Jurat FuentesetalARE2021

Annual Review of Entomology
Mechanisms of Resistance to
Insecticidal Proteins from
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Bacillus thuringiensis
Annu. Rev. Entomol. 2021.66:121-140. Downloaded from www.annualreviews.org
Juan Luis Jurat-Fuentes,1 David G. Heckel,2

and Juan Ferré3
1
Department of Entomology and Plant Pathology, University of Tennessee, Knoxville,
Tennessee 37996, USA; email: jurat@utk.edu
2
Department of Entomology, Max Planck Institute for Chemical Ecology, Jena 07745,
Germany; email: heckel@ice.mpg.de
3
ERI of Biotechnology and Biomedicine (BIOTECMED), Universitat de València,
Burjassot 46100, Spain; email: Juan.Ferre@uv.es
Annu. Rev. Entomol. 2021. 66:121–40 Keywords

The Annual Review of Entomology is online at
Bacillus thuringiensis, Cry protein, Vip3 protein, resistance mechanism,
ento.annualreviews.org
receptor
https://doi.org/10.1146/annurev-ento-052620-
073348 Abstract
Copyright © 2021 by Annual Reviews.
Insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are used
All rights reserved
in sprayable formulations or produced in transgenic crops as the most suc-
cessful alternatives to synthetic pesticides. The most relevant threat to sus-
tainability of Bt insecticidal proteins (toxins) is the evolution of resistance
in target pests. To date, high-level resistance to Bt sprays has been limited
to one species in the field and another in commercial greenhouses. In con-
trast, there are currently seven lepidopteran and one coleopteran species that
have evolved practical resistance to transgenic plants producing insecticidal
Bt proteins. In this article, we present a review of the current knowledge on
mechanisms of resistance to Bt toxins, with emphasis on key resistance genes
and field-evolved resistance, to support improvement of Bt technology and
its sustainability.
121
1 . INTRODUCTION: BACILLUS THURINGIENSIS IN INSECT
PEST CONTROL
Gene pyramiding: The Gram-positive bacterium Bacillus thuringiensis (Bt) is the most economically successful en-
strategy consisting of tomopathogen to date. Its pesticidal activity is mainly due to production of insecticidal proteins,
coexpressing two or most of which are synthesized and packaged during sporulation into a parasporal crystal unique to
more toxins in the
Bt compared to other Bacillus spp. Sprayable mixtures of Bt spores and parasporal crystals are con-
same plant to reduce
resistance risk and sidered safer and more specific than synthetic pesticides and account for 75–95% of the microbial
expand target range biopesticide market (93). Natural strains of Bt registered in the United States include isolates active
against pests in Lepidoptera (subsp. kurstaki, aizawai, and galleriae), Coleoptera (subsp. morrisoni
Enterocytes:
columnar epithelial biovar tenebrionis), and Diptera (subsp. israelensis). In addition, pesticides based on recombinant Bt
cells lining the midgut or Bt toxins expressed in other bacteria have been commercialized (71).
with microvilli on the Insecticidal Bt genes are expressed in transgenic crops as plant-incorporated protectants (PIPs),
apical surface facing the most important contribution of biotechnology to pest control in agriculture to date. These
the lumen for

transgenic Bt crops are planted in approximately 101 million hectares worldwide, representing
absorption of nutrients
>53% of the global cultivated area of genetically modified crops (58). Major benefits driving
Peritrophic matrix: adoption of Bt crops include high efficacy; greater gross margins with higher yields; reduced pes-
tubular mesh of chitin
ticide usage, resulting in increased biodiversity; and, in the case of Bt maize, improved health safety
and cross-linked
proteins that lines the due to lower contamination with mycotoxins (91).
midgut and protects it A large number of pesticidal proteins have been identified in Bt and other bacterial isolates (1),
from damage from the and they are named and classified according to protein sequence identity and structural similar-
food bolus ities. There are currently 391 holotype sequences (23), of which the largest group (271 proteins)
belongs to the three-domain (3-D) Cry (from Crystal) family. The Vip3 family of vegetative in-
secticidal proteins contains 14 holotype proteings. However, only eight natural [Cry1Ab, Cry1Ac,
Cry1Fa2, Cry2Ab2, Cry2Ae, Cry3Bb1, Cry34Ab1 (Gpp34Ab1 in the new nomenclature), and
Cry35Ab1 (Tpp35Ab1 in the new nomenclature)] and three engineered (Cry1A.105, mCry3A,
and eCry3.1Ab) Cry proteins and two natural Vip3 proteins (Vip3Aa19 and Vip3Aa20) are cur-
rently produced by commercialized Bt crops. Evolution of resistance to these crops is hindered
by the production of high toxin doses in the plant and planting of non-Bt refuges to increase
susceptible populations that dilute the frequency of resistance alleles. Combining expression of Bt
genes in the same hybrid (gene pyramiding) also reduces the risk of resistance and expands activity
range.
2. MODE OF ACTION OF INSECTICIDAL CRY PROTEINS

In the Cry proteins with a 3-D structure, domain I consists of a bundle of 7–8 α-helices with
the hydrophobic α-helix 5 located in the center, and the other two domains are composed of an-
tiparallel β sheets arranged in a Greek key motif (domain II) or a jelly roll (domain III) topology
(133). Upon ingestion by an insect of a 3-D Cry protein, the parasporal crystal is solubilized by
the host gut fluids to release an approximately 120-kDa proprotein (protoxin), which is then pro-
cessed mostly from the C terminus by gut digestive proteases to an active 55–65-kDa insecticidal
protein (toxin) core. Shorter 3-D Cry protoxins (approximately 70 kDa) lack the C terminus and
are mostly processed at the N terminus. Insecticidal Cry proteins produced by Bt crops are either
the activated toxin or the full-length form of shorter protoxins. To reach the target enterocytes
in the midgut, the activated Cry proteins have to cross the peritrophic matrix, which contains
carbohydrate moieties that may bind and retain some of the protein (46).
Upon reaching the midgut brush border membrane epithelium, 3-D Cry proteins bind to spe-
cific sites that are considered necessary but not sufficient for toxicity (131). Diverse proteins and
glycoconjugates contain binding sites for Cry toxins (101), and empirical evidence for receptor
122 Jurat-Fuentes • Heckel • Ferré

functionality is reported for cadherin-like, aminopeptidase-N (APN), alkaline phosphatase (ALP),
and ATP-binding cassette (ABC) transporter family proteins (1). Recent findings suggest that Cry
protein binding to ABC transporters is critical for high cytotoxicity, while interaction with the
Cadherins: a
other receptors may increase toxicity and thus play an enhancer role (13, 116). Mechanistic de- superfamily of proteins
tails of the post-binding events resulting in cell death remain controversial (119). Most evidence characterized by
supports insertion of the Cry protein oligomer into the enterocyte membrane to form a cation- variable numbers of an
selective pore that results in osmotic imbalance and cell death promoted by compensatory water extracellular
110-amino-acid
influx through aquaporins (27). Massive enterocyte death destroys the midgut epithelial barrier
domain, the cadherin
and allows invasion of the hemocoel by Bt and other gut-resident bacteria, ultimately causing death repeat
of the host by septicemia (106). There is increasing evidence that C-terminal domains of Cry1A
ABC transporters:
protoxins may contribute to toxicity by an alternative mechanism (104). While having little or no
a superfamily of
insecticidal activity by themselves, spores can contribute to toxicity through additional virulence ATP-binding cassette
factors (70). transmembrane
proteins that transport

small molecules across
3. EXPERIMENTAL EVIDENCE FOR RESISTANCE MECHANISMS
lipid bilayer
TO INSECTICIDAL CRY PROTEINS membranes by binding
3.1. Alteration of Toxin Binding and hydrolyzing ATP
Given the importance for toxicity of Cry protein binding to midgut receptors, much effort has Ligand blot: proteins
are denatured, resolved
been devoted to determining binding parameters and their potential alterations in resistant in-
by gel electrophoresis,
sects. Quantitative binding studies involve monitoring specific and nonspecific binding of a Cry and transferred and
protein labeled with radioactive iodine to brush border membrane vesicles (BBMV), a prepara- fixed to a membrane to
tion of the midgut microvillar layer. Further experimental elaboration allows distinction between be visualized with
reversible and irreversible components of binding (14). Displacement of a labeled Cry protein by labeled probes
an unlabeled one indicates that the proteins share binding sites. Even if the molecular identities GPI anchor:
of these binding sites are not known, these experiments can predict risks of cross-resistance by a glycosyl-
change of a single binding site recognized by distinct Cry proteins. Binding site models derived phosphatidylinositol
covalent linkage
from these displacement assays have been reviewed for several relevant species (53).
between a C-terminal
Alternative binding tests include histological midgut sections probed with fluorescently labeled amino acid of a protein
toxins or antibodies coupled to a visualization technique (28) and real-time binding experiments by and a phospholipid in
surface plasmon resonance (SPR) with a toxin or a binding target immobilized on chips (81). These the cell membrane
methods, however, have often failed to detect the reduced Cry binding expected from BBMV
binding assays (28, 81). Similarly, results from one-dimensional or two-dimensional ligand blots
of BBMV proteins should be taken with caution, as protein denaturation may expose epitopes
that are not accessible in vivo (24). Nevertheless, ligand and Western blotting have been valuable
in detecting altered expression of putative Cry protein receptors in resistant insects (66, 118).
Examples of these binding proteins are discussed below.
3.1.1. Aminopeptidase-N. The insect midgut APNs are covalently attached to lipids in the
epithelial membrane by a glycosylphosphatidylinositol (GPI) anchor and show sequence similarity
to vertebrate zinc-dependent APNs that cleave amino acids from the amino terminus of proteins.
Only some APNs have been described as interacting with Cry toxins (101), and only in a few
cases have alterations in APN structure or expression been associated with resistance (see also
Section 4.2). In Spodoptera exigua, a single APN (APN1) out of four was absent from midguts of a
Cry1Ca-resistant strain (50); however, Cry1Ca binding to APN1 was not examined. In Helicoverpa
armigera, an APN gene differentially expressed in a Cry1Ac-resistant strain exhibited a 22-amino-
acid deletion, which, unlike the full-length susceptible form, failed to bind Cry1Ac (146).
www.annualreviews.org • Mechanisms of Resistance to Bt Proteins 123

