PLOS ONE
RESEARCH ARTICLE
Development of a bioassay method to test
activity of cry insecticidal proteins against
Diatraea spp. (Lepidoptera: Crambidae)
sugarcane stem borers
Juan Sebastián Ángel-Salazar1, Claudia Echeverri-Rubiano1, Jairo Rodrı́guez-Chalarca2,
Jershon López-Gerena 1
I , Rafael Ferreira dos Santos 3
D I , Juan Luis Jurat-Fuentes 3
D ID ,
Alexandra M. Revynthi4, Germán Vargas 4
ID *
1 Colombian Sugarcane Research Center (Cenicaña), Florida, Colombia, 2 Alliance Bioversity-CIAT (The
International Center for Tropical Agriculture), Palmira, Colombia, 3 Department of Entomology and Plant
a1111111111 Pathology, University of Tennessee, Knoxville, Tennessee, United States of America, 4 Department of
a1111111111 Entomology and Nematology, Tropical Research and Education Center, University of Florida, Homestead,
a1111111111 FL, United States of America
a1111111111 * entomogav@gmail.com
a1111111111
Abstract
OPEN ACCESS The genus Diatraea (Lepidoptera: Crambidae) includes stem borers representing the most
critical sugarcane pests in the Americas. Colombia’s most widely distributed and damaging
Citation: Ángel-Salazar JS, Echeverri-Rubiano C,
Rodrı́guez-Chalarca J, López-Gerena J, dos Santos Diatraea species include Diatraea saccharalis, D. indigenella, D. busckella, and D. taber-
RF, Jurat-Fuentes JL, et al. (2023) Development of nella. The reduced efficacy of biological tools commonly used in controlling several species
a bioassay method to test activity of cry insecticidal highlights the importance of evaluating alternative management strategies, such as trans-
proteins against Diatraea spp. (Lepidoptera:
genic plants expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt).
Crambidae) sugarcane stem borers. PLoS ONE
18(10): e0292992. https://doi.org/10.1371/journal. The selection of optimal Bt insecticidal proteins for Diatraea control depends on bioassays
pone.0292992 with purified Bt proteins. Because there is no described artificial diet for borer species other
Editor: Omaththage P. Perera, USDA Agricultural than D. saccharalis and availability of most purified Bt toxins is restricted, this study aimed at
Research Service, UNITED STATES developing a bioassay method using fresh corn tissue and providing proof of concept by
Received: June 23, 2023 testing susceptibility to the Cry1Ac insecticidal protein from Bt. Toxicity was evaluated with a
single Cry1Ac dose applied directly to corn discs. Stem borer mortality after seven days was
Accepted: October 3, 2023
higher than 90% for all four tested Diatraea species, while control mortality was below 8%.
Published: October 18, 2023
In addition, we observed that Cry1Ac caused more than 90% weight inhibition in all survivors
Peer Review History: PLOS recognizes the and delayed development. These results validate the use of this method to determine mor-
benefits of transparency in the peer review
tality and growth inhibition due to the consumption of the Cry1Ac protein in each of the Dia-
process; therefore, we enable the publication of
all of the content of peer review and author traea species. Furthermore, this method could be used to assess other entomopathogenic
responses alongside final, published articles. The substances to control these insect pests.
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0292992
Copyright: © 2023 Ángel-Salazar et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which Introduction
permits unrestricted use, distribution, and
reproduction in any medium, provided the original Sugarcane is an important crop in the tropics and subtropics worldwide, where it is mainly
author and source are credited. grown to produce sucrose, alcohol, electricity, and panela (Jaggery or non-centrifuged sugar)
PLOS ONE | https://doi.org/10.1371/journal.pone.0292992 October 18, 2023 1 / 14
PLOS ONE Bioassay to test activity of cry proteins against Diatraea spp.
