Biological Control 151 (2020) 104408

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Biological Control
journal homepage: www.elsevier.com/locate/ybcon

Use of biocides to minimize microbial contamination in Spodoptera exigua T

multiple nucleopolyhedrovirus preparations
Ehdibaldo Presa-Parraa, Rodrigo Lasaa, , Frédérique Reverchonb, Oihane Simónc,
⁎

Trevor Williamsa,
⁎

a
Red de Manejo Biorracional de Plagas y Vectores, Instituto de Ecología AC, Xalapa, Veracruz 91073, Mexico
b
Red de Estudios Moleculares Avanzados, Instituto de Ecología AC, Centro Regional del Bajío, Pátzcuaro, Michoacán 61600, Mexico
c
Institute for Multidisciplinary Research in Applied Biology, Universidad Pública de Navarra, Mutilva, Navarra 31192, Spain

GRAPHICAL ABSTRACT

ARTICLE INFO ABSTRACT

Keywords: The presence of contaminant microbes in baculovirus-based insecticides is regulated by phytosanitary product
Alphabaculovirus registration authorities. We aimed to determine whether the abundance of microbes in suspensions of
SeMNPV Spodoptera exigua multiple nucleopolyhedrovirus occlusion bodies (OBs) could be reduced by treatment with a
Benzalkonium chloride range of biocidal compounds. The diversity of contaminant bacteria was determined by next-generation se­
Colloidal silver
quencing of the 16S rRNA gene. Overall, 97.9% of sequences detected were Gammaproteobacteria (mostly
Chlorhexidine digluconate
Pseudomonas spp. and Enterobacteriaceae) and 2.1% were Firmicutes (mostly Enterococcus spp.). Colloidal silver,
Occlusion body pathogenicity
Stability benzalkonium chloride and chlorhexidine digluconate were identified as highly effective biocides. Incubation of
Bacterial diversity OB suspensions with high concentrations of colloidal silver (450 mg/l) or benzalkonium chloride (6000 mg/l)
resulted in marked reductions in colony forming unit counts over a 180 day period at 4° or 25 °C.
Benzalkonium chloride and colloidal silver treatments, at either 4 or 25 °C, did not affect the insecticidal activity of
OBs over an 80 day period. However, OB activity decreased following 180 days of treatment by benzalkonium chloride
at either 4 or 25 °C, or by colloidal silver at 25 °C, but not at 4 °C. Counts of OBs revealed a significant decrease in OB
numbers in benzalkonium chloride-treated suspensions after 180 days at both temperatures, whereas colloidal silver-
treated OBs were not affected. Benzalkonium chloride also caused aggregation of OBs at the concentration tested. We
conclude that biocidal compounds can markedly reduce the abundance of contaminant microorganisms in OB sus­
pensions, and can be accompanied by reductions in OB infectivity and OB numbers in some circumstances. Future
studies should focus on lower concentrations of biocides that do not affect OBs in long-term storage.

⁎
Corresponding authors.
E-mail addresses: [email protected] (R. Lasa), [email protected] (T. Williams).

https://doi.org/10.1016/j.biocontrol.2020.104408
Received 29 January 2020; Received in revised form 9 August 2020; Accepted 10 August 2020
Available online 13 August 2020
1049-9644/ © 2020 Elsevier Inc. All rights reserved.
E. Presa-Parra, et al. Biological Control 151 (2020) 104408