3.1.2. Alkaline phosphatase. Soluble or GPI-tethered ALPs are involved in dephosphoryla-
tion reactions. Reduced levels of midgut ALP activity were found in strains of Heliothis virescens
resistant to Cry1Ac and/or Cry2Ab, Cry1F-resistant Spodoptera frugiperda, Cry1Ac-resistant
F1 screen:
field-collected insects H. armigera (66), Cry1Ah-resistant Ostrinia furnacalis (107), and Cry1Ac-resistant Plutella xylostella
are crossed to an (see Section 4.1). In contrast, in a Cry1Ac-resistant strain of Helicoverpa zea, lower ALP levels on
existing resistant BBMV were associated with higher ALP activity in the midgut lumen (14), suggestive of GPI
strain, and the F1 cleavage releasing Cry1Ac-binding ALPs from the enterocyte membrane. Conversely, there was
progeny are screened
no reduction of ALP transcript levels in Cry1Ab-resistant Diatraea saccharalis (141), or Cry1Ac-
for recessive resistance
alleles resistant O. furnacalis (64). While there is evidence that ALPs in Lepidoptera and Diptera are
functional Cry receptors (1), reduction of ALP levels is usually accompanied by another mech-
F2 screen:
anism of resistance. Accordingly, membrane-bound ALPs in vivo may provide binding sites and
field-collected insects
are mated in single potentiate toxicity by increasing Cry protein concentration at the epithelial surface, a function
pairs, their offspring that could also be performed by APNs.
are intercrossed, and

the F2 grand-offspring 3.1.3. Cadherins. The Cry1A-binding cadherins of Lepidoptera have 12 extracellular cadherin
are screened for
domains and single transmembrane and cytoplasmic domains. Cry1A-binding cadherin function
recessive resistance
alleles possibly involves brush border organization, although this has not been confirmed.
Truncation of a cadherin gene by a retrotransposon was linked to resistance against Cry1Ac
and cross-resistance to Cry1Aa and Cry1Ab in the laboratory-selected YHD2 strain of H. virescens
(37). Paradoxically, binding studies showed reduced Cry1Aa, but not Cry1Ab or Cry1Ac, binding
to BBMV from the resistant strain (65, 73). This observation was later explained by an additional
Cry1Ac binding protein in the BBMV that masked any reduced binding due to cadherin disruption
(see Section 3.1.4) (38).
High levels of resistance to Cry1Ac in Pectinophora gossypiella correlated with cadherin muta-
tions and lower levels of cadherin expression in lab-selected colonies in the United States (30, 85)
and with mis-spliced cadherin forms in Indian populations (see Section 4.5). Insertions of trans-
posable elements in the P. gossypiella cadherin gene were recovered from China (126, 128), where a
unique mutation causing deletion of the entire transmembrane domain was also found (127). Cad-
herin mutations and downregulation of cadherin were associated with reduced Cry1Ac binding
and resistance in O. furnacalis (63).
Intensive F2 and F1 screening of field H. armigera populations in China eventually yielded at
least 16 different Cry1Ac-resistant mutations in the cadherin gene (134, 143, 148). Most of these
mutations were recessive and truncated the coding sequence, but one nonrecessive mutant in-
volved deletion of exon 32, causing a shortening of the cytoplasmic domain (143). Nonrecessive
resistance could be more easily selected in the field by Bt-transgenic plants, which may account
for the recovery of multiple independent deletions of exon 32 from the field in China (142). One
unusual mutation involved a single amino acid substitution interfering with protein trafficking
and localization in the epithelial membrane (132). Additional resistance-conferring cadherin mu-
tations were found by screening H. armigera populations from India (89). Confirmation of the
importance of H. armigera cadherin mutations in Cry1Ac resistance came from a CRISPR/Cas9
knockout (125).
Despite there being numerous types of Cry1A resistance–causing cadherin mutations from
H. armigera and P. gossypiella, these mutations are very rare in field populations, and there is little
evidence from other species. A mis-splicing mutant was found in F2 and F1 screens of Australian
Helicoverpa punctigera (122), but similar screens of Australian H. armigera did not detect Cry1A-
resistant mutations, cadherin or otherwise. Exhaustive linkage mapping and mechanistic studies
have also ruled out cadherin involvement in Cry1Ac-resistant strains of P. xylostella (7, 45) and
Trichoplusia ni (147). This paucity of Cry resistance–causing cadherin mutations is puzzling given

the widespread occurrence of Cry-binding cadherins (101). Perhaps one reason is that there is a
high fitness cost for these cadherin alleles, as reported for P. gossypiella (16). More studies on other
species are required to establish the applicability of these findings.
Practical resistance:
a genetically inherited
3.1.4. ABC transporters. The only known integral membrane proteins that participate in the change causing
Cry mode of action are ABC transporters. Increased resistance and reduced Cry1Ac binding in the reduced efficacy and
YHD2 strain of H. virescens were linked to a 22-base-pair deletion in exon 2 of an ABC transporter economic
consequences for pest
subfamily C2 (ABCC2) gene (38). This deletion resulted in a truncated ABCC2 protein, abolishing
control
Cry1Ab and Cry1Ac but not Cry1Aa binding. In Bombyx mori, positional cloning in a Cry1Ab-
resistant strain revealed several amino acid substitutions and one tyrosine insertion in the second
extracellular loop of an ABCC2 gene (2). Germline transformation showed that a single copy
of the susceptible allele could confer Cry1Ab susceptibility in the Cry1Ab-resistant strain (2),
and subsequent heterologous expression in Sf9 cells demonstrated that the tyrosine insertion was
responsible for resistance (116).

Other ABCC proteins have been implicated in Cry1 resistance in Lepidoptera, including prac-
tical resistance to Cry1F in S. frugiperda (see Section 4.4). Mapping of a Cry1Ac-resistant strain of
P. xylostella revealed a 10-amino-acid deletion in the 12th transmembrane domain of ABCC2, pre-
venting expression of a functional protein (6). Additional mapping of Cry1Ac-resistant T. ni (6) and
Cry1F-resistant Ostrinia nubilalis (21) localized resistance to the vicinity of an ABCC2 gene but
did not reveal the resistance-causing mutations. Mapping using bulked segregant analysis identi-
fied a deletion of 82 amino acids in the second nucleotide-binding domain of an ABCC2 protein
in Cry1C-resistant S. exigua, which was associated with a reduction in the irreversible component
of Cry1C binding (98). Knockdown of this ABCC2 significantly reduced susceptibility to Cry1Ac
and Cry1C (98), yet unexpectedly, the ABCC2 mutation did not affect Cry1A receptor function-
ality (103). The neighboring ABCC3 gene may also be involved, as knocking out both ABCC2
and ABCC3 is required for high-level resistance to Cry1Ac in H. armigera (123).
There is mounting evidence supporting the role of ABC transporter genes in resistance to
other Cry protein families. A comprehensive screening of Australian populations of H. armigera
recovered several Cry2Ab resistance alleles consisting of inactivating mutations in another ABC
transporter, ABCA2 (117). The functional significance of ABCA2 for Cry2Ab in H. armigera was
confirmed by CRISPR/Cas9 knockouts of the gene (124). Field and laboratory-evolved resistance
to Cry2Ab in P. gossypiella was linked to mis-spliced alleles of ABCA2 (82). An inactivating trans-
poson insertion into the ABCA2 gene conferred resistance to Cry2Ab in T. ni, and the essential
role of ABCA2 in Cry2Ab toxicity in T. ni was demonstrated by CRISPR/Cas9 knockouts (139).
Another ABC protein, ABCB1 (related to P-glycoprotein), was reported as a functional Cry3Aa
receptor in the coleopteran Chrysomela tremula, and an inactivating frameshift mutation conferred
resistance against Cry3Aa (99). Linkage mapping in Cry3B-resistant Diabrotica virgifera virgifera
identified a genomic locus containing an ABCB1 homolog, explaining part of the resistance phe-
notype (see Section 4.5).
3.2. Alterations in Protoxin Processing

One of the earliest reports of Cry1Ac resistance was in a colony of Plodia interpunctella showing
significantly lower serine protease activity and reduced capacity to activate Cry1Ac proprotein
(96). Absence of a major gut protease was genetically linked to decreased susceptibility to Cry1Ac
(95). Slower activation associated with significantly lower trypsin-like and chymotrypsin-like
activities was reported in Cry1A-resistant strains of H. virescens (35, 36, 72), a DiPel-resistant
strain of O. nubilalis (75), a Cry1Ab-resistant strain of Mythimna unipuncta (43), and field-collected

Cry1Ac-resistant H. zea (145). Improper processing of the N terminus of the Cry1Ac proprotein
associated with downregulation of the HaSP2 protease was reported in a resistant strain of
H. armigera (105). Protoxin processing in these larvae yielded 95- and 68-kDa fragments, instead
Bt subsp. kurstaki
(Btk): a Bt strain in of the expected 65-kDa activated protein observed in susceptible larvae.
many commercial
biopesticides that 3.3. Enhanced Immunity, Toxin Sequestration, Epithelial Regeneration,
produces parasporal
and Other Mechanisms
crystals containing
Cry1Aa, Cry1Ab, Maternally transmitted elevated immune status, denoted by increased melanization and the pres-
Cry1Ac, and Cry2Aa ence of a toxin-binding hexamerin in the hemolymph and gut lumen, was associated with low-level
or Cry2Ab toxins
resistance to Cry1Ac in a strain of H. armigera (78). A claim that pyrethroid-detoxifying esterases