Data Availability Statement: All relevant data are [1, 2]. Colombia has one of the highest global sugarcane productions per area unit, yet stem
within the paper and its Supporting Information borers cause significant yield losses, ranging from 100 to 143 Kg of sucrose per hectare, and
files.
per each percent of bored internodes [3, 4]. Among the lepidopteran species described as sug-
Funding: The authors thank Alliance Bioversity- arcane stem borers [3], most belong to the family Crambidae [5] and to the Diatraea genus in
CIAT (The International Center for Tropical the Western Hemisphere. The most updated report on the Diatraea genus indicates that 41
Agriculture) for providing the logistical support
species are present in the Americas [6, 7].
required to conduct this study and the Colombian
Diatraea stem borers attack sugarcane in all its developmental stages, with production
Sugarcane Research Center (Cenicaña) for
technical and financial support to the first author. losses depending on the crop stage and damage caused [8]. Attacks on young plants can signif-
The NC246 Multistate Hatch project from the USDA icantly damage the leaves and stem tissues and cause the syndrome called “dead heart” (apical
National Institute for Food and Agriculture (NIFA) bud death). However, this syndrome only reduces sugarcane production when most shoots are
and the University of Tennessee provided support affected and if insect feeding continues for at least a month, resulting in reductions up to 30%
for insecticidal protein production. The funders had
of harvested sucrose [9]. In addition, bored holes facilitate infection by the fungi Colletotri-
no role in study design, data collection and
analysis, decision to publish, or preparation of the chum falcatum, resulting in rotting and reduced sucrose content. Losses caused by Diatraea
manuscript. stem borers have been estimated as a reduction in both total biomass production (up to 109
Kg of sucrose per hectare and per each percent of bored internodes) and sucrose content (up
Competing interests: The authors have declared
that no competing interests exist. to 34 Kg of sucrose per hectare and per each percent of bored internodes) [4, 5].
At least six species of the genus Diatraea have been reported to affect Colombia’s sugarcane
crops [7, 10]. Although some sugarcane industries rely on chemical control for the borer’s
management; in Colombia, these pests have been mainly managed using biological control
agents attacking the egg and larval stages [11]. However, decreased effectiveness of several par-
asitoids has been observed on Diatraea species found in the Cauca River Valley (CRV), the
most industrialized area for sugarcane production in Colombia, primarily due to the expres-
sion of host resistance from Diatraea tabernella Dyar and Diatraea busckella Dyar & Heinrich
to the main parasitoids used in the CRV [12]. As a result, searching for new management strat-
egies complementary or alternative to current control tools is needed.
One control option is the use of Cry [13, 14] and Vip3 [15] insecticidal proteins from the
bacterium Bacillus thuringiensis (Berliner) (Bt). These proteins target midgut cells and disrupt
the epithelial barrier, allowing passage of bacteria into the main body cavity resulting in lethal
septicemia [16]. Bt proteins and insecticides’ beneficial hallmarks are their high specificity and
established history of safety to vertebrates and non-target insects [13]. Given the boring activ-
ity displayed by Diatraea spp. in sugarcane, transgenic plants producing Bt insecticidal pro-
teins are the preferred alternative for controlling sugarcane stem borers [17]. The efficacy of
these insecticidal proteins is commonly evaluated in bioassays under controlled conditions
with the purified protein incorporated in an artificial diet and monitoring survival and devel-
opment [18], or by direct observation of their efficacy in plants in the field [19]. Several studies
have assessed the activity of diverse Bt proteins, such as Cry1Ab, Cry1Ac, Cry1F, Cry2Ab, and
Vip3Aa20, on sugarcane stem borers, including Diatraea saccharalis (F.), Diatraea flavipen-
nella Box and Elasmopalpus lignosellus (Zeller) (Lepidoptera: Pyralidae) [15, 17, 20–23]. These
studies were facilitated by the availability of an artificial diet capable of sustaining D. sacchara-
lis development and reproduction [24, 25].
In Colombia, four Diatraea species present the most significant distribution and economic
impact on sugarcane: D. busckella, D. indigenella, D. saccharalis (F.), and D. tabernella [7, 11].
Except for D. saccharalis, no artificial diet is available for testing insecticidal protein activity on
other species. Additional research is warranted in relation to the development of an artificial
diet for the additional Diatraea species (i.e., D. busckella, D. indigenella and D. tabernella).
Investing additional effort will yield appropriate diets for these species. However, a potential
challenge arises from the necessity of utilizing distinct diets for each species, which could com-
plicate the comparability of survival and biological responses in trials where comparisons
among different species are required. Performing experiments using a species-specific diet,
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PLOS ONE Bioassay to test activity of cry proteins against Diatraea spp.
would increase the variation in the set-up, hindering this was straightforward comparisons
among Diatraea species. In solving this current knowledge gap, this study focused on estab-
lishing an alternative method using a fresh food substrate for evaluating susceptibility to Cry
proteins and providing proof of concept by testing Cry1Ac susceptibility in D. busckella, D.
indigenella, and D. tabernella. Results using corn discs as a food source support high suscepti-
bility to Cry1Ac in all tested Diatraea spp. and validate this method for future trials using alter-
native Bt proteins, commercial pesticides, or plant tissues expressing insecticidal proteins.
