soybean defenses is yet ignored. Although gut bacteria could play an essential role in tolerating plant defenses, most studies testing questions related to insect-plant-bacteria interactions have been performed in laboratory condition. Here we performed experiments in laboratory and field conditions with N.viridula and its gut bacteria, studying gut lipid peroxidaxion levels and cysteine activity in infected and unifected nymphs, testing the hypothesis that feeding on field-grown soybean decreases bacterial abundance in stinkbugs. Results: Gut bacterial abundance and infection ratio were higher in N.viridula adults reared in laboratory than in those collected from soybean crops, suggesting that stinkbugs in field conditions may modulate gut bacterial colonization. Manipulating gut microbiota by infecting stinkbugs with Yokenella sp. showed that these bacteria abundance decreased in field conditions, and negatively affected stinkbugs performance and were more aggressive in laboratory rearing than in field conditions. Infected nymphs that fed on soybean pods had lower mortality, higher mass and shorter development period than those reared in the laboratory, and suggested that field conditions helped nymphs to recover from Yokenella sp. infection, despite of increased lipid peroxidation and decreased cysteine proteases activity in nymphs' guts. Conclusions: Our results demonstrated that feeding on field-grown soybean reduced bacterial abundance and infection in guts of N.viridula and highlighted the importance to test functional activities or pathogenicity of microbes under realistic field conditions prior to establish conclusions on three trophic interactions. This article is protected by copyright. All rights reserved.
... On average 6.1 zOTUs were found on the eggs of natural N. viridula populations, while on average only 1.2 zOTU was associated with the internal samples. Similarly, low microbial diversity has been found in the midgut of field-collected N. viridula adults (Medina et al., 2018), suggesting that overall microbial diversity associated with N. viridula is low: no culturable bacteria were found in the V1-V3 midgut sections in more than 54% of N. viridula adults collected in the field, while the rest of the stinkbugs were colonized by only a few culturable bacteria like Bacillus, Enterococcus, Micrococcus, Pantoea, Staphylococcus, and Yokenella (Medina et al., 2018). ...
... On average 6.1 zOTUs were found on the eggs of natural N. viridula populations, while on average only 1.2 zOTU was associated with the internal samples. Similarly, low microbial diversity has been found in the midgut of field-collected N. viridula adults (Medina et al., 2018), suggesting that overall microbial diversity associated with N. viridula is low: no culturable bacteria were found in the V1-V3 midgut sections in more than 54% of N. viridula adults collected in the field, while the rest of the stinkbugs were colonized by only a few culturable bacteria like Bacillus, Enterococcus, Micrococcus, Pantoea, Staphylococcus, and Yokenella (Medina et al., 2018). ...
Article
Full-text available
Although microbial communities of insects from larval to adult stage have been increasingly investigated in recent years, little is still known about the diversity and composition of egg‐associated microbiomes. In this study, we used high‐throughput amplicon sequencing and quantitative PCR to get a better understanding of the microbiome of insect eggs and how they are established using the Southern green stinkbug Nezara viridula (L.) (Hemiptera: Pentatomidae) as a study object. First, to determine the bacterial community composition, egg masses from two natural populations in Belgium and Italy were examined. Subsequently, microbial community establishment was assessed by studying stinkbug eggs of different ages obtained from laboratory strains (unlaid eggs collected from the ovaries, eggs less than 24 h old, and eggs collected 4 days after oviposition). Both the external and internal egg‐associated microbiomes were analyzed by investigating egg washes and surface‐sterilized washed eggs, respectively. Eggs from the ovaries were completely devoid of bacteria, indicating that egg‐associated bacteria were deposited on the eggs during or after oviposition. The bacterial diversity of deposited eggs was very low, with on average 6.1 zero‐radius operational taxonomic units (zOTUs) in the external microbiome and 1.2 zOTUs in internal samples of egg masses collected from the field. Bacterial community composition and density did not change significantly over time, suggesting limited bacterial growth. A Pantoea‐like symbiont previously found in the midgut of N. viridula was found in every sample and generally occurred at high relative and absolute densities, especially in the internal egg samples. Additionally, some eggs harbored a Sodalis symbiont, which has previously been found in the abdomen of several insects, but so far not in N. viridula populations. We conclude that the egg‐associated bacterial microbiome of N. viridula is species‐poor and dominated by a few symbionts, particularly the species‐specific obligate Pantoea‐like symbiont. In this study, we assessed the composition and establishment of the microbiome of insect eggs using the Southern green stinkbug Nezara viridula (Hemiptera: Pentatomidae) as a study object. Our results show that the egg‐associated bacterial microbiome is species‐poor and dominated by a few symbionts, particularly the species‐specific obligate Pantoea‐like symbiont.