Bt subsp. aizawai could sequester Cry1Ac in a strain of H. armigera was complicated by resistance being only de-
(Bta): a Bt strain in tectable under very specific bioassay conditions (44). Similarly, a threefold increase in the level of
many commercial
Cry1Ac-binding glucosinolate sulfatase was unlikely to account for the 100,000-fold increase in
biopesticides that
produces parasporal resistance to Cry1Ac in a strain of P. xylostella (135). As discussed above, soluble Cry1Ac-binding
crystals containing ALP in the gut lumen could sequester Cry1Ac in a strain of H. zea (14).
Cry1Aa, Cry1Ab, In two reports on midgut healing in H. virescens, histopathological evidence supported faster
Cry1C, and Cry1D recovery of epithelial integrity in resistant larvae (36, 80). Additional resistance mechanisms in-
toxins
volving altered processing were also described in these strains (35). Candidate genes to control en-
hanced midgut regeneration include response-to-pathogens (REPAT) (49) and arylphorins (17).
These were highly upregulated in S. exigua resistant to the Bt pesticide XenTari, yet no increased
midgut cell proliferation was detected (48).
An amino acid substitution in a tetraspanin gene confers dominant resistance to Cry1Ac in a
strain of H. armigera (62), but the mechanism is unknown. Polymerase chain reaction screening of
moths collected from northern China from 2006 to 2016 showed an increase in mutant frequency
of up to 8%.
4. MECHANISMS OF FIELD-EVOLVED PRACTICAL RESISTANCE

TO CRY INSECTICIDAL PROTEINS
Practical resistance, the inability of control measures to maintain pest populations below economic
thresholds, has been reported for both Bt pesticides and Bt crops. These cases have been commonly
associated with island effects of isolated populations and lack of effective non-Bt refuges. In this
section, we review the available mechanistic information for these cases. Table 1 presents the
different resistance mechanism to Cry proteins currently identified at the gene or allele level. The
reader is directed to the Supplemental Material for a more detailed compilation of currently
available mechanistic information for cases of insect resistance to Bt toxins (see Supplemental
Table 1) and a discussion of emerging cases of field-evolved resistance to transgenic Bt crops for
which no conclusive mechanistic data are available.
4.1. Resistance to Bt Sprays in Plutella xylostella

The diamondback moth (P. xylostella) remains the only insect that has evolved practical resistance
to Bt sprays based on Bt subsp. kurstaki (Btk) or Bt subsp. aizawai (Bta) under field conditions.
The first mechanistically characterized case of field-evolved resistance to Bt involved a P. xylostella
population from Hawaii resistant to DiPel that was further selected in the laboratory, resulting
in strain NO-QA, which has >1,000-fold resistance to Cry1A proteins but no cross-resistance
to Bta or Cry1B, Cry1C, and Cry1D (112, 114). Inheritance and binding studies showed that a
recessive mutation in a single autosomal locus greatly reduced Cry1Ac binding in NO-QA, and
that this reduced binding was directly associated with resistance (113, 115). Cross-resistance to

Table 1 Published mechanistic reports of alleles genetically linked with resistance to Bt insecticidal proteins in field
populations
Cross-
Mechanism Species (resistance gene) Toxin or event resistance Inheritance References
Mutations in Helicoverpa armigera Cry1Ac Cry1A family Recessive 140, 143,
cadherin gene 148
H. armigera Cry1Ac NA Partly dominant 143
Helicoverpa punctigera Cry1Ac NA Recessive 122
Pectinophora gossypiella Cry1Ac NA Recessive 85, 128
Mutations in Chrysomela tremula (ABCB1) Cry3Aa NA Recessive 99
ABC gene H. armigera (ABCA2) Cry2Ab Cry2Ae Recessive 117

H. punctigera (ABCA2) Cry2Ab Cry2Ae Recessive 117
P. gossypiella (ABCA2) Cry2Ab NA Recessive 82
Plutella xylostella (ABCC2) Cry1Ac Cry1A, Cry1F, Mostly recessive 6

Cry1J
Spodoptera frugiperda Cry1F/TC1507 Cry1Ab, Cry1Ac Recessive 4, 12, 33
(ABCC2)
Trichoplusia ni (ABCA2) Cry2Ab/MON15985 NA Mostly recessive 139
Mutations in H. armigera Cry1Ac NA Dominant 62
tetraspanin
Upregulation of P. xylostella Cry1Ac NA Recessive 45
MAPK4K4
gene
Altered T. ni (APN1, APN6) Cry1Ac Cry1Aa, Cry1Ab Mostly recessive 118
expression of
APN gene
Abbreviations: ABC, ATP-binding cassette; APN, aminopeptidase-N; NA, not applicable.
Cry1Fa and Cry1Ja toxins in NO-QA was attributed to a shared binding site in P. xylostella (3,
114). Complementation tests between NO-QA and seven other independently generated Btk-
resistant P. xylostella strains from the United States, Philippines, China, and Malaysia supported a
common resistance locus in all the strains (7, 22, 42, 113). This locus (called BtR-1) also mapped to
resistance to Javelin (a Btk-based product) in the DBM1Ac-R strain of P. xylostella (45). Resistance
in NO-QA was mapped to a mutation deleting the last transmembrane domain of the ABCC2
transporter gene in the BtR-1 locus (6). In contrast, in the DBM1Ac-R strain, no lesions were
found in ABCC2, and instead, ABCC2, the adjacent ABCC3, and an unlinked alkaline phosphatase
were downregulated under the influence of the tightly linked MAP4K4 gene (45). Knockdown
of the MAP4K4 gene restored ALP and ABCC2–3 expression and Cry1Ac susceptibility. Thus,
mutation and downregulation of ABCC2–3 and ALP are important components of resistance to
Cry1A proteins in P. xylostella.
Other candidate trans-regulators of ABCC and ALP genes that may impact Cry1A resistance
have been recently described. Inhibition of post-transcriptional interaction with a micro-RNA
(miR-998–3p) upregulated ABCC2 and reduced resistance levels in a Cry1Ac-resistant P. xylostella
population (153). Upregulation of ABCC2, cadherin, and ALP genes by the GATAe transcription
factor from H. armigera conferred Cry1Ac susceptibility to cultured insect cell lines (130).
In contrast to Cry1A resistance, there is little reduction in Cry1C binding in Bta-resistant
P. xylostella. Resistance to Cry1C in a strain from South Carolina (United States) was autosomal

and incompletely recessive, yet Cry1C binding was reduced only by 2.5-fold, insufficient to
explain a >12,000-fold resistance ratio (149). Altered protoxin processing was suggested as a
minor mechanism in a strain with 19-fold resistance to Cry1C (77). To date, no mechanistic
description of Cry1C resistance has been empirically tested; further work is needed in this area.
4.2. Greenhouse-Selected Resistance to Btk in Trichoplusia ni from Canada

Up to 160-fold resistance to Btk in T. ni developed frequently and rapidly in commercial vegetable
greenhouses in British Columbia (Canada) (59). The GLEN strain was resistant to both Cry1Ac
and Cry2Ab; the GLEN-Cry1Ac strain selected with Cry1Ac showed monogenic, autosomal, and
incompletely recessive resistance (69). Introgression into a susceptible population generated a

near-isogenic strain (GLEN-Cry1Ac-BCS) with a large reduction in Cry1Ac and Cry1Ab binding
(129), likely due to alteration of a shared receptor (29). The resistance locus was located on a
different chromosome from the cadherin gene (6). Quantitative proteomic analyses detected a
reduction in aminopeptidase APN1, along with upregulation of APN6, and this phenotype was
genetically linked to Cry1Ac resistance (118). However, the cluster of APN genes was unlinked
to the resistance locus (6, 118), suggestive of a trans-regulatory resistance mechanism similar to
the one described in P. xylostella (45). Resistance in GLEN-Cry1Ac-BCS mapped to the ABCC2
linkage group, although the specific mutations and the gene controlling APN1 expression have
not been reported.
A greenhouse-originated T. ni colony resistant to Btk and transgenic cotton producing Cry1Ac
and Cry2Ab (68) was used to isolate Cry2Ab-resistance alleles in a near-isogenic line (GLEN-
Cry2Ab-BCS) of a susceptible T. ni strain (109). Resistance to Cry2Ab was not genetically linked
with cadherin, ALP, APN, or ABCC2 genes and not associated with alterations in midgut pro-
tease, esterase, or alkaline phosphatase activities (109). Instead, resistance was mapped to ABC
transporter subfamily A1 (ABCA1) and A2 (ABCA2) genes on chromosome 17; only ABCA2 was
expressed in the midgut and showed inactivating mutations (139). CRISPR/Cas9 knockouts of
ABCA2, but not of ABCA1, conferred Cry2Ab resistance.
4.3. Resistance to Cry1Fa Maize in Spodoptera frugiperda

in the Western Hemisphere
Practical resistance to transgenic maize producing Cry1Fa2 (event TC1507) was first detected
in Puerto Rico (110) and then isolated in Florida and North Carolina (52); diverse locations in
Brazil (32, 84); and, most recently, Argentina (20). Resistance to Cry1Fa in all of these cases was
autosomal, incompletely recessive, and monogenic and did not incur relevant fitness costs (51, 55,
120). Cross-resistance to Cry1A.105, Cry1Ab, and Cry1Ac was common among Cry1Fa-resistant
strains (9, 56, 94, 121), consistent with alterations in shared binding sites supported by large re-
ductions in Cry1Fa and Cry1A binding in resistant larvae (54, 84).
A common Cry1Fa resistance allele of an ABC transporter subfamily C2 (SfABCC2) gene was
identified in three independently generated S. frugiperda strains from Puerto Rico (4, 33). When
expressed in insect cells, the SfABCC2 protein is a functional receptor for Cry1Fa, Cry1Ab, and
Cry1Ac (4). The resistance allele (SfABCC2mut) consists of a two-base-pair insertion inducing a
nonsense frame shift, resulting in a truncated SfABCC2 protein (4). A second allele coexisting with
SfABCC2mut in a strain from Puerto Rico had an insertion that disrupted SfABCC2 splicing (33).
Complementation tests between Cry1Fa-resistant strains of S. frugiperda from Puerto Rico and
Florida indicated a common locus (15) with different ABCC2 mutations (25). Current haplotyping
evidence supports the idea that invasive S. frugiperda in sub-Saharan Africa and southeast Asia