1. Introduction insecticides in many countries (Smits and Vlak, 1988; Hunter-Fujita

et al., 1998; Bianchi et al., 2000; Lasa et al., 2007).
Lepidopteran nucleopolyhedroviruses (Alphabaculovirus;
Baculoviridae) form the active ingredient in highly effective biological 2. Materials and methods
insecticides for the control of field crop and forest pests (Lacey et al.,
2015). The production of these viruses involves the inoculation and 2.1. Insect and virus production
rearing of massive numbers of larvae, followed by harvesting and
processing of virus-killed insects to extract virus occlusion bodies (OBs). Larvae of S. exigua were obtained from a laboratory colony main­
Considerable efforts have been made to develop efficient systems for tained in the Instituto de Ecología AC, Xalapa, Mexico. The colony was
the in vitro production of these virus with limited success due to the reared on a semi-synthetic diet (Supplementary material, Table S1),
rapid adaptation of the virus to cell culture conditions and the resulting under controlled conditions of 25 ± 2 °C temperature, 70 ± 5% re­
loss of key insecticidal traits (Reid et al., 2016). As such, all virus in­ lative humidity (RH) and 12:12 light–dark photoperiod. Each liter of
secticides are currently produced in vivo using insects reared on semi- larval diet contained 1 g potassium sorbate, 1.6 g methyl paraben and
synthetic diet (Grzywacz et al., 2014). 4.4 ml of 4% formaldehyde solution to reduce microbial proliferation.
Following death, each virus-killed insect is rapidly colonized by the Adults were fed ad libitum on a 10% (wt/vol) sucrose solution. A sample
microbiota of the gut (Grzywacz et al., 1997). These microbes usually of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) occlu­
comprise species from the Proteobacteria and Firmicutes bacterial sion bodies (OBs) was kindly provided by P. Támez-Guerra (Uni­
phyla, with lower numbers of fungi (mainly Ascomycota and Basidio­ versidad Autónoma de Nuevo León, Mexico) and was used to inoculate
mycota) that are members of a highly variable microbial community S. exigua fourth instars by the droplet feeding method (Hughes et al.,
that inhabits the alkaline gut of phytophagous lepidopteran larvae 1986). OBs were extracted from virus killed larvae by trituration in
(Hammer et al., 2017; Chen et al., 2018). However, in the case of la­ sterile distilled water, filtered through a fine metal mesh (80 µm pore
boratory-reared larvae, the use of semi-synthetic diets that often con­ size) and stored at −20 °C until use.
tain antibiotics and preservatives can strongly influence the composi­
tion of the gut microbiota (van der Hoeven et al., 2008). 2.2. Production of experimental batches of OBs
The abundance and type of microbes in OB preparations are subject
to regulation by phytosanitary product registration authorities, who The production of experimental batches of OBs involved the in­
define the upper limits for these microorganisms (usually specified at oculation of three independent batches of 320–400 larvae. Each batch
108 colony forming units (CFU)/mL of formulated product) and restrict comprised four groups of 80–100 S. exigua larvae, at 24–30 h after
the presence of vertebrate and plant pathogens in baculovirus-based molting to the fourth instar. Each group of larvae was inoculated by
insecticides (OECD, 2011). Microbial contamination of OB preparations placing insects in a ventilated plastic box (340 × 190 × 60 mm) with a
can also reduce the stability of virus insecticides in long-term storage strip of diet (230 × 10 × 2 mm) previously treated with 1 × 109 OBs,
(Burges and Jones, 1998). As many bacterial contaminants have a si­ estimated to result in a 95% prevalence of lethal polyhedrosis.
milar size and density as OBs, efforts to remove these by filtration and Following a 24 h period of feeding, when the treated diet had been
differential centrifugation, including zonal centrifugation, can be in­ consumed completely, a new piece of OB-free diet
efficient, time-consuming, costly in terms of labor and lead to sig­ (230 × 10 × 10 mm) was placed in the box and rearing continued at
nificant loss of OBs (Ignoffo and Couch, 1981; Shapiro, 1986; Grzywacz 25 ± 1 °C and 60 ± 10% RH in continuous darkness.
et al., 1997; Hunter-Fujita et al., 1998). An alternative approach, in­ At six days after inoculation, dead larvae were collected using en­
volving the use of ultra-high pressure treatments, was effective but also tomological tweezers and stored in 50 ml plastic tubes at −20 °C until
costly and time-consuming (Butz et al., 1995). processed.
Baculovirus formulations often include bacteriostatic and fungi­ To extract OBs, each group of infected larvae was thawed, homo­
static agents such as benzoates, sorbates or parabens as preservatives genized in sterile distilled water using a ceramic mortar and the re­
(Jones and Burges, 1998), but the use of biocidal compounds to kill sulting slurry was filtered twice through steel mesh (80 µm pore size) to
contaminant microbes has received little attention from researchers in remove debris. The OBs from the four groups of larvae were pooled to
baculovirus production. To our knowledge, a single exception to this is form a single batch, counted in triplicate using a Neubauer Improved
a study in which the abundance of bacteria in preparations of purified chamber (Hawksley, Lancing, UK) and the OB concentration was ad­
Cydia pomonella granulovirus OBs was markedly reduced by treatment justed to 1.0 × 109 –1.1 × 109 OBs/ml, in a total volume of 600 ml,
with 0.4% sodium pyrithione and 0.1% dodecylamine (triamine), using sterile distilled water. Each final OB suspension was placed in a
without significant loss of insecticidal activity, whereas occlusion de­ l000 ml sterile glass jar and stored in darkness at 4 °C for 48 h until used
rived virions released from granulovirus OBs were completely deacti­ in experiments.
vated (Krieg et al., 1979).
Biocides are compounds that kill a broad spectrum of bacterial and 2.3. Metagenomics analysis of microbial community
fungal microbes, and are widely used in cosmetics, pharmaceuticals and
paints, and have numerous clinical, culinary and industrial applications Total DNA was extracted from the OB suspension of each of the
as disinfectants (Rossmoore, 1995). The mode of action of these sub­ three batches. For this, 15 ml of each OB suspension was filtered under
stances differs among the different compounds, but often involves dis­ vacuum through sterile filter paper (Whatman No. 3) under sterile
ruption of microbial cell integrity or cell metabolic processes. conditions. Each filter paper was cut into small pieces, placed in a 1.5
In order to identify biocidal compounds that were highly effective at microcentrifuge tube and DNA extraction was performed using the
reducing microbial contaminants, without adverse effects on the in­ DNeasy Blood & Tissue Kit® (Qiagen, USA) and following the manu­
secticidal activity or stability of OBs, we performed a detailed char­ facturer’s instructions. The quantity and quality of the extracted DNA
acterization of the bacterial community present in OB preparations. We were verified by electrophoresis in 1% agarose and measured in a
then screened a range of different types of biocides for their efficacy spectrophotometer (Biospec-nano, Shimadzu Scientific Instruments,
and examined the effects of high concentrations of the selected com­ Japan).
pounds on the insecticidal activity and stability of OBs stored under Amplicon library preparation and genomic sequencing were per­
refrigeration or at room temperature. For this we used preparations of formed by Macrogen Inc. (Seoul, South Korea). Amplicons of the V3
Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), a virus region of the 16S rDNA gene were amplified using universal primers
that has an established history of use as the basis for biological 27F (5′-AGAGTTTGATCMTGGCTCAG) and 518R (5′-GTATTACCGCGG

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E. Presa-Parra, et al. Biological Control 151 (2020) 104408