... Despite an increased interest in the regulatory and physiological aspects of insect diapause [1,[5][6][7][8][9][10][11][12][13][14], the dynamics of host-microbiome interactions during diapause remain underexplored (but see [15][16][17][18]). This contrasts with a growing body of research on hibernating mammals, demonstrating a seasonal remodelling of the gut microbiome between the hibernating and active phases in various species [19][20][21][22]. ...
... While the dominant members of the microbiome remained largely unaffected, diapause seemed to have an impact on many less abundant taxa. This is similar to observations in stinkbugs, where only a non-transient core microbiome was detected in diapausing individuals [17]. In N. vitripennis, about 20% of all bacterial genera were exclusively observed in nondiapausing larvae, indicating that some bacterial taxa may be lost already during preparation for diapause. ...
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Background The life cycles of many insect species include an obligatory or facultative diapause stage with arrested development and low metabolic activity as an overwintering strategy. Diapause is characterised by profound physiological changes in endocrine activity, cell proliferation and nutrient metabolism. However, little is known regarding host-microbiome interactions during diapause, despite the importance of bacterial symbionts for host nutrition and development. In this work, we investigated (i) the role of the microbiome for host nutrient allocation during diapause and (ii) the impact of larval diapause on microbiome dynamics in the parasitoid wasp Nasonia vitripennis , a model organism for host-microbiome interactions. Results Our results demonstrate that the microbiome is essential for host nutrient allocation during diapause in N. vitripennis , as axenic diapausing larvae had consistently lower glucose and glycerol levels than conventional diapausing larvae, especially when exposed to cold temperature. In turn, microbiome composition was altered in diapausing larvae, potentially due to changes in the surrounding temperature, host nutrient levels and a downregulation of host immune genes. Importantly, prolonged larval diapause had a transstadial effect on the adult microbiome, with unknown consequences for host fitness. Notably, the most dominant microbiome member, Providencia sp., was drastically reduced in adults after more than 4 months of larval diapause, while potential bacterial pathogens increased in abundance. Conclusion This work investigates host-microbiome interactions during a crucial developmental stage, which challenges both the insect host and its microbial associates. The impact of diapause on the microbiome is likely due to several factors, including altered host regulatory mechanisms and changes in the host environment.
... A recent study also showed that a carnivorous diet was preferential for Enterobacteria, including some entomopathogenic bacteria in the gut of plant bugs, compared to an herbivorous diet, which was detrimental to bugs' survival [67]. Enterococcus in stink bugs and other herbivorous insects was reported to be implicated with diet digestion and the detoxification of plant defensive chemicals [68][69][70]. Enterococcus dominated in the gut of generalist herbivore cotton leafworm (Spodoptera littoralis) and was documented to secrete bacteriocin against invading bacteria, providing a defensive function to the host [71]. More empirical tests are needed to validate whether Enterococcus in the gut of assassin bugs plays a similar defensive role for its hosts. ...
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... Some of the bacterial community isolates showed in-vitro β-glucosidase and raffinase activities, which enable the degradation of galactosyl derivatives and increase the digestibility of soybean plants. Enzymes in midgut isolates might also degrade isoflavonoids and deactivate soybean protease inhibitors, helping aphids tolerate soybean defences and feed on the plant (Medina et al., 2018). ...
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... In some systems, diapause has been shown to reduce microbial diversity to a core subset, as seen in both stink bugs (Nezara viridula) and marine copepods (Arias-Cordero et al., 2012;Datta et al., 2018;Medina et al., 2018). Few studies to date have investigated the role of the microbiome during diapause and other overwintering strategies of insects (Almada, 2015;Dittmer and Brucker, 2021;Liu et al., 2016). ...
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Legumes are important for both food security and sustainable agriculture. Given their relatively high protein content, more than 700 million people worldwide rely on them as food or poultry feeds. They are also important in intercropping systems for improving soil quality, due to their nitrogen-fixing ability. One major challenge to the yield of legumes is infestation by insect pests and pathogens both in the field and during storage. Most farmers have responded by planting resistant strains of legume crops and spraying them with insecticide and fungicide. Nevertheless, the continuous use of biocides, despite their cost-effectiveness, results in resistance development by the pests and pathogens, and raises environmental safety concerns for both humans and off-target beneficial species of insects and microbes. In this review, we discuss the most up-to-date thinking on the interactions between legumes and their insect pests and current farming practices, explain the latest techniques used in identifying molecular markers to aid in the breeding of insect-resistant cultivars, and highlight areas that require further development for effective and ecofriendly integrated pest management.
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