possibly originated in the Florida–Puerto Rico area (86), yet DNA-based screening performed to
date has failed to detect the SfABCC2mut allele in Africa or China (87, 144).
Resistance to Cry1F in Brazilian S. frugiperda involved novel mutations in ABCC2. A two-
residue deletion and a substitution in a conserved region of ABCC2 extracellular loop 4 disrupted
the Cry1F receptor role of ABCC2 in cultured insect cells and were linked to Cry1F resistance in
Brazil (12). Pyrosequencing of S. frugiperda from diverse maize-growing regions in Brazil detected
the two-residue deletion allele at high frequency, in addition to many rare alleles also disrupting
loop 4, suggesting the importance of this loop for Cry1F toxicity (12).
4.4. Resistance to Cry1Ac and Cry2Ab2 Cotton in Pectinophora gossypiella

from India
Field-evolved resistance to Cry1Ac in P. gossypiella (pink bollworm) initially involved a 44-fold-
resistant population, described in 2008 in non-Bt cotton from Gujarat, India (26). Later samples
were >2,000-fold resistant to Cry1Ac but not Cry2Ab (83). Resistance was autosomal and mostly
recessive (90). Binding of Cry1Ac estimated by a qualitative dot-blot technique appeared reduced
in samples identified as resistant by survivorship to the third instar after 21 days on 1 ppm of
Cry1Ac (92). Sequencing of cDNA revealed eight novel severely disrupted cadherin alleles in
Cry1Ac-resistant Indian P. gossypiella, seven of which had multiple transcript isoforms, but only
wild-type cadherin alleles in laboratory-selected P. gossypiella from the United States (31).
Reduced field performance of Bollgard II cotton and results from diet bioassays provided evi-
dence of field-evolved resistance to both Cry1Ac and Cry2Ab (88). Larvae resistant to Bollgard II
had truncated cadherin isoforms consistent with resistance to Cry1Ac in addition to alternative
splicing of the PgABCA2 gene, resulting in diverse truncated PgABCA2 proteins (82). The same
mechanism was found in laboratory-selected P. gossypiella from the United States with >10,000-
fold recessive resistance to Cry2Ab (82).
4.5. Resistance to Maize Producing Cry3Bb1, mCry3A, eCry3.1Ab,

or Cry34/35Ab1 in Diabrotica virgifera virgifera in the US Corn Belt
The corn rootworm (D. v. virgifera) has developed field-evolved resistance and cross-resistance
(57) to all Bt proteins used to manage this pest (39). Rootworm resistance differs from resis-
tance to high-dose Bt crops in lepidopteran pests because low resistance levels (<25-fold) can
allow severe feeding injury to Bt corn in the field and may be conferred by multiple loci. Pos-
sibly due to this complexity, there are not many mechanistic data available for resistance in
D. v. virgifera.
Approximately 25% of the genetic variation in field-evolved resistance to Cry3Bb1 was associ-
ated with a single linkage group (LG8) transmitted as an autosomal and nearly completely reces-
sive trait (34). The locus contains 39 genes, including a putative ABC subfamily B (ABCB) trans-
porter gene (34). Heterologous expression demonstrating that this protein is a functional receptor
for Cry3Bb1 was reported in an oral presentation at a meeting but has not been published in a peer-
reviewed journal. Transcriptome profiling by RNA-Seq in susceptible and eCry3.1Ab-resistant
rootworm colonies detected differential upregulation of esterase and dynein in resistant root-
worms, suggesting participation of detoxification and midgut repair processes in resistance (151).
The only published evidence for reduced binding in Cry3-resistant rootworms involves a strain
with 97-fold resistance to the mCry3A protein (150). However, normalization based on APN
activity, which was higher in BBMV from resistant larvae, rather than on BBMV protein content,
could have biased results toward detection of lower mCry3A binding to BBMV from resistant
rootworms.

5. MODE OF ACTION OF INSECTICIDAL VIP3 PROTEINS
The Vip3Aa protein is activated by proteases before binding to the apical membrane of brush bor-
der epithelial cells and forming pores (74). Pore formation by activated Vip3Aa has been shown in
BBMV (76), dissected midguts (74), and planar lipid bilayers (74). Ingestion of Vip3Aa or Vip3Ca
by susceptible Lepidoptera leads to extensive midgut damage, with lysed and swollen cells leak-
ing cellular material to the lumen (40). Sublethal Vip3Aa and Vip3Ca doses trigger apoptosis in
S. exigua larvae (47), and Vip3Aa internalizes and activates apoptosis in Sf9 cultured cells (61).
Processing of Vip3 proteins by trypsin or larval midgut fluids yields approximately a 19-kDa
fragment from the N terminus and a 65-kDa fragment from the C terminus of the protein; these
fragments remain strongly associated (19). The Vip3 proteins spontaneously form homotetramers
in solution, adopting a globular structure (97, 152). Activated Vip3 proteins bind to sites on the
apical microvilli of midgut epithelial cells (40), and these sites are shared among Vip3 proteins but
not with Cry proteins (67), with the exception of Cry1Ia in Spodoptera eridania (8). This observation
explains lack of relevant cross-resistance to Vip3 in Cry1- or Cry2-resistant lepidopteran colonies

(111). However, Vip3A and Vip3C proteins share binding sites, suggesting that a binding site
alteration could confer resistance to these two families of Vip3 proteins (67).
Ribosomal protein S2 (RpS2) interacts with Vip3A in Spodoptera litura, and RNA interference of
RpS2 reduces Vip3A larval mortality (108). The scavenger receptor class C protein from cultured
Sf9 cells has been shown to mediate Vip3Aa endocytosis, which is associated with cytotoxicity
(60). It is possible that, similar to Cry proteins, Vip3 proteins interact with more than one class of
proteins in the insect midgut.
6. LABORATORY-SELECTED MECHANISMS OF RESISTANCE

TO INSECTICIDAL VIP3 PROTEINS
Several Vip3A-resistant colonies have been selected in the laboratory, yet there is scarce informa-
tion on the biochemical mechanisms of resistance. Larvae of a Vip3A-resistant strain of S. litura
lacked two casein-degrading bands in zymograms and had an approximately twofold reduction
in proteolytic activity toward several protease substrates (5). Selection over 12 generations pro-
duced 2,040-fold recessive polygenic resistance in a H. virescens strain, with no cross-resistance
to Cry1Ab or Cry1Ac (100) and no Vip3Aa binding differences (102). The same resistance phe-
notype and lack of binding differences were observed in a H. armigera isofemale line from an F2
screen in Australia (18). Activation kinetics of the Vip3Aa protoxin with midgut fluids showed
slower processing in resistant compared to susceptible larvae, which may explain cross-resistance
to Vip3Ca (41). Current lack of evidence for binding alterations in Vip3-resistant strains may be
due to limitations in detecting Vip3A binding to toxicity-relevant receptors or to post-binding
resistance mechanisms.
7. FIELD-ISOLATED RESISTANCE ALLELES TO VIP3 PROTEINS

There are currently no mechanistic data on field-evolved resistance to Vip3 proteins. Before
commercialization of Vip3A, Vip3A-resistant alleles were detected in Australian populations of
H. armigera (at 2.7% frequency) and H. punctigera (0.8%) using F2 screens (79). These frequencies
had not increased four seasons after commercialization (18). Within each species, alleles from
different F2 families did not complement each other, indicating a single locus for each species.
Resistance was essentially recessive and most probably conferred by a single autosomal gene
and no cross-resistance to Cry1Ac or Cry2Ab was observed (79). F2 screens for major Vip3Aa
resistance alleles in H. zea from Texas (United States) have estimated 0.65% frequency and a
strain with >588-fold resistance to Vip3Aa has been generated (138).

An F2 screen for Vip3Aa resistance in S. frugiperda from Brazil indicated a 0.09% overall fre-
quency of resistance alleles (10). Laboratory selection of one of the lines resulted in >3,200-fold
resistance, which was monogenic, autosomal, and recessive (11). Similar screens in Louisiana and
Florida (United States) resulted in an estimated resistance allele frequency of 0.48% (136, 137). As
in Brazil, resistance was monogenic and autosomal recessive and did not confer cross-resistance
to Cry1Fa, Cry2Ab, or Cry2Ae (137).
SUMMARY POINTS
1. High selection pressure for resistance is commonly exacerbated by island effects of iso-
lated populations and lack of effective non-Bt refuges.
2. Mechanistically characterized field-evolved resistance commonly involves alterations in
Cry binding proteins that prevent receptor functionality and result in cross-resistance
to toxins sharing the modified binding site.

3. Selection with high-dose products containing or producing a single or multiple Bt toxins
but belonging to the same family (i.e., Cry1A) tends to select alteration of binding sites
as a resistance mechanism capable of overcoming exposure to a high toxin dose.
4. Mutations in ABC transporters are important to the high levels of resistance needed to
overcome the high-dose scenario; cadherin mutations in P. gossypiella resistant to Cry1Ac
cotton are also significant.
5. While disruptive mutations in ABCC2 have been identified in several cases, emerging ev-
idence suggests the possibility that insects may develop high levels of resistance through
altered trans-regulators of Cry binding proteins.
6. Complex mechanisms may be selected by pesticides and Bt crops representing low-dose
products, for example, in the case of D. v. virgifera resistant to Cry3 proteins.
7. Despite alleles for Vip3Aa resistance being detected at relatively high frequencies in field
populations of different insect species, no cases of field resistance have been reported to
date.
FUTURE ISSUES
1. What is the role of tetraspanin in the mode of action and resistance to Cry proteins?
Do mutations in this gene lead to dominant resistance in other lepidopterans apart from
H. armigera?
2. What is the significance of reduced ALP levels in multiple Cry-resistant insects?
3. What is the mechanism of resistance to Cry1C proteins?
4. What is the main mechanism by which Vip3 proteins disrupt the insect midgut, and what
is the role of brush border receptors in the mode of action of these proteins?
5. What is the mechanism of resistance to Vip3A proteins?
6. Can available data lead to the development of high-throughput targeted DNA sequenc-
ing as a more sensitive, cost-effective, and predictive tool compared to current screening
methods?

DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
J.L.J.-F. acknowledges support from Agriculture and Food Research Initiative Foundational Pro-
gram competitive grant 2018-67013-27820 and Hatch Multistate NC-246 from the US Depart-
ment of Agriculture, National Institute of Food and Agriculture. D.G.H. was supported by the
Max-Planck-Gesellschaft. J.F. was supported by the Spanish Ministry of Science, Innovation and
Universities (grant RTI2018-095204-B-C21).
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Annual Review
of Entomology
Contents Volume 66, 2021
Preference Provides a Plethora of Problems (Don’t Panic)

Michael C. Singer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
A Century of Synergy in Termite Symbiosis Research: Linking the Past
with New Genomic Insights
Michael E. Scharf and Brittany F. Peterson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p23

Chemical Ecology, Biochemistry, and Molecular Biology of Insect
Hydrocarbons
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The Interplay Between Viruses and RNAi Pathways in Insects
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Growing Up in a Changing World: Environmental Regulation of
Development in Insects
Christen K. Mirth, Timothy E. Saunders, and Christopher Amourda p p p p p p p p p p p p p p p p p p p p p p81
Semiochemicals for Thrips and Their Use in Pest Management
William D.J. Kirk, Willem Jan de Kogel, Elisabeth H. Koschier,
and David A.J. Teulon p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 101
Mechanisms of Resistance to Insecticidal Proteins from Bacillus
thuringiensis
Juan Luis Jurat-Fuentes, David G. Heckel, and Juan Ferré p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 121
Emergence of Maruca vitrata as a Major Pest of Food Legumes and
Evolution of Management Practices in Asia and Africa
Ramasamy Srinivasan, Manuele Tamò, and Periasamy Malini p p p p p p p p p p p p p p p p p p p p p p p p p p 141
Survive a Warming Climate: Insect Responses to Extreme High
Temperatures
Chun-Sen Ma, Gang Ma, and Sylvain Pincebourde p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 163
Honey as a Functional Food for Apis mellifera
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Population Dynamics of Chewing Lice (Phthiraptera) Infesting Birds
(Aves)
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vii
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How Dung Beetles Steer Straight
Marie Dacke, Emily Baird, Basil el Jundi, Eric J. Warrant, and Marcus Byrne p p p p p p p p 243
Laboulbeniomycetes: Intimate Fungal Associates of Arthropods
Danny Haelewaters, Meredith Blackwell, and Donald H. Pfister p p p p p p p p p p p p p p p p p p p p p p p p p 257
Tree Diversity and Forest Resistance to Insect Pests: Patterns,
Mechanisms, and Prospects
Hervé Jactel, Xoaquín Moreira, and Bastien Castagneyrol p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 277

Symbiont-Mediated Digestion of Plant Biomass in Fungus-Farming
Insects
Hongjie Li, Soleil E. Young, Michael Poulsen, and Cameron R. Currie p p p p p p p p p p p p p p p p p p 297
Navigation Along Windborne Plumes of Pheromone and
Resource-Linked Odors
Ring T. Cardé p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 317
Behaviors and Interactions of Insects in Mid-Mesozoic Ecosystems of
Northeastern China
Taiping Gao, Chungkun Shih, and Dong Ren p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 337
Transposable Elements and the Evolution of Insects
Clément Gilbert, Jean Peccoud, and Richard Cordaux p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 355
The Impact of Climate Change on Ticks and Tick-Borne Disease Risk
Lucy Gilbert p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 373
Insect Transmission of Plant Single-Stranded DNA Viruses
Xiao-Wei Wang and Stéphane Blanc p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 389
Engineering the Composition and Fate of Wild Populations with Gene
Drive
Bruce A. Hay, Georg Oberhofer, and Ming Guo p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 407
Evolution of Insect Color Vision: From Spectral Sensitivity to Visual
Ecology
Casper J. van der Kooi, Doekele G. Stavenga, Kentaro Arikawa, Gregor Belušič,
and Almut Kelber p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 435
Biological Control with Trichogramma in China: History, Present Status,
and Perspectives
Lian-Sheng Zang, Su Wang, Fan Zhang, and Nicolas Desneux p p p p p p p p p p p p p p p p p p p p p p p p p p 463
Advancing Undergraduate Laboratory Education Using Non-Model
Insect Species
Christopher W. Beck and Lawrence S. Blumer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 485
viii Contents
Supplemental Material: Annu. Rev. Entomol. 2021. 66:121-140
https://doi.org/10.1146/annurev-ento-052620-073348
Supplemental Material
Mechanisms of Resistance to Insecticidal Proteins from Bacillus thuringiensis
Juan Luis Jurat-Fuentes1*, David G. Heckel2, Juan Ferré3
1
Department of Entomology and Plant Pathology, University of Tennessee, Knoxville,
Tennessee 37996, USA; email: jurat@utk.edu; ORCID 0000-0002-8945-1814

2
Department of Entomology, Max Planck Institute for Chemical Ecology, Jena D-07745,
Germany; email: heckel@ice.mpg.de; ORCID 0000-0001-8991-2150

3
ERI of Biotechnology and Biomedicine (BIOTECMED), Universitat de València, Burjassot
46100, Spain; email: Juan.Ferre@uv.es; ORCID 0000-0001-5535-0612
S.1. EMERGING RESISTANCE WITH NO MECHANISTIC INFORMATION
Here we discuss cases of practical or emerging resistance to Cry toxins in which resistance
mechanisms have not been empirically tested and are unknown, and we provide Supplemental
Table 1 summarizing mechanistic data on resistance to Bt insecticidal proteins since the review
of Ferré and Van Rie (29).
S.1.1. Resistance to Cry1Ab Maize in Busseola fusca in South Africa
Transgenic maize event MON810 producing Cry1Ab does not represent a high-dose product
against Busseola fusca (Fuller), but it was commercialized in South Africa because it provided
valuable control of other stem borers, especially Chilo partellus (Swinhoe) (114). Field-evolved
resistance was confirmed initially in 2006 in the Christiana area (115) and later expanded
throughout the maize production regions in South Africa (113). Resistance of B. fusca to
Cry1Ab maize was not recessive (14), in line with its rapid spread. Frequency of amplified
fragment length polymorphism (AFLP) markers identified four candidate loci associated with
Cry1Ab resistance, yet variability in susceptibility to Cry1Ab maize depending on the
geographic origin of the resistant parent suggested non-uniform resistance and independent
evolution of diverse resistance alleles (13). Further work is needed to identify the mechanism
and gene/s involved in this resistance. Although Cry protein binding site models are currently
not available for B. fusca, sharing of binding sites between Cry1Ab and Cry1A.105 proteins is
common in lepidopteran maize pests (51). Putative alterations in Cry1Ab receptors in resistant
B. fusca could challenge the sustainability of maize event MON89034 producing Cry1A.105
and Cry2Ab2 proteins currently deployed in South Africa to control MON810-resistant B.
fusca, if redundant killing is lost due to existing cross-resistance to Cry1A.105.
S.1.2. Resistance to Cry1Ab Maize in Helicoverpa zea in USA
The existence of practical Bt resistance in field populations of the corn earworm Helicoverpa
zea (Boddie) in the USA has been controversial. Baseline studies for Cry1Ab susceptibility
(88) detected no resistance, and F1 screens on Cry1Ac or Cry2Ab-incorporated diet (10, 50)
estimated a very low frequency (< 0.04%) of resistance alleles. An influential review in 2008
claimed that H. zea had evolved field resistance to Cry1Ac-expressing cotton (103); this
conclusion was criticized on the basis of an inadequate baseline (75) but defended by the
authors (102). A population was selected with activated Cry1Ac toxin to a resistance ratio of
>100-fold and cross-resistance to Cry1Ab but not Cry2Ab2 (2). Brush border membrane
vesicles (BBMVs) from this strain did not exhibit altered Cry1Ac binding or pore formation,
but had lower amounts of an ALP (12). Another strain collected from Cry1Ab-expressing maize
in Georgia and further selected with MVPII showed evidence of reduced proteolytic activation
of Cry1Ac protoxin (141). However, in this study similar levels of reduced Cry1Ac activation
were detected in strains with 14-fold differences in resistance levels, suggesting an association
with low levels of field-evolved resistance. Damage to ears of sweet corn expressing Cry1Ab
or Cry1A.105 + Cry2Ab2 by H. zea increased from 1996 to 2016 in Maryland, and elimination
2
of possible contributing factors left evolution of field-evolved resistance as a likely explanation
(23). A study in Texas found no difference in ear damage on Cry1A.105 + Cry2Ab2 or Cry1Ab
+ Cry1F compared with non-Bt maize (132). Populations recently collected from North and
South Carolina were 13-4,000-fold resistant to Cry1A.105 and up to 33-fold resistant to
Cry2Ab2 in diet overlay assays (7), yet a study in Mexico found no increase in tolerance to
Cry1Ac or Cry2Ab from 1998 to 2015, when Bollgard and Bollgard II cotton were in use (1).
The resistance situation in field populations of H. zea is highly dynamic and no resistance
mechanism is convincingly established.
S.1.3. Range Expansion and Cry1F Resistance in Striacosta albicosta
The western bean cutworm Striacosta albicosta (Smith), a pest of maize and dry beans in the
Great Plains of the USA, has expanded its range eastward and northward to become an emergent
pest on maize since the late 1990s. South Dakota Cry1Ab-expressing maize was infested in
2000 (16, 17). Baseline Bt susceptibility data for S. albicosta are not available because efficient
laboratory rearing methods were not developed until 2013 (26), but it is likely that unselected
populations originally had much higher tolerances to Cry1Ab, Cry1F, and Vip3A than Ostrinia
nubilalis and H. zea. Bean cutworm larvae are effective intra-guild competitors with those
species on Cry1Ab-expressing maize (24). A meta-analysis estimated that the median lethal
concentration of Cry1F increased by a factor of 5.2 over ten years prior to 2014 in Nebraska
and other states (79). Kernel damage of maize hybrids expressing Cry1F and Cry1A.105 +
Cry2Ab2 increased from 2011 to 2015 in Ontario (93). The mechanism of resistance is
unknown, but due to the univoltine habit of this pest, geographic spread and relatively short
time frame, it is likely that any selected resistance was building on a tolerance for Cry toxins
that was innately higher than O. nubilalis, for example, which has been effectively completely
controlled by Cry1Ab-expressing maize for years.
3
S.1.4. Emerging Resistance in Diatraea saccharalis
Genetically-based resistance to Cry1Ab-expressing maize was detected in Louisiana
populations of the sugarcane borer Diatraea saccharalis (F.) using the F2 screen (46, 48). The
one resistant family out of 213 tested carried a recessive resistant allele with a frequency of
about 0.23% in the population (46) conferring about 100-fold resistance to Cry1Ab (47).
Additional F1 and F2 screens provided similar frequency estimates and resistance levels. There
were negligible fitness costs on wild-type maize (124). Silencing by RNA interference of three
APNs (137) and a cadherin but not ALPs (136) in a susceptible D. saccharalis strain reduced
its mortality on a low concentration of Cry1Ab by 2-3-fold. Continued laboratory selection
produced three strains with a common genetic basis and resistance ratio to Cry1Ab of about
550 (131). Practical resistance has not been reported from USA field populations, possibly
because the assumptions of recessive resistance and a high dose to susceptibles is satisfied in
this case (125). Moreover, the Cry1Ab-resistant strain was effectively controlled by maize
expressing Cry1A.105 and Cry2Ab2 (33). Nonetheless, an F2 screen of 28 families collected
from Louisiana produced one family with at least 10-fold resistance to Cry2Ab2 (45). Field
strains from Argentina showed practical resistance to Cry1F and Cry1A.105 but not Cry1Ab or
Cry2Ab2 (38).
S.5 Durable Control of Ostrinia nubilalis by Cry1Ab but not Cry1F
More than 20 years of deployment of Cry1Ab-expressing maize has greatly suppressed
population densities of the European corn borer, Ostrinia nubilalis (Hübner), in the corn belt
of the USA, benefitting non-Bt maize growers (49) as well as vegetable growers (22) by the so-
called "halo effect" (97). However, practical resistance to Cry1F-expressing maize was
recently reported from Nova Scotia, 12 years after Cry1F-expressing hybrids were introduced
there (92). While the LC50 of susceptible larvae is about 10 ng/cm2 of Cry1F in laboratory
bioassays, second-generation offspring of field-collected insects showed about 5% mortality to