CTGCTGG). Sequencing was performed on a GS-FLX Titanium platform, insecticidal activity of OBs. For this, the three biocides were each mixed
generating over 163,000 paired-end reads with an average length of with triplicate samples of 500 µl of OB suspension (1 × 109 OBs) at the
422 bp. Sequencing datasets were deposited in the Sequence Read concentrations that proved most effective for suppression of microbial
Archive Database of the NCBI with the accession number contaminants in the previous screening assay: 6000 mg/l benzalkonium
PRJNA603778. chloride, 450 mg/l colloidal silver and 2000 mg/l chlorhexidine di­
Quality analysis of raw paired-end reads was performed with gluconate. Following treatment, OB + biocide suspensions were in­
PRINSEQ v.0.20.4 (Schmieder and Edwards, 2011). Reads shorter than cubated at 30 ± 1 °C for 24 h in continuous agitation at 150 rpm and
50 bp or with a quality score lower than 20 were discarded from the were then used for CFU counts and insect bioassays. The addition of 1%
analysis. High quality paired-end reads were joined using QIIME v.1.8.0 Tween 80 was used to overcome the aggregation of OBs in the ben­
(Caporaso et al., 2010). Detection and removal of chimeras were con­ zalkonium chloride treatment.
ducted with MOTHUR v.1.25.0 (Schloss et al., 2009). Sequences were Aerobic CFU counts on biocide-treated OB suspensions were eval­
then clustered in Operational Taxonomic Units (OTUs) at 97% simi­ uated following the methodology described in the initial screening
larity, using QIIME under the default settings (Caporaso et al., 2010). A process, using LB agar plates inoculated with 100 µl of OB suspension
representative sequence was randomly selected within each OTU and a that had been diluted 10-fold and 100-fold. All dilutions were prepared
taxonomic category was assigned to each OTU using the Greengenes and plated using triplicate samples. Plates were incubated at 30 ± 1 °C
13_8 reference database (DeSantis et al., 2006). The OTUs corre­ for 24 h and CFU counts were performed. Control OB suspensions
sponding to chloroplast, mitochondrial and archaeal DNA were re­ without biocides were treated identically.
moved from the OTU table. A rarefaction curve based on Chao1 index Biocide-treated OB suspensions were assayed in second instar S.
was drawn from the filtered OTUs in QIIME v.1.8.0. exigua larvae inoculated by the droplet feeding technique (Hughes
et al., 1986). Groups of 30 newly molted larvae were starved overnight
2.4. Biocide effects on microbial contamination: Initial screening and then allowed to feed on droplets of 10% (wt/vol) sucrose, 0.01%
(vol/vol) fluorella blue food dye and a concentration of 6.2 × 103 OBs/
Ten different commercial biocidal products were assessed for their ml. This OB concentration was previously determined to result in 50%
activity against aerobic microbes present in OBs suspensions: i) ben­ mortality for this virus strain. A similar group of control larvae were
zalkonium chloride (alkylbenzyldimethylammonium chloride, 80%, treated identically but fed on a solution of sucrose and food dye alone.
Golden Bell, Mexico), ii) iodopovidone (10%, Laboratorios Jaloma SA de Groups of 24 larvae that ingested the suspension within 15 min were
CV, Mexico), iii) colloidal silver (0.35%, Mercancías Salubles SA de CV, transferred individually to a 24 well tissue culture plate containing
Mexico), iv) copper oxide nanopowder, < 50 nm particle size (Sigma- pieces of diet and reared at 24 ± 1 °C. Virus induced mortality was
Aldrich, USA), v) chlorhexidine digluconate (≥99.5%, 1,6-Bis(N5-[p- observed at seven days post-inoculation and confirmed by microscopic
chlorophenyl]-N1-biguanido)hexane, Sigma-Aldrich, Switzerland), vi) observation of OBs. The bioassay was performed on three occasions for
methylisothiazolinone (2-methyl-4-isothiazolin-3-one, ≥95%, Sigma- each batch of OB suspension.
Aldrich, Germany), vii) Triclosan (5-chloro-2-[2,4-dichlorophenoxy]
phenol, > 99%, Sigma-Aldrich, USA), viii) azadirachtin (3%, Biokrone 2.6. Effects of biocide treatment on OB suspensions stored at two
SA de CV, Mexico), ix) phthaldialdehyde solution (benzene-1,2-di­ temperatures
carbaldehyde, ≥99.9%, Sigma-Aldrich, USA), x) peracetic acid (39% in
acetic acid, Sigma-Aldrich, USA). Two effective biocides, benzalkonium chloride and colloidal silver,
The range of concentrations of each biocide tested was defined by were selected to evaluate their effects on microbial contaminants and
reference to published literature that quantified their efficacy against insecticidal activity of OBs suspension stored for six months at re­
Gram positive and Gram negative bacteria. Once a reference con­ frigerated or ambient temperatures. Chlorhexidine digluconate was
centration had been identified, we performed preliminary tests invol­ excluded from this test due the high cost of this substance. For ben­
ving 24 h treatment of newly-prepared OB suspensions with a ten-fold zalkonium chloride and colloidal silver treatments, a new batch of OBs
lower concentration and ten-fold to one hundred-fold higher con­ was produced with a concentration of 1.05 × 109 OBs/ml, as described
centrations of each biocide. If a biocide was found to be ineffective in in the section on production of experimental batches of OBs. Before
preliminary tests, it was still included in the detailed tests in order to biocide treatment, the microbial load (CFU/ml) and insecticidal activity
demonstrate its reduced efficacy compared to that of the other com­ in second instars were evaluated in triplicate samples following the
pounds tested. methodology described in Section 2.5. Two samples of 10 ml were
Detailed screening involved the following procedure. A 500 µl vo­ treated with biocide, one with 6000 mg/l benzalkonium chloride and
lume of OB suspension was placed in a 1.5 ml microtube and mixed the other with 450 mg/l colloidal silver. A third sample of 10 ml was
with the predetermined concentration of each biocidal compound. In all used as an untreated control. The microbial load and insecticidal ac­
cases an identical sample of OB suspension without biocide was in­ tivity of these three 10 ml samples were again incubated at 30 ± 1 °C
cluded as control. Microcentrifuge tubes were placed in an orbital for 24 h after treatment. Each OB suspension was then divided into two
shaker at 150 rpm and a temperature of 30 ± 1 °C for 24 h, after which 5 ml samples that were stored at 4 ± 1.3 °C in a laboratory refrigerator
each sample was diluted (1/10 and 1/100 in sterile water) and a 100 µl or 25 ± 1 °C in a laboratory incubator. Microbial contaminants and
volume of each dilution was inoculated on LB agar (Broth Lennox, BD insecticidal activity were evaluated in each 5 ml sample after 80 and
Bioxon Agar, Sigma-Aldrich) in Petri plates. Sterile glass beads (5 mm 180 days. The number of OBs in each sample was counted in triplicate
diameter) were used to spread bacteria. Plates were incubated at using a Neubauer Improved chamber using a phase contrast microscope
30 ± 1 °C for 24 h and the numbers of colony forming units were then at the beginning and end of the experiment.
counted. CFU counts that exceeded 100 colonies per plate were clas­
sified as > 100 CFU. Three plates (replicates) were inoculated si­ 2.7. Statistical analysis
multaneously for each sample.
Larval mortality in controls and biocide treatments was compared
2.5. Biocide effects on microbial contaminants and insecticidal activity of by t-test for benzalkonium chloride and chlorhexidine digluconate and
OBs by Wilcoxon rank sum test with continuity correction for colloidal
silver. CFU counts were also subjected to a Wilcoxon test for colloidal
Three biocides that significantly reduced the microbial load in the silver, but were zero in the other treatments that were not subjected to
initial screening assay were tested to evaluate their influence on the statistical analysis. The effects of biocide treatment, temperature and