Materials and methods
Insects
The entomology laboratory at the Colombian Sugarcane Research Center (known by the Span-
ish acronym Cenicaña), located in Florida, Valle del Cauca, Colombia, supplied the insects
used in this study. All insect colonies were obtained from sugarcane collections in the CRV
and maintained in a climate-controlled room at a temperature of 25 ± 1˚C, 70 ± 4% RH, and
12:12 L:D photoperiod; on slices of tender corn cob previously disinfected by flame steriliza-
tion using laboratory burners for 15–20 seconds. All insects used in bioassays corresponded to
first generation individuals from field-collected insects. Corn cobs for rearing larval feeding
were sliced into pieces (0.5 ± 0.1 cm thick) using an 11–1/4–inch Alligator Slicer (Alligator of
Sweden, Upplands Vasby, Sweden) and subsequently cut into discs with a hole punch (1.4 cm
diameter). The maize cultivar SV 1035 was grown conventionally (mineral fertilization with
NPK and manual weed control) under field conditions, but without using insecticides, while
ensuring it was situated 2 kilometers away from other commercial corn crops. This strategic
separation aimed to prevent any potential cross-contamination. The harvesting process was
done manually at 70 days of the crop cycle. Coincidentally, this same source of corn was also
employed as a substrate to sustain the various Diatraea species stock colonies at the Cenicaña
entomology laboratory. To confirm the absence of any expression of Bt proteins, we conducted
regular assessments of survival within the stock colonies. Corn was used instead of sugarcane
because it allows the development of all borer species and presents a lower level of fermenta-
tion [26].
Insecticidal protein purification
The Cry1Ac protoxin was produced in cultures of Bacillus thuringiensis strain HD73, obtained
from the Bacillus Genetic Stock Center (Columbus, OH, USA) in 1/3 tryptic soy broth (TSB)
medium at 28˚C with shaking for 72h. After collecting spores and Cry1Ac protein crystals by
centrifugation at 6,000rpm for 30min, pellets were washed in 1M NaCl plus 0.1% Triton-X-
100, and this collection and washing cycle was repeated twice until the final pellet was washed
with MilliQ water. The Cry1Ac crystals were solubilized at 30˚C in carbonate buffer (50mM
Na2CO3, 0.1M NaCl, 0.1% 2-mercaptoethanol, pH10.5), and the solubilized Cry1Ac protoxin
activated by digestion with bovine TPCK-treated trypsin (Sigma,�10,000 BAEE units/mg) at
a final concentration of 0.2mg/ml for 1 hour. Activated Cry1Ac toxin was purified by anion
exchange in a Hitrap Q HP column (GE Healthcare) connected to an ÄKTA chromatography
system (GE LifeSciences), using a linear gradient of 1M NaCl for elution. The purified Cry1Ac
toxin was quantified using BSA as a standard [27] and shipped frozen to Cenicaña for testing.
Bioassay
Tests were performed on four Diatraea species, Diatraea saccharalis, D. indigenella, D. taber-
nella, and D. busckella, by exposing neonates (< 24 h after hatching) to cob corn discs
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PLOS ONE Bioassay to test activity of cry proteins against Diatraea spp.
prepared as described before, 70–75 days after planting, when kernels were at the milk stage
(R3), using same cultivar and growing conditions described above. In general, the corn kernel
at the milk stage is on the cusp of maturity, yet it remains soft and brimming with a milky
fluid. This fluid is rich in carbohydrates, mainly in the form of sugars. At the start of the R3
stage, approximately 80% of its content comprises water. Non-peeled cob corns were store at
8˚C for no more than one week.
