United States Environmental Protection Agency

Ecological Risk Assessment for the Proposed New Chemical Registration of GS-U-ACTX-Hv1a-SEQ2 (Versutide™ peptide) on Vegetables and Cole Crops; Herbs, Spices, and Mints; Pasture and Hay Crops; Fruit, Nut, and Vine Crops; Field Crops; Commercial Flowers and Ornamental Plants; Forest, Shade Tree, and Nursery Stock; and Turf

ASSOCIATED BARCODE: D411507

By Meghan Radtke, Ph.D., Biologist

Peer Reviewers Faruque Khan, Ph.D., Senior Scientist Edward Odenkirchen, Ph.D., Senior Scientist Sujatha Sankula, Ph.D., Lead Biologist

Acting Branch Chief Edward Odenkirchen, Ph.D.

Date of Approval May 30, 2013

1. PROBLEM FORMULATION

1.1 Nature of the Registration Action

This environmental risk assessment evaluates the potential ecological risks of the new broad spectrum insecticide, GS-U-ACTX-Hv1a-SEQ2 (Versutide™ peptide). The insecticide is proposed for use on a number of agricultural and home/garden uses; general categories include: vegetable and cole crops; herbs, spices, and mints; fruit, nut, and vine crops; field crops; commercial flowers and ornamental plants; forest, shade tree, and nursery stock; and turf. The insecticide is listed as being effective to control numerous insects including: alfalfa looper, army worm, Colorado potato beetle larvae, hornworms, spotted cucumber beetle, tobacco budworm, European corn borer, cankerworm, western tent caterpillar, cutworms, corn earworm, and gypsy moth. Two formulations (20% and 30% ai) are being registered. The proposed maximum single application rate is 0.8 lb ai/A with a maximum yearly application rate (assumed) of 0.8 lb ai/A. The products are foliar sprays that are applied via ground or aerial application equipment.

1.2 Nature of the Chemical Stressor

GS-U-ACTX-Hv1a-SEQ2 is a peptide that is derived from the venom of a species of Australian funnel spider (Hadronyche versuta). Specifically, it is derived from the u-ACTX-HV1 peptide, which represents a small fraction of the funnel spider’s venom (< 0.5% by dry weight); there are only two amino acid differences between the u-ACTX-HV1 peptide and GS-U-ACTX-Hv1a- SEQ2. The protein structure consists of a solvent-accessible beta-hairpin protruding from a disulphide-bonded globular core comprising four beta-turns. The three intramolecular disulphide bonds form a cystine knot motif similar to that seen in several other neurotoxic peptides. The u- ACTX-HV1 peptide acts by inhibiting insect voltage-gated calcium channel currents (Fletcher et al. 1997) and by inhibiting the calcium activated potassium channels (Gunning et al., 2008). GS-U-ACTX-Hv1a-SEQ2 is mass produced via transgenic yeast.

1.3 Use Characterization

GS-U-ACTX-Hv1a-SEQ2 is proposed for registration on a wide variety of uses.

Vegetable and Cole Crops: artichoke, arugula, asparagus, beans, beets, bok choy, broccoli, broccoli raab, Brussels sprouts, cabbage, cardoni, carrots, cassava, cauliflower, cavalo, celeriac, celery, chayote root, chervil, chick peas, chicory, China greens, Chinese artichoke, Chinese broccoli, Chinese cabbage (napa), Chinese mustard cabbage, collards, corn salad, cress, cucumber, cucurbits, dandelion dock, dry bulb onions, edible burdock, edible chrysanthemum, edible gourds, eggplant, escarole, endive, garlic, ginger, ginseng, green onions, greens (beet, China, dandelion, mustard, turnip, rape), ground cherry, horseradish, Jerusalem artichoke, kale, kohlrabi, leeks, lentils, lettuce (head, leaf, romaine), lupine, malanga, melons, mizuna, mustard spinach, okra, olives, onions, parsley, parsnips, peas, pepino, peppers, potatoes, pumpkins, purslane, radicchio, radishes, rhubarb, rutabaga, salsify, shallots, skirret, soybean foliage, soybean, spinach, squash, sugar beets, sweet potatoes, swiss chard, tomatillo, tomatoes, turmeric, turnips, turnip-rooted chervil, turnip-rooted parsley, watercress, yams