4
200 ng/ cm2 (92). The mechanism of resistance is unknown; although Cry1F resistance in a
laboratory-selected strain >3,000-fold resistant to Cry1F (84) was found to be linked to ABCC2
(19).
5
Supplemental Table 1 Published mechanistic reports of resistance to Bt insecticidal proteins since the review by Ferré and Van Rie (29). When
clearly stated by the author, “pro” is used when the protoxin form of the Cry protein was used for selection or bioassays. Broad “–omic” studies
identifying multiple potential mechanisms are not included.
Species Strain Origin Selection agent Source Genetics Toxin/crop RR Mechanism Refs
name resisted
Aedes LiTOX Bora-Bora Btia Lab Cry4Aa 60.2 Increased proteolytic (80,
aegypti Laboratory Cry4Ba 4.0 activities and faster 96,
colony Cry11Aa 7.4 processing, reduced ALP 111)
Cyt1A 4.3 activity
Bti 2.6
LR4A Bora-Bora Cry4Aa Lab Cry4Aa 1,018 Reduced ALP activity, up- (96)
Laboratory Cry4Ba 2.7 regulation of chitin
colony Cry11Aa 3.4 metabolism,
Bti susceptible transcriptomic changes
in APN, ALP and alpha-
amylase
LR4B Bora-Bora Cry4Ba Lab Cry4Aa 34 Reduced ALP activity, up- (96)
Laboratory Cry4Ba 226 regulation of chitin
colony Cry11Aa 13 metabolism,
amylase
LR11 Bora-Bora Cry11Aa Lab Cry4Aa 18 Reduced ALP activity, up- (96)
Laboratory Cry4Ba 36 regulation of chitin
colony Cry11Aa 70 metabolism,
amylase
Bombyx C440 Strain stock Guardjet Lab Incompletely Guardjet 2,017 Resistance linked to (86)
mori (Cry1Ac pro) dominant chromosome 15
6
Chinese Strain stock Cry1Ab pro Lab Autosomal Cry1Ab pro 315 Tyrosine insertion in an (3)
No 2 (C2) recessive Cry1Aa susceptible outer loop of ABCC2
monogenic
Chrysomela Cry3Aa- Vatan Bt27b F2 screen Autosomal Cry3Aa >6,400 4 bp deletion in ABCB1 (4, 81)
tremula resistant (France) recessive Bt27 resistant gene
(R#60) monogenic
Diatraea Cry1Ab-RR Louisiana MON810c F2 screen Recessive MON810 Resistant No alteration in trypsin (46,
saccharalis (USA) single locus Cry1Ab ~100 and chymotrypsin 136)
activities, reduced
expression of cadherin
Helicoverpa XJ10 Selection lab Cry1Ac Lab Cry1Ac pro 146 Downregulated protease (15,
armigera strain (XJ-F) Cry1Ac 45 genes 56)
from
Shangdong
province
(China)
96CAD Backcross Cry1Ac Lab Autosomal Cry1Ac 516 D172G cadherin (126)
BtR and recessive substitution leading to
susceptible monogenic localization away from
(96S) cell membrane
SP15 New South Cry2Ab F2 screen Autosomal Cry2Ab 6,830 Reduced Cry2Ab binding (11,
Wales recessive, Cry2Aa 9,640 74)
(Australia) monogenic Cry2Ae >1,923
Cry1Ab Susceptible
Cry1Ac Susceptible
DiPeld Susceptible
Sicala V-2e Resistant
LF120 Susceptible MVP IIf Lab Cry1Ac pro 1,600 No changes in cadherin (69,
strains from Cry1Ac 1,200-5,000- or APN, no changes in 122)
Henan oligomer or pore
7
Province formation, reduced
(China) trypsin activity,
LF5 Susceptible Cry1Ac pro Lab Cry1Ac pro 110 Promoter mutations (70)
strain (LF) Cry1Ac 39 resulting in down-
from Hebei regulation of trypsin
Province gene
(China)
SP85 Queensland Vip3A F2 screen Recessive Vip3A >400 Slower processing of (18,
(Australia) Cry1Ac Susceptible Vip3A, no binding 73)
Cry2bA Susceptible differences
AY2 northern Cry1Ac F1, F2 Dominant Cry1Ac 1,200 L31S substitution in (54,
China screen Cry1Ab 69 tetraspanin 55)
Cry2Ab 5.9
GYBT Hebei Cry1Ac Field + Lab Autosomal Cry1Ac 103 Disruption of cadherin (130,
Province recessive Cry1Ab 46 gene (allele r1) 134)
(China) Btkg 5
Cry2Aa 1.4
15sel Hebei Cry1Ac F1 screen Autosomal, Cry1Ac ~100 Disruption of cadherin (135)
Province, recessive gene (r2 & r3 alleles)
(China)
Maharash- Cry1Ac Field + Lab Cry1Ac 72 Reduced proteolytic (85)
tra (India) activation
SCD-r1 Hebei Cry1Ac Introgressi Recessive Cry1Ac 438 Disruption of cadherin (138)
Province on from Cry1Aa >41 gene (r1)
(China) GYBT strain Cry1Ab 31
Cry2Aa susceptible
r4, r5, r6, Hebei Cry1Ac F1 screen Recessive Cry1Ac Disruption of cadherin (142)
r7, r8 Province gene (alleles r4, r5, r6,
(China) r7, 48)
8
AY335 Anyang Cry1Ac F2 screen Recessive Cry1Ac 89 Substitutions in cadherin (139)
(China) gene (r10)
AY423 Anyang Cry1Ac F2 screen Partly Cry1Ac 660 Not cadherin-based (139)
(China) dominant
AY440 Anyang Cry1Ac F2 screen Recessive Cry1Ac 47 Substitutions in cadherin (139)
(China) gene (r11)
AY441 Anyang Cry1Ac F2 screen Partly Cry1Ac 95 Substitutions in cadherin (139)
(China) dominant gene (r12)
SC23 Shache Cry1Ac F2 screen Recessive Cry1Ac 39 Substitutions in cadherin (139)
(China) gene (r13)
SW34 Shawan Cry1Ac F2 screen Recessive Cry1Ac 31 Substitutions in cadherin (139)
(China) gene (r14)
XJ-r15 Xiajin (China) Cry1Ac F2 screen Partly Cry1Ac 140 Deletion in cadherin (140)
dominant Cry1Aa 27 gene (r15)
Cry1Ab 6.3
Cry2Ab 1.4
AY-r15 Anyang Cry1Ac F2 screen Partly Cry1Ac 82 Deletion in cadherin (140)
(China) dominant gene (r15)
Ha-rr; rr Cry2Ab Inter-cross Autosomal Cry2Ab ~6,800 Two unlinked genes (117)
Vip3A strains resistant Vip3A >>400
from unlinked
separate
F2 screens
LF60 Henan MVP II Lab Cry1Ac 1100 Mis-splicing of ABCC2 (127)
province Cry1Ac pro 1400 gene
(China)
SCD-Cad Cote D'Ivoire CRISPR/Cas recessive Cry1Ac 549 Truncation of cadherin (119)
9 Cry2Ab susceptible
SCD- Cote D'Ivoire CRISPR/Cas recessive Cry2Aa >120 Knockout of ABCA2, exon (118)
A2K01 9 Cry2Ab >100 2
9
Cry1Ac susceptible
SCD- Cote D'Ivoire CRISPR/Cas recessive Cry2Aa >120 Knockout of ABCA2, exon (118)
A2K02 9 Cry2Ab >100 18
Cry1Ac 3.9
Helicoverpa Hp4-13 Queensland Cry2Ab F2 screen Autosomal Cry2Ab >4,100 Reduced Cry2Ab binding, (11,
punctigera (Australia) recessive Cry2Ae >3,100 disruption of ABCA2 25,
Cry1Ab Susceptible gene 110)
Cry1Ac susceptible
Hp9-3784 New South Cry1Ac pro F2 screen Autosomal Cry1Ac pro 113-fold No difference in Cry1Ac (116)
Wales completely binding, alternative
(Australia) recessive splicing of cadherin gene
monogenic
Hp-rr;rr Queensland Cry2Ab Inter-cross Autosomal Cry2Ab >4,000 Two unlinked genes (117)
(Australia) Vip3A strains resistant Vip3A >>215
from unlinked
separate
F2 screens
Helicoverpa AR (AR1) Monsanto Cry1Ac Lab Cry1Ab resistant No differences in Cry1Aa (2, 12)
zea Lab strain Cry1Ac ~100 or Cry1Ac binding, or
Cry2Aa2 susceptible Cry1Ac pore formation.
MVP II 10.3 Reduced ALP levels in
Vip3A Susceptible midgut brush border
Cry1AbModh Resistant
Cry1AcModh Resistant
GA (GA-R) Georgia MON810 Field MVP II 7.8 No difference in ALP (9,
(USA) Cry1Ac 55 activity, reduced trypsin 141)
Cry2Ab 14 and chymotrypsin
activities, slower Cry1Ac
processing
10
GA-R GA strain Cry1Ac pro Lab MVP II 110 No difference in ALP (9,
MVP II Cry1Ac 560 activity, reduced trypsin 141)
Cry2Ab 28 and chymotrypsin
activities, slower Cry1Ac
processing
Heliothis CPNxCP73 North Cry1Ac pro Lab Partially Cry1Aa Low No binding differences (31,
virescens -3, CP73-3 Carolina recessive/ad Cry1Ab 12 (Cry1Aa, Cry1Ab, Cry1Ac, 37, 58,
(CXC) (USA) ditive Cry1Ac 50-289 Cry2Aa), altered 62)
(depending Cry2Aa 53->250 proteolytic band pattern,
on dose) Cry1B Low reduced ALP
Cry1C Low
Cry1F Resistant
KCB North Cry1Ac Lab Cry1Ac 187-400 Altered proteolytic band (31,
(KCBhyb) Carolina Cry2Aa Cry2Aa >250 pattern, enhanced 58, 62)
(USA) Cry1F Resistant healing, slower protoxin
processing, reduced
Cry1Aa binding (not
Cry1Ab, Cry1Ac, Cry2Aa,
Cry1F)
YHD2-B YHD2 strain Cry1Ac Lab Cry1Aa >55 Reduced Cry1Aa, Cry1Ab (32,
(YHD3) Cry1Ab >900 and Cry1Ac binding. 57)
Cry1Ac >2,000 Cadherin gene disruption
Cry1Fa >130 and ABCC2 gene
frameshift deletion
YFO YHD2-B Lab Reduced Cry1Aa but not (32)
strain Cry1Ab or Cry1Ac
binding. Cadherin gene
disruption
YEE YHD2-B Lab Reduced Cry1Ab and (32)
strain Cry1AC but not Cry1Aa
11
binding. ABCC2 gene
frameshift deletion
Leptinotarsa R Michigan Btti Lab Cry3Aa pro 60 Additional proteolytic (72,
decemlineata (USA) NewLeafj bands in zymograms, 123)
higher APN activity, no
differences in processing
or Cry3Aa binding
Mythimna MR Huesca MON810 Lab MON810 resistant Incomplete processing of (36)
unipuncta (Spain) protoxin, reduced
trypsin, chymotrypsin
and elastase activities,
no binding differences
Ostrinia ACB-AbR Selected Cry1Ab Lab Cry1Ab 40 Potential increased levels (128,
furnacalis from Cry1Ac 37 of V-ATPase A and heat 129)
susceptible Cry1Ah 132 shock 70 kDa protein in
strain from Cry1F 6 2D ligand blots
Central China Cry1Ie susceptible
Mon810 resistant to
silk but not
leaves
ACB-AcR Cry1Ac Lab Cry1Ab 4-6 Mutations in cadherin (41)