Bioassays were performed using 128-well bioassay trays (CD International Pitman, NJ),
with a 2% agar plug poured and solidified at the bottom of each well to retain moisture and
turgidity of the corn material used as feeding substrate. A corn disc with a surface area of 1.54
cm2 and 0.5 ± 0.1 cm thick was incorporated into each well. The Cry1Ac protein in water con-
taining 0.1%Triton-X 100 (total volume) was added to the surface of the discs. To account for
any effects of moisturizing the cob corn discs with water and detergent (Triton-X 100), control
larvae were exposed to discs treated with water and detergent. A concentration of 619.5 μg/ml
in a final volume of 60 μl per well was used over an area of 1.54 cm2, representing 24.1 μg/cm2
of Cry1Ac. This lethal diagnostic dose was selected in assessing toxicity based on previously
reported LC50 values for Cry1Ac from diet surface bioassays with D. saccharalis [28–30] and
D. flavipennella [21], but also based on trial-and-error observations trying to guarantee cover-
age and efficacy of the application over the fresh tissue.
The volume of 60 μl was uniformly applied onto the corn tissue using a 100 μl Eppendorf
pipette. The tray was then balanced after each application to facilitate protein dispersion and
absorption within the tissue. The corn disc substrate was allowed to absorb the solution for 1
h, then one first instar larva was added per disc. Wells were then sealed with air-permeable lids
(CD International Pitman, NJ) and trays maintained at laboratory conditions mentioned pre-
viously. Mortality was scored 7 days after infestation, by assessing any level of activity in
treated larvae. Larvae that survived the protein treatment were subsequently transferred to a
control diet (corn cob discs treated with water and detergent) to observe further development.
Considering insect availability in the colony, Diatraea species were tested in separate weeks
against their control. Each combination of Diatraea species and the protein were evaluated
separately using a tray containing 16 wells. Simultaneously, a separate tray was designated as
the control, at it contained water and detergent. As an air-permeable lid separated each group
of 16 wells, it was considered an independent replicate, and the 16 wells were considered sub-
samples. Hence each combination of species and treatment had eight replicates, for a total of
128 larvae evaluated per species and treatment.
Study variables
Variables were recorded 7 days after infestation, including larval mortality, weight, and instar.
Percentage mortality (%M) was estimated from the number of dead individuals considering
the number of individuals established in the bioassay using the formula %M = (m / N) * 100,
where m is the total number of dead insects in the treatment, and N is the total number of
insects established in the treatment. Surviving larvae were weighted using a Mettler Toledo
AB204 (Switzerland) analytical balance (precision ± 0.1 mg). These surviving individuals were
then transferred to untreated corn to determine if they presented late mortality or if they man-
aged to complete development.
The Growth Index for Survival (GIS) and Relative Growth Index (RGI) [31] were estimated
considering the developmental stage of live and dead individuals. Instar information was gath-
ered based on morphological characteristics for all the borer species under study [26]. Under
the laboratory conditions described above, larvae of the four borer species tested took 3 days to
grow from the first larval instar (L1) to the second instar (L2) and 4 days to grow from L2 to
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PLOS ONE Bioassay to test activity of cry proteins against Diatraea spp.
the third instar (L3), totaling 7 days from L1 to L3. The GIS was estimated using the formula:
GIS ¼ ½ðn1 � L1Þ þ ðn2 � L2Þ þ � � � þ ðnx � LxÞ�=ðN� LxÞ;
Where n1 was the number of individuals in instar L1, n2 was the number of individuals in
instar L2, and so forth, N corresponds to the total number of insects established in the treat-
ment, and Lx was the maximum instar reached in the control treatment. After estimating the
GIS for the control and treatment, the RGI for each species was calculated as the division
between the GIS of the treatment and that of the control, where RGI < 1 indicates that the
individuals survived or died in the early stages, RGI = 0 no development (or all killed in the
first stage), and RGI = 1 complete development of individuals in the test group (or no differ-
ence from the control group) [32].
Percentage Growth Inhibition for Weight (%GI) [33] was calculated using the average larval
weight from each replicate of the Cry1Ac treatments relative to the average weight in respec-
tive control treatments, calculated as follows:
%GI ¼ 1  ðTWIT=TNITÞ=ðTWIC=TNICÞ x 100;
Where TWIT is the total weight of insects in the treatment, TNIT is the total number of
insects in the treatment, TWIC is the total weight of insects in the control treatment, and
TNIC is the total number of insects in the control treatment [33].