Herbs, Spices, and Mints: angelica, balm, basil, borage, burnet, catnip, chamomile, chervil, chives, cilantro, clary, coriander, costmary, curry, dill, dill weed, fennel, horehound, hyssop, lavender, lemongrass, lovage, marjoram, mint, nasturtium, oregano, parsley, peppermint, rosemary, sage, savory, spearmint, sweet bay, tansy, tarragon, thyme, wintergreen, woodruff, wormwood

Pasture and Hay Crops: alfalfa (hay and seed), hay and other forage crops, pasture (grasses and hay), silage

Pome and Stone Fruit Trees: apples, apricots, crabapple, cherries, figs, loquat, mayhaw, nectarines, peaches, pears, plums, prunes, quince

Nut Trees: almonds, cashew, chestnuts, filberts, macadamia nut, pecans, pistachios, walnuts

Tree Fruits: acerola, atemoya, biriba, black sapote, canistel, cherimoya, custard apple, feijoa, guava, llama, jaboticaba, kiwi, longan, lychee, mamey sapote, mango, papaya, passion fruit, pulasan, rambutan, sapodilla, soursop, Spanish lime, star apple, starfruit, sugar apple, ti palm, wax jambu, white sapote

Citrus: grapefruit, lemons, limes, organs, tangerines

Small Fruit and Berries: blackberries, blueberries, boysenberries, cranberries, currants, elderberry, gooseberry, huckleberry, juneberry, ligonberry, loganberries, raspberries, salal, strawberries, grapes

Tropical and Other Fruit: bananas, kiwi, persimmon, pomegranate, pineapple

Tropical/Sub-Tropical Fruits: avocado, guava, lychee, sugar apple

Field Crops: canola/rape seed, evening primrose, meadow foam, corn (field, sweet, popcorn, seed), cotton, hops, jojoba, peanuts, rice safflower

Small Grains: barley, buckwheat, grain amaranth, millet, milo, oats, pearl, proso millet, rye, triticale, wheat, sorghum, soybeans, sunflower (oil and seed), tobacco, coffee

Commercial Flowers and Ornamental Plants: bedding plants, flowers (greenhouse and field), greenhouse ornamentals, greenhouse vegetables, container stock

Forest, Shade Tree, and Nursery Stock: deciduous, forest, shade trees, nursery trees, ornamental trees, conifers (including Christmas trees)

Turf: turf and turf grown for seed

According to the label, for all uses, a single application can range from 0.025 to 0.8 lb ai/A; the single maximum application rate is 0.8 lb ai/A. The target pests include: achema sphinx moth, alfalfa caterpillar, alfalfa looper, alfalfa webworm, amorbia, armyworm, artichoke plume moth,

asparagus beetle, avocado leaf roller, azalea caterpillar, azalea moth, bagworm, banana skipper, banana moth, banded sunflower moth, beet armyworm, bertha armyworm, blackheaded budworm, blackheaded fireworm, blueberry leaf roller, bollworm, browntail moth, cabbage budworm, cabbage looper, cabbage webworm, California oakworm, cankerworm, celery leaftier, cereal leaf beetle, cherry fruitworm, citrus cutworm, citrus peelminer, codling moth, Colorado potato beetle larvae, corn earworm, corn rootworm, cotton leaf perforator, cottonwood leaf beetle, cranberry fruitworm, cranberry girdler, cross-stripped cabbageworm, cutworms, diamondback moth, douglas fir tussock moth, eastern tent caterpillar, ello moth, elm spanworm, European corn borer, European grapevine moth, European skipper, fall webworm, filbert leaf roller, filbert webworm, fireworm, Florida fern caterpillar, fruit tree leaf roller, grape berry moth, grape leaffolder, grapeleaf skeletonizer, green cloverworm, green fruitworm, greenstriped mapleworm, gummosos-batrachedra commosae, gypsy moth, headworm, heliothis, hemlock looper, hickory shuckworm, hornworm, imported cabbageworm, jack pine budworm, leaf rollers, light brown apple moth, lo moth, looper, melonworm, mimosa webworm, navel orangeworm, oblique banded leaf roller, oleander moth, omniforous leaftier, omnivorous leaf roller, orange tortix, orangedog, oriental fruit moth, pandemic leaf roller, peach twig borer, pecan nut casebearer, pickleworm, pine butterfly, pine tip moths, plum curculio, podworm, red-neck peanut worm, redbanded leaf roller, redhumped caterpillar, rindworm complex, roughskinned cutworm, saddleback caterpillar, saddle prominent caterpillar, saltmarsh caterpillar, silverleaf whitefly nymphs, sod webworm, southwestern corn border, soybean looper, spanworm, sparganothis, spotted asparagus beetle, spotted cucumber beetle, spotted cutworm, spring and fall cankerworm, spruce budworm, striped cucumber beetle, sunflower moth, tent caterpillar, thecla- thecla basilides, tobacco budworm, tobacco hornworm, tomato fruitworm, tomato hornworm, tomato pinworm, tortix, tortix moth, tropical sod webworm, tufted apple budmoth, tussock moth, twig borer, variegated cutworm, variegated leaf roller, velvetbean caterpillar, viburnam beetle, walnut caterpillar, webworm, western bean cutworm, western corn cutworm, western raspberry fruitworm, western tent caterpillar, western tussock moth, yellow margined leaf beetle larvae, and yellowstriped armyworm.