(ACB- Cry1Ac 49 gene
Ac200) Cry1Ah 15
Cry1Ie susceptible
Ostrinia KS-SC Kansas (USA) DiPel Lab Incompletely DiPel 47-70 Reduced trypsin-like (44,
nubilalis dominant Cry1Aa 170 activity and protoxin 64-68)
Cry1Ab 205 hydrolysis. No binding
Cry1Ac 524 alterations
Cry2Aa >640
12
Cry1Ba 36
MON810 susceptible
Bt11k susceptible
Europe-R Lombardy Cry1Ab pro Lab Cry1Ab pro 5.7-9.8 Reduced cadherin levels (89-
(Italy) Cry1Ab 39-107.8 and Cry1Aa binding (but 91)
Cry1Ac 35 not Cry1Ab or Cry1Ac)
Cry1F 4.7
Cry9C susceptible
RSTT-R Lombardy Cry1Ab pro Lab Cry1Ab pro 9-15.2 No binding alterations (89,
(Italy) Cry1Ab 32-483.7 91)
Cry1Ac 52
Cry1F 3.4
Cry9C susceptible
Cry1F US Corn Belt Cry1F Lab Autosomal Cry1Ab susceptible No differences in Cry1F (82-
resistant recessive Cry1Ac 6.89 binding or proteases 84)
monogenic Cry1F >3,000
Cry9C susceptible
TC1507 resistant
SKY-R Minnesota Cry1Ab pro Field + Lab Autosomal Cry1Aa pro >37,000 Reduced affinity for (20,
(SKY) (USA) MON810 incompletely Cry1Aa >2,775 Cry1Aa but not Cry1Ab 21, 63)
Cry1Ab recessive Cry1Ab pro 3,470 or Cry1Ac binding,
polygenic Cry1Ab 720-4,278 mutations in
Cry1Ac >535 aminopeptidase (OnAPP)
Cry1F 5.8
Pectinophora APHIS-98R Arizona Deltapine 50Bl Lab Recessive Cry1Aa resistant Mutations in cadherin (71,
gossypiella (USA) MVP II Cry1Ab resistant gene 105,
Cry1Ac >100 107)
Cry1Bb susceptible
Cry1Ca susceptible
13
Cry1Da susceptible
Cry1Ja susceptible
Cry2Aa susceptible
Cry9Ca susceptible
AZP-R Arizona MVP II Lab Autosomal Cry1Aa resistant Reduced Cry1Ab binding (35,
(USA) Recessive Cry1Ab resistant but not Cry1Ac, reduced 77,
Cry1Ac 3,100 Cry1Ac oligomerization, 100,
Cry1Bb susceptible mutations in cadherin 105,
Cry1Ca susceptible gene, 107)
Cry1Da susceptible
Cry1Ja susceptible
Cry2Aa susceptible
AQ65 Anhui Cry1Ac pro F1 screen Autosomal Cry1Ac 300 Degenerate transposon (120)
Province recessive Cry2Ab 3 insertion in cadherin
(China) monogenic gene resulting in mis-
localization of cadherin
protein
MOV97-R Arizona MVP II Lab Recessive Cry1Ac 1,700 Mutations in cadherin (98,
(USA) Deltapine resistant gene (r1 and r3 alleles) 99)
33Bm
SAF97-R Arizona MVP II Lab Recessive Cry1Ac 520 Mutations in cadherin (99)
(USA) Deltapine 33B resistant gene (r1 and r2 alleles)
Bt4R Arizona Deltapine 33B Lab Autosomal Cry1Ac 240 Deletion in cadherin (27)
(USA) MVP II Recessive gene (r4 allele)
TX01 Texas (USA) None Field Cry1Ac 14% survival Mutations in cadherin (76)
at diagnostic gene
dose
Lab Maharashtra MVP II Field MVP II 64 Reduced Cry1Ac binding (78)
selected (India)
14
BX-R (BX- Southwester Cry2Ab pro Lab Autosomal Cry2Aa 43 Disruption of cadherin (28,
R1 + BX- n USA recessive Cry2Ab 240 gene by retrotransposon 109)
R2) Cry1Ac 420 insertion (Cry1Ac
Deltapine 33B susceptible resistance)
Plutella NO-QA Hawaii (USA) Btk (DiPel) Field + Lab Autosomal Dipel 3,300 Reduced binding of (6,
xylostella recessive Cry1Aa >100 Cry1Ab and Cry1Ac. 101,
Cry1Ab 100->750 Deletion in ABCC2 gene 106,
Cry1Ac >100 108)
Cry1Fa 100->240
Cry1Ja >140
Cry1Bb 6
Cry1D 3
Cry1I 3
NO-QAGE Hawaii (USA) Cry1Ac Derived Autosomal Cry1Aa >20,000 Reduced binding of (6, 42,
from NO- recessive Cry1Ab >10,000 Cry1Fa. Deletion in 104)
QA Cry1Ac >40,000 ABCC2 gene
Cry1Fa >10,000
Cry1Ja >2,000
DBM1Ac- Florida (USA) Cry1Ac broccoli Field + Lab Autosomal, Cry1Ac >3,500 Reduced Cry1Ac binding. (39)
R Cry1Ac pro incompletely Cry1Ac pro 5,100 Down-regulation of
recessive ABCC2, ABCC3 and ALP
genes caused by over-
expression of MAPK4K4
gene
NIL-R DBM1Ac-R Cry1Ac Backcross Autosomal Btk >2,800 Reduced Cry1Ac binding. (39)
to DBM recessive Cry1Ac >5,000 Down-regulation of
1AC-S single locus Cry1Ab >2,000 ABCC2, ABCC3 and ALP
selection Cry1Ca susceptible genes caused by over-
Cry1Ie susceptibe expression of MAPK4K4
Cry1Ah >52 gene
15
SZ-R (T2- Shenzhen Cry1Ac pro Field + Lab Cry1Ac 458 Reduced Cry1Ac binding. (39)
R) (China) Down-regulation of
ABCC2, ABCC3 and ALP
gene
SH-R Shanghai Btk Field + Lab Btk 1,890 Reduced Cry1Ac binding. (39)
(China) Down-regulation of
ABCC2, ABCC3 and ALP
gene
GX-R Guangxi Cry1Ac Field +Lab Cry1Ac 33.5 Down-regulation of (143)
(China) ABCC2 gene
Karak Malaysia Btk Field Autosomal DiPel 770 Reduced binding of (87)
recessive Cry1Aa 845 Cry1Ab and Cry1Ac
single locus Cry1Ab 164
Cry1Ac >5,710
Cry1Fa 414
Cry1Ca 59
Cry1Ab- Serdang Cry1Ab Incompletely Cry1Ab 3,848 Reduced binding of (87)
SEL (Malaysia) dominant Cry1Ac 64,210 Cry1Ab and Cry1Ac
more than
one locus,
one of them
sex
influenced
16
SZBT Guandong Btk Field + Lab Autosomal Btk 17 Lack of Cry1Ac binding (34)
(China) Cry1Ac incompletely Cry1Aa 31
recessive Cry1Ab 1,900
single locus Cry1Ac 1,200
Cry1Fa >33
ABCC2KO Not Not applicable CRISPR/Cas Autosomal, Cry1Ac 724 Reduced Cry1Ac binding. (40)
applicable 9 incompletely Deletion in ABCC2 gene
recessive
single locus
ABCC3KO Not Not applicable CRISPR/Cas Autosomal, Cry1Ac 413 Reduced Cry1Ac binding. (40)
applicable 9 incompletely Deletion in ABCC3 gene
recessive
single locus
Spodoptera Xen-R Alabama Xentarin Xentari >1,000 Up-regulated Repat5 and (43)
exigua (USA) arylphorin genes
Spodoptera 456 Santa Isabel TC1507o Field + Lab Autosomal TC1507 Resistant Reduced Cry1F and (5, 52,
frugiperda (456LS3) (Puerto Rico) recessive Cry1Ab 12 Cry1A binding, but not 53, 59)
Cry1C Susceptible Cry1C, 2 bp frameshift
DiPel Susceptible insertion in ABCC2 gene
Xentari Susceptible
JuanaDiaz Juana Diaz Cry1F Field + Lab Autosomal Cry1F >579 2 bp frameshift insertion (30)
R (Puerto Rico) recessive Cry1A.105 >87 in ABCC2 gene (as in 456)
and insertion near 4th
exon of ABCC2 gene
resulting in mis-splicing
Sf_Cor Bahia (Brazil) Field Field Autosomal Cry1F >490 GY deletion from the (8)
recessive Xentari 4 extracellular loop 4 of
ABCC2
17
Sf_Des Bahia (Brazil) Field Field Autosomal Cry1F >490 GY deletion from the (8)
recessive Xentari 5 extracellular loop 4 of
ABCC2
Trichoplusia GLEN- Originated Cry1Ac pro Greenhous Autosomal Cry1Aa pro 8.2 Reduced Cry1Ab and (61,
ni Cry1Ac, from GLEN- e + Lab incompletely Cry1Ab pro 938 Cry1Ac binding. Trans- 112,
(GLEN- DiPel recessive Cry1Ac pro 8710-1,281 regulated down- 121)
Cry1Ac- monogenic Cry1Bb pro 3.3 regulation of APN1 and
BCS) Cry1C pro 2.5 up-regulation of APN6
Cry1D pro 2.4
Cry1E pro 6.7
Cry1F pro 7.8
Cry1J pro 3.3
Cry1Ab pro 2.2
Cry9C pro 2.1
GLEN-BGII Originated Bollgard IIp Greenhous Autosomal Cry2Ab 228->1,467 Mutations in ABCA2 (60,
(GLEN- from GLEN- e +Lab incompletely transporter 95,
Cry2Ab- DiPel recessive 133)
BCS) monogenic
a
Bacillus thuringiensis (Bt) subsp. israelensis
b
Transgenic poplar event producing the Cry3Aa protoxin
c
Transgenic corn event producing the Cry1Ab toxin
d
Pesticide based on Bt subsp. kurstaki
e
Transgenic cotton event producing Cry2Ab protoxin
f
Formulation of Cry1Ac protoxin inclusion bodies encapsulated in Pseudomonas fluorescens cells
g
Bt subsp. kurstaki
h
Modified Cry1Ab and Cry1Ac proteins described elsewhere (94).
i
Bt subsp. tenebrionis
j
Transgenic potato event producing the Cry3Aa protoxin
k
Transgenic corn event producing the Cry1Ab toxin
l
Transgenic cotton producing the Cry1Ac toxin
m
Transgenic cotton producing the Cry1Ac toxin
18
n
Pesticide based on Bt subsp. aizawai
o
Transgenic corn event producing the Cry1F toxin
p
Transgenic cotton event producing the Cry1Ac and Cry2Ab toxins
19
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Annual Review of Entomology