Contamination is not an issue in bioassays when using artificial diets as feeding substrates
because they typically contain antimicrobial compounds. Our bioassays used fresh corn tissue
(tender corn) as substrate, which is susceptible to fungal contamination. Therefore, we moni-
tored and quantified fungal contamination levels. The percentage of corn contamination (%C)
in each treatment was evaluated based on the presence of fungal mycelia on the corn surface as
follows:
%C ¼ ðc=NÞ ∗ 100;
where c was the number of wells contaminated in the treatment, and N corresponds to the
total number of wells established for that treatment.
Statistical analyses
Experiments were arranged under a completely randomized design, with a factorial struc-
ture considering Diatraea species and treatments (Cry1Ac protein and control) as fixed
effects and analyzed using generalized linear mixed models (PROC GLIMMIX, SAS 8.2)
[34]. The 16 wells within each of the eight replicates were considered subsamples, and well
effects were considered random. Each combination of Diatraea species and the protein was
made performed once using a single borer generation. Mortality was analyzed as a propor-
tion of dead larvae within each replicate, assuming binomial distribution, whereas indexes
of the stage of development (GIS and RGI) and weight (%GI) were analyzed with available
(alive) individuals, assuming Gaussian distribution. The mean weight of surviving larvae
was also analyzed assuming Gaussian distribution. Fungi contamination of wells was esti-
mated as the proportion of wells contaminated from the total 16 wells per replicate (sub-
sample), and analyzed assuming a binomial distribution. Means were separated by Tukey
tests (α = 0.05). In addition, linear regressions comparing the proportion of contaminated
wells with the proportion of dead larvae in the controls and the proportion of contaminated
wells with the proportion of larvae killed in the treatment were analyzed by linear regression
(PROC REG, SAS 8.2) [34].
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Fig 1. Mean percent mortality for a diagnostic Cry1Ac dose (24.1 μg/cm2) coated on corn discs against Diatraea stem borer species: D. busckella, D.
indigenella, D. saccharalis, and D. tabernella. Analyses detected no effect attributable to borer species, but statistically different treatments are separated
with uppercase letters within each species (Tukey, α = 0.05).
https://doi.org/10.1371/journal.pone.0292992.g001
Results
Mortality
Percentage mortality attributable to Cry1Ac (24.1 μg/cm2) consumption was higher than 90%
for all the four evaluated species, while control mortality was less than 8% (Fig 1). Analyses
detected no effect attributable to borer species (F = 2.18; df = 3, 56; P = 0.099) nor the interac-
tion between borer species and treatment (F = 2.30; df = 3, 56; P = 0.087); however, there was
evidence of a treatment effect, with mortality being higher in the Cry1Ac treatment
(F = 319.21; df = 1, 56; P < 0.001).
Growth Index for Survival (GIS) and Relative Growth Index (RGI)
Growth index for survival (GIS) analysis detected an effect attributable to borer species, with
D. indigenella presenting the highest growth (F = 4.84; df = 3, 56; P = 0.004). A treatment effect
was also evidenced, with the GIS being higher in control (F = 3,652.74; df = 1, 56; P< 0.001),
as well as an interaction between borer species and treatment, with the control treatment of D.
indigenella presenting the highest GIS (F = 3.85; df = 3, 56; P = 0.014) (Fig 2). Many larvae
reached the second and third instar in the control treatment, while in the Cry1Ac protein
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PLOS ONE Bioassay to test activity of cry proteins against Diatraea spp.
Fig 2. Mean Growth index for Survival (GIS ± SEM) considering the developmental stage of live individuals (between L1 and L3) of Diatraea larvae
surviving 7 days after treatment with corn discs coated with Cry1Ac (24.1 μg/cm2), compared with control treatment. Statistically different means for all
pairwise comparisons among species and treatments are separated with uppercase letters (Tukey, α = 0.05).
https://doi.org/10.1371/journal.pone.0292992.g002
treatment, almost all larvae showed growth inhibition and remained in the first instar (Fig 3).
Among D. busckella larvae treated with the protein, only six reached the second instar, while
control treatments exhibited 89.3% and 90.5% more larvae in the second and third instar,
respectively. Similarly, in the same species, 95% more larvae progressed to the second and
third instar in control treatments compared to those treated with the protein and observed in
the first instar. For D. indigenella, control treatments showed 68% and 88% more larvae
advancing to the second and third instar, respectively compared to those individuals treated
with the protein and observed in the first instar. In D. saccharalis, control treatments showed
between 93% and 91% more larvae reaching second and third instar, respectively in contrast to
those treated with the protein and observed in the first instar. Likewise, in D. tabernella, con-
trol treatments exhibited between 96% and 93% more larvae moving between the second and
third instar, respectively, compared to the individuals treated with the protein and observed in
the first instar (Fig 3).
