**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** UNDERSTANDING THE DISPERSAL AND NON-TARGET IMPACT OF GENETICALLY ENGINEERED (GE) ORGANISMS AND THEIR BYPRODUCTS IS A CRITICAL COMPONENT OF THE SAFE AND RESPONSIBLE USE OF TRANSGENIC TECHNOLOGY IN THE ENVIRONMENT. HOWEVER, WE CURRENTLY LACK THE ABILITY TO TRACK THE MOVEMENT AND NON-TARGET EFFECTS OF GE ORGANISMS AND THEIR BYPRODUCTS IN THE ENVIRONMENT, EVEN THOUGH PREVIOUS WORK HAS DEMONSTRATED THAT GE ORGANISMS CAN ESCAPE, OR BE UNINTENTIONALLY TRANSPORTED, FROM THEIR INTENDED RANGE. FOR EXAMPLE, GE CROP BYPRODUCTS CAN DISPERSE OFF OF AGRICULTURAL FIELDS TO BE TRANSPORTED THROUGHOUT RIVER NETWORKS, AND THESE BYPRODUCTS CAN HAVE NON-TARGET EFFECTS ON AQUATIC ORGANISMS.AS ONE EXAMPLE, INSECT-RESISTANT GE MAIZE IS NOW PLANTED THROUGHOUT NORTH AMERICA AND GLOBALLY, WITH &GT;80% OF MAIZE IN THE US PLANTED IN BT OR STACKED VARIETIES. BT MAIZE IS ONE OF THE MORE COMMON INSECT-RESISTANT GE MAIZE VARIETIES, WHICH EXPRESSES THE INSECTICIDAL CRY1AB PROTEIN FROM BACILLUS THURINGIENSIS TO RESIST CROP DAMAGE BY THE EUROPEAN CORN BORER (OSTRINIA NUBILALIS), A MOTH IN THE FAMILY CRAMBIDAE (LEPIDOPTERA). THE CRY1AB PROTEIN IS EXPRESSED THROUGHOUT THE TISSUES OF BT MAIZE, AND AFTER MAIZE IS HARVESTED, THE PROTEIN REMAINS DETECTABLE IN DETRITAL MATERIAL FOR UP TO SEVEN MONTHS. THE POTENTIAL EFFECTS OF BT MAIZE DETRITUS ON NON-TARGET TERRESTRIAL ORGANISMS AND ECOSYSTEMS IS OF GREAT INTEREST TO STAKE HOLDERS, FEDERAL AGENCIES, AND THE GENERAL PUBLIC. IN ADDITION, BT MAIZE DETRITUS ENTERS, IS PROCESSED, AND CAN BE TRANSPORTED WITHIN STREAMS AND RIVERS FLOWING ADJACENT TO CROP FIELDS. LABORATORY TRIALS SUGGESTED THAT CONSUMPTION OF BT MAIZE DETRITUS MAY AFFECT STREAM-DWELLING INVERTEBRATES, BUT MAY BE DEPENDENT ON DURATION OF LEACHING OF SUBMERGED DETRITUS. THUS, THE POTENTIAL EFFECTS OF BT MAIZE ON AQUATIC ECOSYSTEMS HAVE ATTRACTED A GREAT DEAL OF ATTENTION, DESPITE ONLY A HANDFUL OF STUDIES EXPLORING THIS TOPIC.OUR MULTIDISCIPLINARY TEAM, WITH TRAINING IN EVOLUTIONARY BIOLOGY, ENTOMOLOGY, STREAM ECOLOGY, AND BIOGEOCHEMISTRY, HAS DEVELOPED A NOVEL APPROACH TO ADDRESS MULTIPLE PROGRAM AREAS IN THE BIOTECHNOLOGY RISK ASSESSMENT RESEARCH GRANTS PROGRAM (BRAG), INCLUDING METHODS TO MONITOR DISPERSAL OF GE-ORGANISMS (AREA #2) AND ASSESSING IMPACTS OF GE-ORGANISMS AND THEIR BYPRODUCTS ON NON-TARGET ORGANISMS (AREA #4). OUR NOVEL APPROACH USES EVOLUTION AS A MONITORING