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E. Presa-Parra, et al. Biological Control 151 (2020) 104408

sample time on log CFU counts, log OB counts and percentage of larval (19.1%) families. The most prevalent organism by far was Pseudomonas
mortality in bioassays were examined by three-way analysis of variance viridiflava that represented 59.7–71.0% of all 16S rRNA gene sequences
in an R-based package (Jamovi, 2019). The normality of log-trans­ detected (Fig. 1). Other relatively abundant organisms were Morganella
formed and percentage mortality values was assessed by Shapiro–Wilk morganii, Pseudomonas spp. and Providencia spp. that each represented
test. Treatment means were compared by Tukey's test (P < 0.05). approximately 10% of the bacterial community. The other organisms
were present at low levels (mostly < 2% of sequences detected)
(Fig. 1).
3. Results In terms of richness, 95 of the 214 identified OTUs were assigned to
Pseudomonads, in particular to unidentified Pseudomonas spp. (43
3.1. Metagenomics analysis of the bacterial community OTUs identified in total), P. viridiflava (25 OTUs) and P. veronii (11
OTUs) (Fig. 2). In contrast, the highest richness of Enterobacteriales, a
After quality checks and OTU filtering, we obtained a total of typically dominant bacterial order in the digestive tract of insects, was
51,266 reads for batch 1, 48,321 reads for batch 2, and 46,514 reads for observed for M. morganii (32–34 OTUs identified in each batch of OBs),
batch 3. The rarefaction curves based on Chao1 index confirmed that followed by unidentified Enterobacteriaceae, Providencia spp. and En­
sequencing depth was sufficient (Supplementary Figure S1). terococcus haemoperoxidus and E. casseliflavus.
The richness of operational taxonomic units (OTUs), akin to bac­
terial molecular species, was 163, 156 and 183 for batches 1, 2 and 3,
respectively, with a total of 214 OTUs across all batches. Bacteria 3.2. Biocide effects on microbial contamination: initial screening
present in the OBs suspensions mainly belonged to the phyla
Proteobacteria (97.9% of sequences), specifically to the Of the ten biocides assessed, only 6000 mg/l benzalkonium
Gammaproteobacteria class, and in lesser proportion to Firmicutes chloride, 450 mg/l colloidal silver and 2000 mg/l chlorhexidine di­
(2.1%). The Firmicutes phylum was dominated by the Enterococcaceae gluconate resulted in a marked reduction or complete elimination of the
family (1.6% of sequences) whilst the Proteobacteria phylum was aerobic microbial load (Table 1). All three compounds showed similar
dominated by the Pseudomonadaceae (78.4%) and Enterobacteriaceae efficacy at the dilutions of 1/10 and 1/100 with CFU counts of less than

Fig. 1. Relative abundance of operational taxonomic units (OTUs) of bacteria detected by 16S rRNA gene sequence analysis on three batches of SeMNPV OB
suspensions. Values next to bars indicate percentage values.

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Fig. 2. Numbers of operational taxonomic units (OTUs) of bacteria detected by 16S rRNA gene sequence analysis on three batches of SeMNPV OB suspensions.