Based on GIS estimates for both treated and control larvae, RGI values in larvae treated
with Cry1Ac protein showed levels of growth below 5% compared to those from the controls;
with mean values of 0.01 (± 0.01 SEM) for D. tabernella, 0.02 (± 0.01 SEM) for D. saccharalis,
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Fig 3. Number of surviving larvae after feeding on corn discs coated with Cry1Ac (24.1 μg/cm2) and in control. L1, L2, and L3 refer to the three larval
instars observed.
https://doi.org/10.1371/journal.pone.0292992.g003
0.03 (± 0.01 SEM) for D. indigenella, and 0.05 (± 0.02 SEM) for D. busckella; but no significant
differences were detected among species (F = 2.04; df = 3, 28; P = 0.134).
Growth Inhibition for weight (%GI)
The percentage of growth inhibition for the weight (%GI) varied between 93.2% (D. busckella),
95.7% (D. indigenella), 97.7% (D. saccharalis), and 96.3% (D. tabernella), with no significant
differences between species (F = 2.77; df = 3, 11; P = 0.091). Only 4% (5 larvae) of D. saccharalis
individuals that consumed Cry1Ac survived and weighted approximately 47-fold lower than
the average in control treatment (Fig 4). These larvae did not survive more than one day after
the 7-day exposure to treatment when they were then moved to a control diet. Lower weight
inhibition was observed in surviving individuals of D. tabernella (about 28-fold) and D. indi-
genella (approximately 24-fold) that consumed Cry1Ac, compared to controls (Fig 4), with a
survival of only 2% (11 larvae) and 8% (3 larvae), respectively. However, these larvae did not
survive more than one day (D. tabernella) or survived up to four days (D. indigenella) after the
7-days exposure to treatment, when they were then moved to a control diet; in addition, larvae
did not show growth or molting during this period. The weight of surviving D. busckella indi-
viduals was the least affected, with a 16-fold lower weight than in controls (Fig 4), yet the
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Fig 4. Mean larval weight (± SEM) of Diatraea larvae surviving 7 days after treatment with corn discs coated with Cry1Ac (24.1 μg/cm2), compared with
control treatment. Statistically different means for all pairwise comparisons among species and treatments are separated with uppercase letters (Tukey, α =
0.05).
https://doi.org/10.1371/journal.pone.0292992.g004
survival of these larvae was only 7% (9 larvae) and they did not survive more than two days
after the 7-days exposure to treatment when they were then moved to a control diet. A general
weighted mean among the species showed that larvae survived 1.2 days after the 7-days expo-
sure to treatment.
Analysis of mean larval weights detected an effect of borer species, with D. saccharalis being
the species that presented the highest weight increase over time and D. tabernella the lowest
(F = 32.68; df = 3, 56; P < 0.001). Results also showed a treatment effect, with the control treat-
ment presenting a higher average weight than the Cry1Ac treatment (F = 966.69; df = 1, 56;
P< 0.001). The interaction between borer species and treatment was also evident, with D. sac-
charalis presenting the highest average weight (F = 32.13; df = 3, 56; P < 0.001) in the control
treatment concerning the other species and treatments. In contrast, no differences were found
in the weight of surviving larvae on the Cry1Ac treatment (Fig 4).
Bioassay contamination
The contamination analysis in the corn discs showed an effect of the borer species, with the
lowest percentage of contaminated wells occurring in D. indigenella (F = 4.40; df = 3, 56,
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Fig 5. Percentage of contaminated wells (± SEM) 7 days after coating corn discs with Cry1Ac (24.1 μg/cm2), compared with control treatment with no
protein. Statistically different species are separated with upper case letters within each treatment, while statistically different treatments are separated with
lowercase letters within each species (Tukey, α = 0.05).
https://doi.org/10.1371/journal.pone.0292992.g005
P = 0.007). A treatment effect was also found, with the percentage of contaminated wells being
higher in the Cry1Ac treatment (F = 5.22; df = 1, 56; P = 0.026) and no evidence of interaction
between borer species and treatment was detected (F = 1.31; df = 3, 56; P = 0.279) (Fig 5).