TOOL IN FRESHWATER ECOSYSTEMS.WE WILL APPLY THIS NOVEL MONITORING TOOL TO AN AQUATIC KEYSTONE SPECIES, THE CADDISFLY (ORDER TRICHOPTERA), AS OUR FOCAL ORGANISM, AND ITS RESPONSE TO THE CRY-PROTEINS PRODUCED BY BT-MAIZE THAT HAVE BEEN SHOWN TO LEACH INTO AQUATIC ENVIRONMENTS AS A PSEUDO-PERSISTENT CONTAMINANT. WE WILL MEASURE PREDICTED CHANGES ACROSS THE CADDISFLY GENOME IN RESPONSE TO THE NON-TARGET EFFECTS OF CRY-PROTEINS AS THE METRIC TO ASSESS THE INCURSION AND IMPACT OF GE-ORGANISMS AND THEIR BY-PRODUCTS IN NATURAL SYSTEMS. WE ARE CONFIDENT THAT THIS NOVEL APPROACH WILL ALLOW US TO ASSESS THE EFFECT OF CRY-PROTEINS IN FRESHWATER ECOSYSTEMS ACROSS THE AGRICULTURAL MIDWEST, UNIQUELY DISENTANGLING IT FROM OTHER ENVIRONMENTAL STRESSORS (E.G., HABITAT DEGRADATION, EUTROPHICATION), AND PIONEER A NOVEL METHOD FOR THE DETECTION OF GE-ORGANISMS AND THEIR IMPACT ON NATURAL SYSTEMS.PREVIOUS LAB EXPERIMENTS HAVE DEMONSTRATED THAT CRY-PROTEINS HAVE NON-TARGET EFFECTS ON CADDISFLIES, WHICH ARE CLOSELY RELATED TO THE CRY-PROTEINS' TARGETED PESTS FROM THE INSECT ORDER LEPIDOPTERA. SPECIFICALLY, THE TARGET ORGANISMS FOR CRY-PROTEINS ARE MOST OFTEN PEST CATERPILLARS IN THE INSECT ORDER LEPIDOPTERA, WHICH INCLUDES MOTHS AND BUTTERFLIES, BUT THE CLOSEST RELATIVE OR SISTER GROUP TO THESE TARGETS IS THE INSECT ORDER TRICHOPTERA, WHICH ENCOMPASSES THE AQUATIC KEYSTONE SPECIES, THE CADDISFLIES. THEREFORE, BASED ON THEIR EVOLUTIONARY RELATIONSHIPS, CRY-PROTEINS ARE PREDICTED TO ACT AS A SELECTIVE PRESSURE IN AGRICULTURALLY-IMPACTED FRESHWATERS, JUST AS IT HAS ACTED ON TERRESTRIAL AGRICULTURAL LEPIDOPTERAN PESTS. THIS EVOLUTIONARY LINKAGE HIGHLIGHTS THE GENERAL APPROACH OF THIS PROPOSAL USING EVOLUTION AS A MONITORING TOOL IN AN APPLIED ENVIRONMENTAL CONTEXT. HERE, BY INFORMING OUR APPROACH USING THE EVOLUTIONARY RELATIONSHIPS BETWEEN INTENDED TARGETS OF BT-CRY AND POTENTIAL NON-TARGET ORGANISMS, WE CAN ZERO IN ON THE SPECIES GROUPS OF GREATEST CONCERN.OUR OVERARCHING HYPOTHESIS IS THAT CRY-PROTEINS WILL GENERATE UNIQUE GENOMIC SIGNATURES OF SELECTION IN GENES ASSOCIATED WITH CRY-PROTEIN RESISTANCE IN AQUATIC CADDISFLIES JUST AS IT HAS DONE IN ITS CLOSELY RELATED TERRESTRIAL COUNTERPARTS AND THAT WE CAN USE THAT SIGNATURE ACROSS THE GENOME AS A TOOL FOR GE DETECTION IN NATURE. THUS, WE WILL USE AN EXPERIMENTAL GENOMICS APPROACH TO MONITOR THE POTENTIAL IMPACT OF CRY-PROTEINS ON KEYSTONE TAXA COMMON IN AGRICULTURAL STREAMS.