DOES TOXIN SEQUESTRATION TRANSLATE TO PESTICIDE RESILIENCE IN LEPIDOPTERA? INSIGHTS FROM HEMOLYMPH PHYSIOLOGY AND FLIGHT BEHAVIOR

Growers regularly employ chemical compounds such as herbicides and insecticides to counter competing plants and insects that may reduce crop yield. Pesticides in agricultural environments can have non-target effects, including sub-lethal impacts on pollinators, potentially causing damage to insects and ecosystems. While pesticide use has clear and necessary benefits for crop production, exposure to pesticides has unknown and detrimental affects on insect populations, individuals, and colony-level behavior.Sublethal doses dramatically alter development and behavior (i.e. emergence and mating)in ways that remain largely unexplored.However in natural environments insects are also affected by toxins. Plants ward off insect pests with specialized defenses that can be chemical (i.e. latex and cardenolides produced by milkweed). In turn, highly adapted insects may be immune to some plant toxins or even sequester them for their own benefit. Toxic compounds produce downstream effects on an insect's physiology and how the insect interacts with its host plant or specific crop plant. In an effort to better understand the accumulations of pesticides and plant toxins in insects, this proposal works to link: 1) how insects functionally sequester toxins, 2) how natural and applied toxins affect insect physiology such as circulation, and 3) whether insects can be effectively monitored as sentinels in the field.More than 250 species of insects have been studied to sequester plant chemicals into their tissues, which in turn, deter predators, complementing aposematic or "warning" coloration. Of course sequestration likely occurs in hundreds of thousands of species; it is general phenomena. Well recognized for sequestering toxins is the monarch butterfly (Danaus plexippus) from its two primary host plants, the common and tropical milkweed (Asclepias syriaca and A. curassavica, respectively). Milkweeds, the only host plants for the monarch butterfly, produce cardenolides, steroidal compounds that are dynamically produced in the plant and sequestered by monarchs. Cardenolides are produced throughout milkweed tissues and when consumed affect the critical and universal cellular enzyme, the potassium-sodium pump (or Na-K-ATPase). Despite its toxicity, monarch butterflies have evolved to consume milkweed such that plant-produced cardenolides do not severely inhibit potassium-sodium pump found throughout the insect nervous system.Sequestering insects are specialists that can consume plant toxins at intermediate levels with little detrimental effect, while non-sequestering insects are typically only toxin-tolerant at low levels (although they may have other generalized mechanisms of protection, such as gut barriers). Monarch caterpillars may frequently consume a diversity of pesticides as they inhabit widespread areas in North America and very common in highly agriculturalized environments. In a recent study of milkweed patches near cropland in Indiana of over 1500 analyzed milkweed leaves, 14 pesticides were detected. The neonicotinoid clothianidin, a common agriculture insecticide, is the only pesticide for which toxicity are known and measured in monarchs. More studies are focusing on monarch development and how sub-lethal clothianidin affects development, transferring through soil and plants to the insect.Insects actively pump hemolymph into their wings, which may have implications for toxin sequestration in the wings. In monarchs, high sequestration of cardenolides occurs in the wings. Concentration of cardenolides in whole monarchs changes along the annual southern migration and is affected by the cardenolide content of milkweed species. How cardenolides accumulate in the wings is unknown, yet may be driven by pumping of hemolymph and active circulation in the wing veins. Flow in the wing veins is not passive, but actively pumped into and out of the wing, and likely plays an important role during flightand in opening the wings (for insects with folded wings). While many insects use a circuitous pattern (hemolymph flows in a loop in the wing), an undescribed pattern of unidirectional flow, suggests an evaporation-driven mechanism where hemolymph evaporates out of the veins and a semi-porous wing membrane.Insect wings are living structures and represent a nexus between environmental toxins that impact insect physiology and circulation of hemolymph. Wings also contain structures that need a continuous supply of hemolymph such as: sensory sensilla, mechanosensors, and tympanal organs on the wing. Nonetheless, we know little of how circulation (and thus downstream effects on behavior) can be disrupted by pesticides such as neonicotinoids. Loss of beneficial insects and pollinators has direct and negative effects on agricultural yields, human health, and food security.Given that few keystone plant genera support the majority of lepidopterans, understanding relationships between insects that are highly specialized to their host plant (monarch to milkweed) - and their physiology - is critical to their survival. It is particularly important to identify how sub-lethal effects that may occur via pesticide residues from croplands localize within insects. Further, work that ties both basic insect structures (i.e., insect body and appendages) with active physiology (circulation and respiration) under conditions of applied toxins is rarely done.The overarching goal of this proposed research is to identify the localized effects of toxicity on key pollinators. Whether cardenolide sequestration confers a benefit to insects by also sequestering pesticides (Aim 1 and 2) will give critical information on how insects in natural and croplands are dealing are processing toxins (Aim 3). The following experiments focus on two closely related pairs of Lepidoptera that all feed on toxic milkweed, where one in the pair sequesters cardenolides and the other does not. These relationships, during experiments, will establish strong and replicated comparisons to understand the distribution of both natural and applied toxins on insect systems. This will be accomplished by extensive lepidopteran rearing experiments combined with controlled application to the insects of host plant toxin (cardenolides) and a commonly encountered neonicotinoid (clothianidin) along with subsequent sampling of where the toxins localized (Aim 1). Localization of toxins depends on a suite of factors, but a major driver is the circulatory system. By examining insect circulatory flows, with a focus on the wing, and paired with sampling, this research will determine if sequestration occurs in the wings for both cardenolides and clothianidin (Aim 2). The subsequent impact of the circulatory system as a driver for sequestration of toxins (Aim 1 and 2) indicates that broad sampling of insects in diverse environments could signal the healthiness of that ecosystem. As part of Aim 3, collecting, sampling and testing insects and their host plants for pesticides in natural and farmed areas will elucidate the relationship between physiology, toxins, and how applied agriculture practices feed back into this plant-insect interaction.This interdisciplinary work relies on fundamental, yet understudied physiology, activity and robustness of the insect circulatory system, and how insects localize toxins that enter their bodies. By synthesizing insect physiology with its biological flows and how toxins enter the plant-insect system, has implications for insect health in natural and managed systems.