However, no correlation was found between the percentage of contaminated wells and the per-
centage of mortality when all species were combined, neither in the control (F = 0.14; df = 1,
30; P = 0.706) nor in the Cry1Ac treatment (F = 1.01; df = 1, 30; P = 0.323).
Discussion
Bioassay experiments are critical to identifying active insecticidal proteins against Diatraea
spp., a genus including the most widely distributed boring pests of sugarcane in the western
hemisphere [7]. Previous reports presented data evaluating insecticidal proteins from B. thur-
ingiensis against D. saccharalis [15, 16, 23, 28, 29, 30] and Bt isolates against D. flavipennella
[21] using artificial diets. The lack of available artificial diets supporting larvae growth has pre-
vented testing susceptibility to these insecticidal proteins in other relevant Diatraea species. In
resolving this knowledge gap, we developed a new method using corn leaf discs coated with
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PLOS ONE Bioassay to test activity of cry proteins against Diatraea spp.
treatments. We tested its utility in determining susceptibility to the Cry1Ac insecticidal protein
in the most widely distributed sugarcane borer species in Colombia. Mortality in control treat-
ment in the developed bioassay protocol did not exceed 10%, as recommended by Beegle [18]
when working with artificial diets. Interestingly, we detected weight and developmental differ-
ences between the tested species in the control treatment, with D. saccharalis presenting the
highest average weight and D indigenella with the highest number of third instar larvae. This
variation is also observed under rearing conditions [26], showing that the bioassay can sustain
normal biological function. In contrast to controls, high mortality was observed in Cry1Ac
treatments with larvae of D. saccharalis, as expected from the increased susceptibility to that
protein reported from bioassays using artificial diets for this species [21, 28, 30].
The new bioassay method allowed, for the first time, determining that high susceptibility to
Cry1Ac in D. saccharalis extends to other relevant sugarcane borer species in Colombia: D.
busckella, D. indigenella, and D. tabernella. These observations support the use of this bioassay
for testing insecticidal protein activity in Diatraea species lacking an artificial diet. However, it
is essential to note that if we compare this new tissue bioassay with the one using an artificial
diet [21], where a concentration of 619.5 μg/ml would be applied over an area of 1.77 cm2, the
use of 30 μl in each well will represent 10.5 μg/cm2 of Cry1Ac, meaning that more than double
of the protein is required for coating the tissue (i.e., 24.1 μg/cm2) and effectively expose the
insects. Given the challenges and expenses associated with producing Cry1Ac, ensuring a
streamlined and cost-effective screening process would necessitate dedicated efforts towards
formulating an artificial diet that sufficiently supports the development of different Diatraea
species.
Bioassays with fresh tissue were previously reported as successful set-ups for testing the
activity of Bt insecticidal proteins. Examples include Cry3 and Cry7Aa activity in Coleoptera
using potato tubers and potato flour as substrate [35]. However, plant derivatives, such as
carotenoids, have been shown to influence Cry1Ac toxicity in bioassays [36]. Using fresh tissue
offers the possibility of fungal contamination, as we detected in our method. Treatment with
Cry1Ac and subsequent larval death could increase the likelihood of observing fungal contami-
nation, yet we did not detect a correlation between treatment, mortality, and fungal contami-
nation. Contamination was significantly influenced by species, suggesting that some Diatraea
species may be more prone to harbor/distribute fungal contaminants.
Information on growth inhibition complements mortality data, allowing the detection of
possible sublethal effects of Cry proteins. Individuals from all tested species surviving Cry1Ac
exposure presented severe growth inhibition, reflected in a significant decrease in weight and
stunting as indicated by RGI values, wherein the best of cases, larvae grew only 5% of the
expected size considering size in controls. Similar weight reduction was previously reported
for D. saccharalis exposed to Cry1Ab [23] and Vip3Aa20 [15]. Observations with larvae of S.
frugiperda [29] and Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) [28] suggested that
heavily stunted larvae (not growing beyond first instar and weighing less than 0.1 mg) after
exposure to Bt insecticidal proteins should be considered as dead because they eventually die
without completing development. Our observations support this idea, as all surviving larvae
presented stunting after Cry1Ac treatment for all species and died shortly after concluding the
bioassay.