1000 CFU/ml in all cases, i.e., < 10 CFU in a 100 µl volume of the 1/ compared to a complete elimination of microbial contaminants in the
100 dilution. In contrast, OB preparations treated with any of the seven 25 °C treatment at the end of the 180 day incubation period (Fig. 3AB).
remaining biocides retained counts of over 100 CFU per plate, so were
not considered for additional experiments. 3.4.2. Insecticidal activity
Bioassays performed using S. exigua second instars that consumed
3.3. Biocide effects on microbial contaminants and insecticidal activity of an LC50 concentration of inoculum (1.05 × 109 OBs/ml), prior to
OBs biocide treatment of OB preparations, resulted in a mean ( ± SE)
mortality of 47.2 ± 3.0%. Virus-induced mortality was significantly
The aerobic microbial load in OB preparations was quantified and affected by biocidal treatment (F = 13.473; df = 2, 36; P < 0.001),
observed to decrease from approximately 5 × 108 CFU/ml in control and sample time (F = 41.5; df = 2, 36; P < 0.001), but not by in­
preparations to between zero and 10 CFU/ml in preparations treated cubation temperature (F = 2.771; df = 1, 36; P = 0.105). At 24 h post-
with benzalkonium chloride, colloidal silver, or chlorhexidine diglu­ treatment, the prevalence of mortality did not differ significantly in any
conate, at the concentrations identified as effective in the screening of the biocide treatments or the control (Fig. 4AB). The control OB
assay (Table 2). preparations retained insecticidal activity without significant decreases
None of the biocides had a significant effect on the insecticidal ac­ at 4 °C and 25 °C during the 180 day period. Benzalkonium-treated OBs
tivity of OBs after 24 h of incubation. In all cases, the prevalence of were significantly less pathogenic (18.9 ± 2.8% mortality) at 180 days
lethal polyhedrosis of second instars varied between 40.3 and 60.6% in post-treatment compared with the control treatment. Colloidal silver-
control groups (Table 2), and was similar to the mortality (41.2–52.3%) treated OBs at 4 °C retained insecticidal activity similar to that of the
of larvae that consumed OBs that had been treated with benzalkonium control, but not at 25 °C at which larval mortality at 180 days was
chloride (t = 1.412; df = 16; P = 0.177), colloidal silver (Wilcoxon reduced to 26.4 ± 4.2% (Fig. 4AB).
W = 53.5; P = 0.251; N = 18), or chlorhexidine digluconate
(t = 0.913; df = 16; P = 0.375). 3.4.3. Occlusion body counts
OB counts were performed at the start (24 h post-treatment) and
3.4. Storage stability of OB preparations with selected biocides following 180 days incubation (Fig. 5AB). OB counts were significantly
affected by biocidal treatment (F = 88.0; df = 2, 24; P < 0.001),
3.4.1. Microbial contaminants sample time (F = 168.5; df = 1, 24; P < 0.001), and incubation
The mean ( ± SE) aerobic CFU count of OB preparations prior to temperature (F = 8.136; df = 1, 24; P < 0.009). OB counts in the
treatment was 7.4 ( ± 1.3) × 108 CFU/ml. Microbial load was sig­ control were unchanged following incubation at 4 °C, but were slightly
nificantly affected by biocidal treatment (F = 3446.5; df = 1, 36; reduced in the control at 25 °C (Fig. 5AB). OB counts were reduced by
P < 0.001), sample time (F = 101.5; df = 1, 36; P < 0.001), and approximately half a logarithm (~65% reduction) in the benzalkonium
incubation temperature (F = 31.2; df = 1, 36; P < 0.001). The mi­ chloride treatment at both temperatures after 180 days. This reduction
crobial load of control OB preparations increased slightly after 24 h was apparently due to the disappearance of OBs during the experiment.
incubation at 30 °C (1.6 × 109 CFU/ml, shown as log 109.193 ± 0.059 in In contrast, OB counts remained unchanged in the colloidal silver
Fig. 3AB), and did not change significantly during 180 days of in­ treated preparations for the duration of the study.
cubation at 4 °C, whereas at 25 °C microbial load decreased by ap­
proximately two logarithms during the same period (Fig. 3B). The 4. Discussion
benzalkonium chloride treatment reduced CFU counts to zero at all
time points and was not included in the statistical analysis. The mi­ The bacterial composition of SeMNPV OB suspensions was char­
crobial load of colloidal silver-treated OB preparations was reduced to acterized by analysis of 16S rRNA gene sequences. Three highly effec­
2.5 × 103 CFU/ml (log 103.399 ± 0.059) at 24 h post-treatment and de­ tive biocidal compounds were identified that controlled bacterial con­
creased by approximately one logarithm in the 4 °C treatment, taminants for the duration of the study. The insecticidal activity of

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Table 1
Colony forming units (CFU) counts in plates inoculated with OB suspensions
alone or previously treated with different biocide and concentrations. OB sus­ A.
pensions were diluted 1/10 or 1/100 prior to plating.
Biocide Concentration (mg/l) CFU/plate

1/10 1/100

Benzalkonium chloride Control > 100 > 100

6 > 100 > 100
60 > 100 > 100
600 > 100 > 100
6000 0 0*
Iodopovidone Control > 100 > 100
6.2 > 100 > 100
62 > 100 > 100
620 > 100 > 100
6200 > 100 > 100
Colloidal silver Control > 100 > 100
0.45 > 100 > 100
4.5 > 100 > 100
45 > 100 > 100