The label does not specify a yearly maximum application rate. Therefore, it is assumed that 0.8 lb ai/A is the maximum annual application rate for GS-U-ACTX-Hv1a-SEQ2. For single application rates below 0.8 lb ai/A, the application interval is 3 to 10 days.

1.5 Analysis Plan

The screening-level risk assessment is used as the approach to determine risk to non-target organisms from the proposed new uses of GS-U-ACTX-Hv1a-SEQ2. Given that the toxicity data for this chemical are non-definitive and that limited fate data are provided, the assessment will qualitatively analyze the anticipated effects of GS-U-ACTX-Hv1a-SEQ2 on non-target organisms in the environment. The qualitative analysis is done by comparing the estimated environmental concentrations (EECs) with the toxicity value generated during testing. The environmental fate of proteins will also be explored.

2. ENVIRONMENTAL FATE CHARACTERIZATION

Limited fate data were submitted in support of the registration of GS-U-ACTX-Hv1a-SEQ2. The active ingredient is a peptide derived from one of the toxicologically active components of

Australian funnel spider venom (Figure 1). Its molecular weight is 4565 kDa and the pH is 4.89- 5.95 (MRIDs 48779002 and 48779101). It is a large molecule that is made up of a chain of 41 amino acids. Its surface is covered in hydrophilic residues, making it highly soluble in water, but insoluble in ethanol (specific solubility data were not submitted). The formulation contains paracellular transport mechanisms (derived from inert ingredients on EPA’s list) to facilitate the translocation of the peptide from the gut into the insect’s hemocoel and ultimately the nervous system. Upon entering the environment, GS-U-ACTX-Hv1a-SEQ2 is readily biodegradable and breaks down into its amino acid components. Its conformation (quaternary structure) determines its toxicity; thus, even a minor change to its structure may render it non-toxic. Its terrestrial half- life is approximately 4 days, extrapolated from simulated sunlight exposures in terrestrial settings where degradation occurred after 48-hours (MRIDs 48779002 and 48779003).

Figure 1. Richardson schematic of calculated 3-D structures of u-ACTX-Hv1a peptide from Protein Data Bank number 2H1Z (MRID 48779001)

The following required fate guideline studies, as stipulated by the Code of Federal Regulation, are not available and are consequently identified as data gaps:

• 835.2129 (hydrolysis) • 835.2240 (photodegradation in water) • 835.2410 (photodegradation in soil) • 835.2370 (photodegradation in air – conditional, depending on chemical’s properties) • 835.4100 (aerobic soil metabolism) • 835.4200 (anaerobic soil metabolism) • 835.4300 (aerobic aquatic metabolism) • 835.4400 (anaerobic aquatic metabolism) • 835.1230/835.1240 (leaching adsorption/desorption) • 835.6100 (terrestrial field dissipation) • 835.1410 (volatility laboratory – conditional, depending on chemical’s properties)

3. EXPOSURE ESTIMATION

3.2 Aquatic Exposure Modeling

Given the limited amount of data available for this assessment, a simplified approach will be taken to estimate environmental exposure. The standard pond used in the Tier I GENEEC or Tier II the Pesticide Root Zone Model, (PRZM, Carousel et al. 1997) and the Exposure Analysis Modeling System (EXAMS, Burns 2000) is 1 ha in size and contains 20,000,000 L of water. Rather than using the models to calculate an estimated environmental concentration (EEC), the EEC is calculated by dividing the amount of GS-U-ACTX-Hv1a-SEQ2 that is applied within a 1 ha area by the 20,000,000 L contained in the pond. The application rate is 0.8 lb ai/A. This is converted to mg/ha by:

(0.8 lb/A)(362874 mg/lb)(1 A/0.404686 ha) = 896,680 mg/ha

Dividing 896,680 mg by 20,000,000 L yields 0.044 mg/L, which is the concentration of GS-U- ACTX-Hv1a-SEQ2 that is expected in the standard pond. This represents the peak concentration in the pond; concentrations over longer periods of time are expected to be much lower.