Juan Luis Jurat-Fuentes,1 David G. Heckel,2

Annu. Rev. Entomol. 2021. 66:121–40 Keywords

the lumen for

2. MODE OF ACTION OF INSECTICIDAL CRY PROTEINS

122 Jurat-Fuentes • Heckel • Ferré

proteins that transport

www.annualreviews.org • Mechanisms of Resistance to Bt Proteins 123

are intercrossed, and

124 Jurat-Fuentes • Heckel • Ferré

responsible for resistance (116).

3.2. Alterations in Protoxin Processing

www.annualreviews.org • Mechanisms of Resistance to Bt Proteins 125

resistance to Cry1Ac in a strain of H. armigera (78). A claim that pyrethroid-detoxifying esterases

4. MECHANISMS OF FIELD-EVOLVED PRACTICAL RESISTANCE

4.1. Resistance to Bt Sprays in Plutella xylostella

126 Jurat-Fuentes • Heckel • Ferré

ABC gene H. armigera (ABCA2) Cry2Ab Cry2Ae Recessive 117

Plutella xylostella (ABCC2) Cry1Ac Cry1A, Cry1F, Mostly recessive 6

Abbreviations: ABC, ATP-binding cassette; APN, aminopeptidase-N; NA, not applicable.

www.annualreviews.org • Mechanisms of Resistance to Bt Proteins 127

4.2. Greenhouse-Selected Resistance to Btk in Trichoplusia ni from Canada

incompletely recessive resistance (69). Introgression into a susceptible population generated a

4.3. Resistance to Cry1Fa Maize in Spodoptera frugiperda

128 Jurat-Fuentes • Heckel • Ferré

4.4. Resistance to Cry1Ac and Cry2Ab2 Cotton in Pectinophora gossypiella

4.5. Resistance to Maize Producing Cry3Bb1, mCry3A, eCry3.1Ab,

www.annualreviews.org • Mechanisms of Resistance to Bt Proteins 129

explains lack of relevant cross-resistance to Vip3 in Cry1- or Cry2-resistant lepidopteran colonies

6. LABORATORY-SELECTED MECHANISMS OF RESISTANCE

7. FIELD-ISOLATED RESISTANCE ALLELES TO VIP3 PROTEINS

130 Jurat-Fuentes • Heckel • Ferré

to toxins sharing the modified binding site.

www.annualreviews.org • Mechanisms of Resistance to Bt Proteins 131

Universities (grant RTI2018-095204-B-C21).

132 Jurat-Fuentes • Heckel • Ferré

www.annualreviews.org • Mechanisms of Resistance to Bt Proteins 133

134 Jurat-Fuentes • Heckel • Ferré

www.annualreviews.org • Mechanisms of Resistance to Bt Proteins 135

136 Jurat-Fuentes • Heckel • Ferré

www.annualreviews.org • Mechanisms of Resistance to Bt Proteins 137

138 Jurat-Fuentes • Heckel • Ferré

www.annualreviews.org • Mechanisms of Resistance to Bt Proteins 139

140 Jurat-Fuentes • Heckel • Ferré

Contents Volume 66, 2021

Preference Provides a Plethora of Problems (Don’t Panic)

Michael E. Scharf and Brittany F. Peterson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p23

Spider Diversification Through Space and Time

Hervé Jactel, Xoaquín Moreira, and Bastien Castagneyrol p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 277

Mechanisms of Resistance to Insecticidal Proteins from Bacillus thuringiensis

Juan Luis Jurat-Fuentes1*, David G. Heckel2, Juan Ferré3

Tennessee 37996, USA; email: jurat@utk.edu; ORCID 0000-0002-8945-1814

Germany; email: heckel@ice.mpg.de; ORCID 0000-0001-8991-2150

46100, Spain; email: Juan.Ferre@uv.es; ORCID 0000-0001-5535-0612

S.1. EMERGING RESISTANCE WITH NO MECHANISTIC INFORMATION

of Ferré and Van Rie (29).

S.1.1. Resistance to Cry1Ab Maize in Busseola fusca in South Africa

and Cry2Ab2 proteins currently deployed in South Africa to control MON810-resistant B.

fusca, if redundant killing is lost due to existing cross-resistance to Cry1A.105.

S.1.2. Resistance to Cry1Ab Maize in Helicoverpa zea in USA

South Carolina were 13-4,000-fold resistant to Cry1A.105 and up to 33-fold resistant to

mechanism is convincingly established.

S.1.3. Range Expansion and Cry1F Resistance in Striacosta albicosta