Conclusions
This study proposes an alternative, novel method to evaluate Bt insecticidal proteins using
fresh corn tissue to determine their toxicity on Diatraea species. The bioassay is based on the
technical criteria recommended for protocols using artificial diets, such as maintaining control
PLOS ONE | https://doi.org/10.1371/journal.pone.0292992 October 18, 2023 11 / 14
PLOS ONE Bioassay to test activity of cry proteins against Diatraea spp.
treatment mortality below 10%. However, sanitation of the work area and careful operations
are fundamental to reducing the potential for contamination of the corn substrate during the
bioassays, thus helping to obtain consistent results. The high susceptibility to Cry1Ac detected
for all tested species, reflected as mortality and heavy stunting of survivors, confirms the poten-
tial of Cry proteins for Diatraea spp. control. Future work is focused on performing dose-
response bioassays and determining toxicity parameters (LD50 and LD90) for the different Dia-
traea species. These results can serve as a starting point for genetically engineering sugarcane
varieties with highly active insecticidal proteins to prevent stem borer attacks. However, con-
sidering the challenges and expenses involved in Cry1Ac production, establishing an efficient
and economical screening procedure would require focused endeavors in formulating an arti-
ficial diet that adequately nurtures the growth of various Diatraea species. Future develop-
ments in the area would expedite alternatives to the use of chemical insecticides in the control
of the stem borers, and it would also need to consider its integration with sugarcane stem bor-
ers IPM programs, where biological control is considered a significant pest management factor
in several sugar industries in the Americas.
Supporting information
S1 Data.
(XLSX)
S2 Data.
(XLSX)
Acknowledgments
The authors thank Sandra Valencia from the International Center for Tropical Agriculture
(CIAT), Orlando Rojas and Alvaro Urresti from the Colombian Sugarcane Research Center
(Cenicaña) for providing the logistical and technical support required to conduct this study.
We also thank staff from the Insect Molecular Pathology and Resistance laboratory from the
Department of Entomology and Plant Pathology of the University of Tennessee, for their work
in the insecticidal protein production.
Author Contributions
Conceptualization: Juan Sebastián Ángel-Salazar, Claudia Echeverri-Rubiano, Jairo Rodrı́-
guez-Chalarca, Jershon López-Gerena, Juan Luis Jurat-Fuentes, Germán Vargas.
Data curation: Juan Sebastián Ángel-Salazar, Claudia Echeverri-Rubiano, Germán Vargas.
Formal analysis: Juan Sebastián Ángel-Salazar, Claudia Echeverri-Rubiano, Germán Vargas.
Funding acquisition: Jershon López-Gerena, Germán Vargas.
Investigation: Juan Sebastián Ángel-Salazar, Claudia Echeverri-Rubiano, Jairo Rodrı́guez-
Chalarca, Jershon López-Gerena, Rafael Ferreira dos Santos, Germán Vargas.
Methodology: Juan Sebastián Ángel-Salazar, Claudia Echeverri-Rubiano, Jairo Rodrı́guez-
Chalarca, Jershon López-Gerena, Germán Vargas.
Project administration: Jershon López-Gerena, Germán Vargas.
Resources: Jairo Rodrı́guez-Chalarca, Jershon López-Gerena, Rafael Ferreira dos Santos, Juan
Luis Jurat-Fuentes, Germán Vargas.
Software: Germán Vargas.
PLOS ONE | https://doi.org/10.1371/journal.pone.0292992 October 18, 2023 12 / 14
PLOS ONE Bioassay to test activity of cry proteins against Diatraea spp.
Supervision: Jershon López-Gerena, Juan Luis Jurat-Fuentes, Germán Vargas.
Validation: Claudia Echeverri-Rubiano, Jershon López-Gerena, Rafael Ferreira dos Santos,
Alexandra M. Revynthi, Germán Vargas.
Visualization: Juan Luis Jurat-Fuentes, Alexandra M. Revynthi, Germán Vargas.
Writing – original draft: Juan Sebastián Ángel-Salazar.
Writing – review & editing: Juan Sebastián Ángel-Salazar, Claudia Echeverri-Rubiano, Jairo
Rodrı́guez-Chalarca, Jershon López-Gerena, Rafael Ferreira dos Santos, Juan Luis Jurat-
Fuentes, Alexandra M. Revynthi, Germán Vargas.
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