Copper oxide
450
Control
6
> 100
1*
> 100
B.
1.6 > 100 > 100
16 > 100 > 100
160 > 100 > 100
1600 > 100 > 100
Chlorhexidine digluconate Control > 100 > 100
2 > 100 > 100
20 > 100 > 100
200 > 100 > 100
2000 0 0*
Methylisothiazolinone Control > 100 > 100
5 > 100 > 100
50 > 100 > 100
500 > 100 > 100
5000 > 100 > 100
Triclosan Control > 100 > 100
3 > 100 > 100
30 > 100 > 100
300 > 100 > 100 Fig. 3. Logarithm of counts of colony forming units (mean CFU ± SE) in
3000 > 100 > 100 control and colloidal silver-treated OB suspensions following 24 h, 80 d, or 180
Azadirachtin Control > 100 > 100 d incubation at (A) 4 °C or (B) 25 °C. Columns headed by identical letters did
15 > 100 > 100 not differ significantly for comparisons of biocide treatment (upper case) and
150 > 100 > 100 incubation period (lower case) (ANOVA, Tukey P > 0.05). CFU counts were
1500 > 100 > 100 zero in all benzalkonium chloride-treated samples (values not shown).
15,000 > 100 > 100
Phthaldialdehyde Control > 100 > 100
5 > 100 > 100 biocide-treated OBs declined after 180 d incubation except in one
50 > 100 > 100 treatment combination (colloidal silver, 4 °C).
500 > 100 > 100
5000 > 100 > 100
Previous estimates of the abundance of aerobic microbial con­
Peracetic acid Control > 100 > 100 taminants in OB suspensions have been in the region of 107–108 CFU/
6 > 100 > 100 ml based on standard culturing methods (Podgwaite et al., 1983;
60 > 100 > 100 Grzywacz et al., 1997; Lasa et al., 2008; Ruiz et al., 2015). Numbers of
600 > 100 > 100
bacteria may increase rapidly after death as the larval cadaver becomes
6000 > 100 > 100
colonized (Grzywacz et al., 1997; Ramírez-Arias et al., 2019), so that
* Reduction of aerobic microorganisms to less than 1 × 103 CFU/ml based the abundance of aerobic microbes measured in control OB suspensions
on a sampled volume of 100 µl diluted 1/100 prior to plating onto agar. in the present study (~5 × 108 CFU/ml) likely reflects the fact that
virus-killed cadavers were collected after all insects had died from virus

Table 2
Microbial load in colony forming units (CFU)/ml and percentage of larval mortality for control (OBs alone) and OB preparations treated with biocides for 24 h.
Biocide Concentration (mg/l) Microbial load ± SE (CFU/ml) Mortality ± SE (%)

OBs alone OBs + biocide OBs alone OBs + biocide

8
Benzalkonium chloride 6000 5.2 ( ± 1.4) × 10 0 ± 0 60.6 ± 3.1a 52.3 ± 5.0a
Colloidal silver 450 4.8 ( ± 2.2) × 108a 10.0 ± 3.3b 44.0 ± 2.1a 41.2 ± 3.1a
Chlorhexidine 2000 5.1 ( ± 3.7) × 108 0 ± 0 40.3 ± 2.7a 43.5 ± 2.3a

Values followed by different letters differ significantly for pairwise comparisons of treatment and controls, Wilcoxon test on CFU values (P < 0.05, colloidal silver)
or t-test on mortality values (P < 0.05). Statistical analyses were not performed for microbial loads with zero values, i.e., benzalkonium chloride and chlorhexidine
digluconate.

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A.
A.

B.
B.

Fig. 5. Logarithm of virus occlusion body counts (mean ± SE) in control and
Fig. 4. Percentage of mortality (mean ± SE) in larvae that consumed control biocide-treated OB suspensions following 24 h or 180 d incubation at (A) 4 °C or
and biocide-treated OB suspensions that had been subjected to 24 h, 80 d, or (B) 25 °C. Columns headed by identical letters did not differ significantly for
180 d incubation at (A) 4 °C or (B) 25 °C. Columns headed by identical letters comparisons of biocide treatments (upper case) and incubation period (lower
did not differ significantly for comparisons of biocide treatments (upper case) case) (ANOVA, Tukey P > 0.05).
and incubation period (lower case) (ANOVA, Tukey P > 0.05).
the insect’s immune response (Crava et al., 2015; Caccia et al., 2016),
disease. All previous studies, without exception, have reported yeasts and the presence of parasites and pathogens (Caccia et al., 2016;
and molds as minor components in the microbiota of OB preparations, Polenogova et al., 2019), including nucleopolyhedroviruses
typically in the range 103–105 CFU/ml (Grzywacz et al., 1997; Lasa (Jakubowska et al., 2013).
et al., 2008; Ruiz et al., 2015). For this reason we focused on the Analysis of 16S rRNA gene sequences directly from OB suspensions
dominant bacterial components of OB suspensions for the 16S rRNA- provided a culture-independent method for characterizing the bacterial
based analyses, rather than fungal components that would have re­ community. Overall, 97.9% of sequences were Gammaproteobacteria
quired analysis of 18S rRNA gene sequences. dominated by Pseudomonadaceae and Enterobacteriaceae families. The
Contaminants are considered a quality control issue as phytosani­ most prevalent organism, Pseudomonas viridiflava, represented two-
tary product registration authorities set limits on the quantity and type thirds of all 16S rRNA gene sequences detected. Other relatively
of microbial contaminants that are permitted in virus-based insecticides abundant organisms were Morganella morganii, Pseudomonas spp. and
(Shapiro, 1986; Jenkins and Grzywacz, 2000; Grzywacz and Moore, Providencia spp. (Fig. 2). Interestingly, P. viridiflava is a well-recognized
2017). Most of the microbial community present in OBs suspensions is pathogen of multiple plant species (Sarris et al., 2012) that has been
likely to be derived from the gut microbiome that colonizes the larval isolated from lepidopteran larvae (Khoja et al., 2006) and the oral se­
cadaver as the tissues break down following death. The midgut of cretions of a beetle (Cardoza et al., 2006). Given its phytopathogenic
phytophagous lepidopteran larvae is highly alkaline (Berenbaum, status, it would be interesting to determine whether P. viridiflava is a
1980) and represents a specific habitat for a community of microbes common component of OB suspensions used to produce biological in­
that are adapted to such conditions (Broderick et al., 2004). The com­ secticides. Other pseudomonads have been reported from the larval
position of the lepidopteran gut community depends on multiple cuticle (Tang et al., 2012) and gut (Shao et al., 2014) of Spodoptera
dietary, physiological and environmental factors, many of which in­ littoralis, whereas Morganella morganii is a normally harmless com­
teract. These include insect species (Phalnikar et al., 2018; Jones et al., mensal species present in the gut of humans and other vertebrates, al­
2019), development stage and niche along the digestive tract (Tang though it may cause opportunistic clinical infections on occasions (Liu
et al., 2012; Jones et al., 2019), the food plant species and associated et al., 2016). This organism has been isolated from the dipteran larval
microbial community (Broderick et al., 2004; Mason and Raffa, 2014; gut (Jeon et al., 2011) and as a lethal pathogen of mass-reared fruit flies
Jones et al., 2019), plant defensive compounds (Mason et al., 2019), the (Salas et al., 2017).
soil microbiome in which the food plant grows (Hannula et al., 2019), The Gram-positive Firmicutes phylum represented just 2.1% of 16S

