Review analyzes 25 years of studies on spinosyns

Since their introduction in 1997, spinosyn insecticides have transformed agricultural pest control. Spinosad and spinetoram act on the nervous system of insects. Both belong to Group 5 of the Insecticide Resistance Action Committee (IRAC) classification, characterized as allosteric modulators of the nicotinic acetylcholine receptor (nAChR), acting specifically at the so-called "Site I."

A recently published scientific review by Corteva scientists summarizes 25 years of research on the molecules' mode of action. It also highlights findings on their effectiveness and the emergence of resistance.

Origin and application of spinosyns

Spinosyns are products obtained from the fermentation of the microorganism Saccharopolyspora spinosa, originally isolated from soil samples in the Virgin Islands.

The semi-synthetic chemical modification of two of these molecules—spinosyns J and L—led to the development of spinetoram, which is more potent and stable under ultraviolet light. Spinosad and spinetoram have a broad spectrum of action against chewing insects of the orders Coleoptera, Diptera, Lepidoptera, and Thysanoptera.

Today, they are used in more than 250 agricultural crops across 130 countries. Their global acceptance is also due to their environmental profile and low toxicity to mammals, characteristics that earned both compounds Green Chemistry awards from the U.S. Environmental Protection Agency.

Neurophysiological effects and symptomatology

The action of spinosyns on insects begins with neural excitation, resulting in involuntary muscle contractions, leg extension, wing flapping, and loss of motor coordination. The compounds cause paralysis, even in decapitated insects, as demonstrated in American Periplaneta.

Unlike traditional insecticides, spinosyns induce symptoms that keep individuals on the leaves. This is relevant for integrated pest management programs, as it affects the dynamics of exposure to natural predators.

Molecular target: the alpha-6 subtype of the nAChR receptor

nAChRs are pentameric acetylcholine-activated ion channels. Spinosyns act as allosteric modulators of a specific combination of these receptors containing the alpha-6 subunit. This subunit has been shown to be highly conserved among different insect orders. Studies of loss-of-function (LOF) mutations in the alpha-6 subunit in Drosophila melanogaster revealed resistance greater than 100 times to spinosad, an effect replicated in other species such as Plutella xylostella e Frankliniella occidentalis.

The most common mutation in the field is the G275E substitution, located in the third transmembrane helix of the receptor. This modification confers resistance greater than 350-fold in populations of F. occidentalis and was validated via gene editing with CRISPR/Cas9.

Functional expression and technical challenges

To study the interactions of spinosyns with alpha-6 receptors, scientists employed heterologous expression systems using oocytes from Xenopus laevis. Receptor functionality required the coexpression of auxiliary proteins such as RIC3, UNC-50, and TMX-3. Even with these chaperones, response levels remain inconsistent, suggesting the existence of unknown cofactors.

The functional expression of a;fa-6 receptors in species such as Rhipicephalus microplus (bovine tick) and Apis mellifera was obtained without the need for exogenous chaperones.

Interestingly, R. microplus receptors exhibit a strong response to acetylcholine, with a modulatory action of spinosyns. And fast-desensitizing depolarization currents, contrasting with the persistent effects in Drosophila spp.

Specificity and absence of cross-resistance

Studies with nAChR mutants in D. melanogaster indicate that only the α6 subunit is directly involved in the action of spinosyns. Mutations in subunits such as alpha-1, beta-1, or alpha-7 do not confer resistance. Not even the simultaneous deletion of the alpha-5 and alpha-7 subunits, which share structural similarity with α6, alters the efficacy of spinosad.

Comparisons between different resistance mechanisms—point mutations such as G275E and LOF mutations—reveal similar effects in terms of resistance, which reinforces the functional importance of the α6 subunit as a single target.

Gene editing studies in agricultural pests

Besides D. melanogaster, gene editing studies have confirmed the essential function of alpha-6 in several agricultural pests. Knockouts of this subunit in Helicoverpa armigera, Spodoptera exigua, Aedes aegypti e P. xylostella conferred resistance between 320 and 1760 times greater. G275E mutations introduced into S. exigua replicated the resistance pattern.

Em F. occidentalis, experiments with simulated laboratory populations showed that the alpha-6 knockout allele disappears rapidly due to fitness costs, while the G275E allele persists. The loss of alpha-6 function, in addition to causing resistance, also affects visual functions in insects, interfering with synapses in the optical system.

Mode of action

The uniqueness of spinosyns is evident by the absence of cross-resistance with other insecticides that also act on nAChRs, such as neonicotinoids (group 4A), sulfoximines (4C), mesoionics (4E), and peptides such as ω-Hexatoxin-Hv1a (group 32). Radioligands confirmed that the binding site of spinosyns is distinct, with no overlap with other compounds.

Although an initial study suggested a secondary interaction with GABA receptors, subsequent analyses refuted this hypothesis. The resistance observed in the field and the molecular binding results demonstrate that spinosyns' action is concentrated exclusively on nAChR receptors with functional α6.

Recent research using intracellular calcium ionometers (GCaMP) and fluorescent proteins suggests that spinosad exposure induces selective degradation of the alpha-6 subunit, interfering with neuronal signaling balance. Although the exact mechanism remains unclear, these findings indicate that the impact of spinosyns goes beyond allosteric modulation.

Expanded use of genomic tools and improved heterologous expression systems may reveal new details about the effects of spinosyn binding to the alpha-6 receptor, including possible conformational interactions and implications for the channel's pentameric assembly. Obtaining the crystallographic structure of the alpha-6 subunit would be a crucial step toward understanding these interactions with atomic precision.

Further information at doi.org/10.1016/j.pestbp.2025.106575