Introduction
Insect populations commonly face exposure to pesticides applied to
target them or another species in their environment, leading to a high
prevalence of pesticide resistance among insect species . At the same
time, insects are frequently infected with a variety of parasites and
pathogens, including bacteria, fungi, and viruses, and these agents can
complement or even replace the use of chemical pesticides . The
physiological effects of chemical pesticides and pathogens are not
independent, however, as both can activate stress, detoxification, and
immune responses . Since the evolution of pesticide resistance also
often arises through these mechanisms , host responses to chemical and
microbial control agents could mediate facilitation or antagonism among
stressors on both proximate and evolutionary time scales . A better
understanding of the molecular basis and phenotypic outcomes of these
interactions would provide fundamental insight into the integration and
evolution of organismal stress and immune responses while also improving
the design and predictive power of pest and vector control strategies .
Pesticides target a diverse set of physiological functions in insects
ranging from neurotoxic activity to the regulation of growth and
development. The mechanisms of pesticide resistance show a similarly
diverse set of solutions within and among pesticide classes, including
target-site modifications, increased metabolic detoxification, and
cuticular modifications . Organophosphates (OP), which inhibit
acetylcholinesterase (AChE) to overexcite cholinergic synapses , and
pyrethroids (Pyr), which disrupt voltage-gated sodium channel (vgsc)
function , are two pesticide classes widely used in agricultural systems
and against disease vectors and also impact important honeybee and
pollinator populations (Berenbaum & Liao, 2019; Schuhmann et al. 2022).
Target-site mutations and AChE gene duplications have been described for
several OP-resistant insects , while Pyr resistance has been associated
with mutations in the voltage-gated sodium channel . Target site
mutations are not the only path to resistance, however. OP and Pyr
pesticides are mainly detoxified through oxidation and hydrolysis, and
resistance associated with differential expression of diverse canonical
detoxification genes, including cytochrome P450s, esterases, and
glutathione S-transferases (GSTs), has also been described for both
pesticide types . Moreover, recent studies have implicated changes in
cuticle and serine endopeptidase gene expression in increased resistance
to penetration by OP and Pyr pesticides .
Recent evidence suggests that the mechanisms of pesticide resistance
could also impact immune and physiological responses against parasites .
For example, esterase-mediated pesticide resistance in Culex
pipiens is associated with changes in immune gene expression including
constitutive upregulation of antimicrobial peptide (AMP) and nitric
oxide synthase (NOS) genes . Moreover, resistance-associated
constitutive changes in metabolic detoxification mechanisms like
cytochrome P450s or GSTs can alter concentrations of damaging reactive
oxygen species (ROS) that a pathogen would encounter within the host and
influence the success of pathogen colonization, growth, and transmission
. For example, changes in cap‘n’collar transcription factor expression
have been shown to alter detoxification gene expression and increase ROS
levels, thereby conferring pesticide resistance in several species while
also modifying vector competence in Aedes aegypti . Populations
of OP and Pyr resistant mosquito strains in possession of target-site
mutant ace-1 (AchE) and kdr (vgsc) alleles, respectively,
support a higher prevalence of Plasmodium falciparum parasites,
but kdr mutations were also associated with reduced midgut oocyst
burden in infected individuals .
Even in the absence of evolved resistance, host exposure to pesticides
may impact pathogen growth directly through contact with toxins or
indirectly through the induction of insect detoxification enzymes .
Exposure to pesticides can also have complex effects on components of
the cellular, humoral, and oxidative stress responses of host immunity .
For example, exposure to OP pesticides has been associated with
increased hemocyte numbers and phenoloxidase (PO) and encapsulation
activity in wax moth (Galleria mellonella ) and Colorado potato
beetle (Leptinotarsa decemlineata ) larvae . However, dual
exposure to OP and a pathogenic virus in silkworm larvae (Bombyx
mori ) resulted in differential expression of oxidative stress and AMP
genes and increased mortality . Exposure to Pyr pesticides, meanwhile,
has been associated with increased melanization responses and decreased
replication of Escherichia coli bacteria and decreased P.
falciparum infection prevalence and intensity in A. gambiae .
Pyr exposure is also hypothesized to impact the production of other
immune responses including serine proteases, lytic enzymes (esterases
and lysozymes), and reactive oxidative stress responses . Moreover,
exposure to neurotoxic pesticides (e.g., neonicotinoids, butenolides) in
bees has been associated with reduced PO activity (Czerwinski and Sadd,
2017) and increased viral loads (Harwood et al., 2022). However, the
combined effects of multiple stressors on bee mortality are variable
(Calhoun et al., 2021; Harwood & Dolezal, 2020).
Clearly, both pesticide resistance and exposure independently have
important effects on insect immunity against pathogens. However, it
remains an open question whether resistance and exposure influence
host-microbe interactions in the same direction, and whether this
influence arises through the same or different mechanisms. To address
this gap in knowledge, we experimentally evolved resistance to two
different classes of pesticides (OP and Pyr) in the red flour beetle
(Tribolium castaneum ), an emerging model for studies on insect
genomics, immunity, and resistance . As a stored-product pest, T.
castaneum may be exposed directly or indirectly to the pesticides used
to combat a variety of pests that co-inhabit stored grain facilities and
that impose selection for resistance.
To investigate the interactive effects of pesticide resistance,
exposure, and infection, we exposed our evolved lines to Bacillus
thuringiensis (Bt), an entomopathogenic Gram-positive bacterium that
has been developed into a widely used biopesticide against many insect
species . While much of the research on host-Bt interactions focuses on
the lethal biocontrol aspects, natural strains also occur in the
environment and vary in lethality, thereby representing a selective
force within natural populations . Insect resistance to Bt commonly
results from changes in specific toxin-receptor interactions , providing
little expectation of cross-resistance with chemical pesticides
(Siegwart et al., 2015). However, Bt-resistance has been associated with
increased susceptibility to bacteria-derived pesticides , and exposure
to Bt has been shown to increase susceptibility to viruses and
entomopathogenic nematodes . The immune responses of susceptibleT. castaneum to oral Bt infection involve significant
upregulation of a suite of immune, stress, and developmental genes that
potentially overlap with the response to chemical pesticides and the
mechanisms of pesticide resistance. However, the mode of infection may
have important implications for interactions with pesticide resistance,
as T. castaneum exhibited contrasting patterns of expression of
infection-related genes dependent on the route of infection,i.e., oral compared to septic infection .
To explore the complex interactions between pesticide resistance and
exposure to pesticides and pathogens, we first investigated the main and
interactive effects of selection regime (pesticide resistant and
susceptible) and pesticide exposure on host fitness-associated
phenotypes after Bt infection. Having established these phenotypes, we
turned to transcriptional data to identify potential mechanisms that
could explain the observed phenotypes. We first asked whether evolution
regime, i.e. , evolved pesticide resistance, alone influences
constitutive gene expression and the transcriptional response to
infection in the absence of pesticides. We next included pesticide
exposure into the regime-by-infection interaction to investigate whether
pesticides facilitate or antagonize the host response to infection, and
whether differential gene expression depends on the experimental
evolution regime. For both investigations, we compared the results of OP
and Pyr treatments to determine whether evolved resistance or exposure
to pesticides with different physiological targets exert different
effects on host-pathogen interactions. Our study provides a
comprehensive window into the physiological and evolutionary processes
that shape interactions among two important ecological stressors.