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E. Presa-Parra, et al. Biological Control 151 (2020) 104408

rRNA gene sequences and was dominated by the Enterococcaceae fa­ treatment at 4 °C (Fig. 4).
mily. Indeed, Enterococcus spp. have been reported to be abundant and The clumping of OBs in the benzalkonium chloride treatment was
constant members of the gut community in Lymantria dispar, Manduca an issue that may have significantly influenced the infectivity of OBs.
sexta and S. littoralis larvae (Broderick et al., 2004; Brinkmann et al., We overcame this issue by adding Tween 80 to the OB suspension prior
2008; Teh et al., 2016), and from other insect orders (Cox and Gilmore, to bioassays and for accurate OB counting. OB counts after 180 days of
2007; Lehman et al., 2009). incubation were reduced by approximately 65% (half a logarithm) in
We detected no major human pathogens, such as species of benzalkonium chloride treated suspensions at both 4 °C and 25 °C,
Salmonella, Shigella, Vibrio, or occasional pathogens such as Bacillus compared to a slight reduction in the control at 25 °C and no significant
cereus or Staphylococcus aureus, a common inhabitant of human skin. In changes in the colloidal silver treatment (Fig. 5). The reduction in in­
contrast, previous studies have emphasized the abundance of secticidal activity of benzalkonium-treated OBs appears to be a direct
Enterococcus spp., Enterobacteriaceae and Bacillus spp. (Grzywacz et al., result of the presence of fewer OBs in treated OB suspensions. Ben­
1997), but are in agreement with our findings in reporting very low zalkonium chloride may have caused OB clumping through its inter­
levels of S. aureus, and an absence of Salmonella, Shigella, and Vibrio action with the polyhedron envelope or OB-associated proteins. In a
species (Grzywacz et al., 1997; Lasa et al., 2008). We presume that the previous study we observed a loss of insecticidal activity, a ~35% re­
principal differences among these studies are derived from the different duction in OB counts and a break down of viral genomes in SeMNPV OB
origins of the insect colonies, experimental virus inocula used and the preparations stored at 25 °C for 18 months, whereas insecticidal activity
marked differences in bacterial identification techniques, based on was fully retained under refrigerated and frozen storage conditions
conventional culture methods and biochemical reactions (Grzywacz (Lasa et al., 2008).
et al., 1997; Lasa et al., 2008), compared to our direct molecular ap­ The reasons why OBs were lost in the benzalkonium chloride
proach that would not necessarily be expected to reflect closely the treatment are unclear, although we postulate that the stability of the
community composition determined from the culture-based methods polyhedrin matrix may have been reduced by exposure to this com­
reported previously. pound, which was mildly alkaline (pH 8.8) at the concentration tested,
The bacterial community composition is also likely to reflect the and the presence of chloride anions that facilitate the dissolution of
addition of antibiotics and bacteriostatic substances to reduce the pre­ polyhedrin in alkaline solution (Whitt and Manning, 1987). In contrast,
valence of opportunistic bacterial pathogens and spoilage of the semi- in the control, the loss of OBs at 25 °C may have been due to de­
synthetic diets used to rear larvae. The diet used in the present study gradation by the alkaline protease enzyme present in OBs produced in
contained sorbate, methyl paraben and formaldehyde to control mi­ insects (Wood, 1980; Boogaard et al., 2017), an enzymatic process that
crobial contaminants (Supplemental material, Table S1), whereas that is likely to have been slowed or halted at 4 °C. In the colloidal silver
of Lasa et al. (2008) contained sorbate, methyl paraben, and two an­ treatment the OB-associated alkaline protease may have been in­
tibiotics (streptomycin, tetracycline), and that of Grzywacz et al. (1997) activated as this biocide attacks key functional groups of enzymes (Jung
contained only methyl paraben (McKinley et al., 1984). et al., 2008), so that silver-treated OBs remained intact for the duration
Of the ten biocidal products screened, three stood out in terms of of the study. Clearly these hypotheses can only be validated through
their antimicrobial activity across a range of concentrations, namely further study.
colloidal silver, benzalkonium chloride and chlorhexidine diglutonate. Previous attempts to control microbial contaminants have focused
These compounds differ in their mode of action. Colloidal silver binds on clean practices during rearing of infected larvae, harvesting infected
to key functional groups of enzymes, particularly thiol groups, resulting insects prior to or immediately following death and the use of bacter­
in the disruption of the cell membrane, the release of potassium ions iostatic agents such as sorbate and benzoate salts or glycerol in OB
and cell death (Jung et al., 2008), whereas benzalkonium chloride is a formulations (Behle and Birthisel, 2014; Grzywacz and Moore, 2017).
cationic surfactant that compromises cellular permeability by dis­ An additional, but largely overlooked factor, is the presence of con­
sociation of cell membrane lipid bilayers, resulting in leakage of cellular taminants in the OB inoculum used to infect larvae, which can mark­
contents followed by the disruption of proteins and nucleic acids and edly affect the abundance and community composition of contaminant
cell death (Tezel and Pavlostathis, 2015). Chlorhexidine digluconate microorganisms that subsequently colonize virus-killed insects (G.
appears to collapse the bacterial cell membrane causing leakage of low Bouwer, pers. comm.).
molecular weight cell components and rapid death (Cheung et al., In conclusion, it was clear that including high concentrations of
2012). However, we did not evaluate this compound in detail due to its benzalkonium chloride or colloidal silver in OB suspensions dramati­
elevated cost. To treat one liter of OB suspension with chlorhexidine cally reduced the abundance of contaminant microorganisms, but did
digluconate would cost approximately US$14.20 compared to US$2.28 not improve the retention of insecticidal activity of OBs in storage
for treatment with colloidal silver and US$0.24 for treatment with compared to that of the control suspensions, which had not lost activity
benzalkonium chloride (Mexican peso - US dollar exchange rate was after 180 days. Indeed, the results of the present study are consistent
22.5:1 at time of publication). The concentrations of benzalkonium with prior assertions that microbial contaminants are not detrimental to
chloride (6000 mg/l) and colloidal silver (450 mg/l) that we used were the stability of mature OBs for periods of months (Grzywacz et al.,
over ten-fold higher than the concentrations at which these compounds 1997; Lasa et al., 2008), although immature OBs lacking a polyhedron
are typically used (Tien et al., 2008; Tezel and Pavlostathis, 2015). The envelope may be more susceptible to microbial degradation (Ramírez-
present study therefore represents a stringent, worst-case test of biocide Arias et al., 2019). However, both biocidal compounds reduced mi­
compound effects on OB activity and stability over an extended period crobial contaminants for at least 180 days and may find applications as
of incubation. Future studies should focus on their efficacy at lower adjuvants in OB formulations, or biocide coatings inside bioinsecticide
concentrations. product containers. The use of lower concentrations of the biocides
Both benzalkonium chloride and colloidal silver had immediate should reduce costs and might reduce the prevalence of OB aggregation
antimicrobial activity that was sustained over the 180 day incubation observed in benzalkonium chloride-treated suspensions.
period, with zero CFU counts in the benzalkonium chloride treatment
and decreasing counts in the colloidal silver treatment, irrespective of CRediT authorship contribution statement
temperature (Fig. 3). Control OB suspensions did not show a significant
reduction in insecticidal activity during the 180 day period at either Ehdibaldo Presa-Parra: Investigation, Formal analysis, Writing -
temperature, whereas the biocide-treated OBs were as active as the original draft. Rodrigo Lasa: Methodology, Resources, Supervision,
control suspensions up to 80 days, but resulted in significantly reduced Writing - review & editing. Frédérique Reverchon: Methodology,
mortality in the 180 day samples, except for the colloidal silver Investigation, Supervision, Writing - review & editing. Oihane Simón:

8
E. Presa-Parra, et al. Biological Control 151 (2020) 104408

Supervision, Writing - review & editing. Trevor Williams: Spodoptera littoralis nuclear polyhedrosis virus produced in insects in Egypt. J.
Methodology, Formal analysis, Supervision, Writing - review & editing. Invertebr. Pathol. 69 (2), 151–156.
Grzywacz, D., Moore, D., Rabindra, R.J., 2014. Mass production of entomopathogens in
less industrialized countries. In: Morales-Ramos, J.A., Rojas, M.G., Shapiro-Ilan, D.I.
Declaration of Competing Interest (Eds.), Mass Production of Beneficial Organisms. Academic Press, San Diego, pp.
519–561.
Grzywacz, D., Moore, S., 2017. Production, formulation, and bioassay of baculoviruses for
The authors declare that they have no known competing financial pest control. In: Lacey, L.A. (Ed.), Microbial Control of Insect and Mite Pests: from
interests or personal relationships that could have appeared to influ­ Theory to Practice. Academic Press, San Diego, pp. 109–124.
Hammer, T.J., Janzen, D.H., Hallwachs, W., Jaffe, S.P., Fierer, N., 2017. Caterpillars lack
ence the work reported in this paper. a resident gut microbiome. Proc. Natl. Acad. Sci. USA 114 (36), 9641–9646.
Hannula, S.E., Zhu, F., Heinen, R., Bezemer, T.M., 2019. Foliar-feeding insects acquire
Acknowledgements microbiomes from the soil rather than the host plant. Nat. Commun. 10 (1). https://
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formatics analyses and Ofelia Ferrera and Gabriel Mercado for technical Invertebr. Pathol. 48 (2), 187–192.
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