This approach is considered conservative because the pesticide is being directly applied to the water rather than arriving in the pond via runoff. Furthermore, the pond does not have any inputs or outputs that would allow for exchange of water.

3.3 Terrestrial Exposure

Terrestrial wildlife exposure estimates are typically calculated for birds and mammals emphasizing a dietary exposure route for uptake of pesticide residues on vegetative matter and insects. Terrestrial wildlife exposure estimates are calculated with T-REX v1.5.1 model (August 20, 2012). The residues, or estimated environmental concentrations (EECs), on food items are compared with sub-acute dietary toxicity data, acute oral data, and chronic dietary or oral toxicity data. Foliar application exposures are estimated for birds in Table 1 and mammals in Table 2. In this case, a single application rate of 0.8 lb ai/A was modeled. The risk assessment uses a default foliar dissipation half-life estimate of 35 days. This default value is used in lieu of representative foliar dissipation data for GS-U-ACTX-Hv1a-SEQ2, because no suitable data were provided that evaluated foliar dissipation (although a 4-day terrestrial half-life is provided, it is unclear if it is a foliar dissipation half-life or if it is for another medium).

Table 1. Avian EECs for the Proposed GS-U-ACTX-Hv1a-SEQ2 Uses Dietary-based Dose-based EECs (mg/kg-bw) Feeding Category EECs (mg/kg- Small Medium Large food item) (20 g) (100 g) (1000 g) All Uses 1 app at 0.8 lb ai/A Short grass 192.00 218.67 124.69 55.83 Tall grass 88.00 100.22 57.15 25.59 Broadleaf plants 108.00 123.00 70.14 31.40 Fruits/pods 12.00 13.67 7.79 3.49 Arthropods 75.20 85.65 48.84 21.87 Seeds - 3.04 1.73 0.78

Table 2. Mammal EECs for the Proposed GS-U-ACTX-Hv1a-SEQ2 Uses Dietary-based Dose-based EECs (mg/kg-bw) Feeding Category EECs (mg/kg- Small Medium Large food item) (15 g) (135 g) (1000 g) All Uses 1 app at 0.8 lb ai/A Short grass 192.00 183.06 126.52 29.33 Tall grass 88.00 83.90 57.99 13.44 Broadleaf plants 108.00 102.97 71.17 16.50 Fruits/pods 12.00 11.44 7.91 1.83 Arthropods 75.20 71.70 49.55 11.49 Seeds - 2.54 1.76 0.41

For insects, only two EECs are calculated. The tall grass EEC represents the exposure through dietary intake whereas the arthropod EEC represents exposure through contact (Table 3) (see USEPA 2012 for further details about the EECs chosen to represent honeybee exposures). Given that honeybee larval toxicity data were not available, only GS-U-ACTX-Hv1a-SEQ2 doses for adult bees are considered.

Table 3. Adult Insect EECs for the Proposed GS-U-ACTX-Hv1a-SEQ2 Uses EEC based on EEC based on tall grass arthropod Use mg/kg-diet mg/kg-diet (dose in µg ai/bee*) (dose in µg ai/bee**)1 All uses (1 app at 0.8 ai/A) 88.00 (25.7) 75.20 (9.6) *Conversion made by multiplying EEC by food consumption rate of adult bee (0.292 g/day) **Conversion made by multiplying EEC by body weight of adult bee (0.128 g)

Terrestrial and semi-aquatic plants may be exposed to pesticides from runoff and spray drift. Semi-aquatic plants are those that inhabit low-lying wet areas that may be dry at certain times of the year. Given that the solubility of GS-U-ACTX-Hv1a-SEQ2 is not available, TerrPlant (v.1.2.1) cannot be used to calculate EECs for terrestrial plants. In lieu of this, the risks to plants

7 will be qualitatively assessed using toxicity information from the vegetative vigor and seedling emergence limit tests.

4. ECOLOGICAL EFFECTS CHARACTERIZATION

Ecotoxicity data are limited to acute mammal, acute avian oral, acute freshwater fish, acute freshwater invertebrate, acute honeybee contact, terrestrial plant seedling emergence, and terrestrial plant vegetative vigor for GS-U-ACTX-Hv1a-SEQ2. According to the Federal Code of Regulations, a number of studies are missing and are considered data gaps. Below is a list of the studies and a description of the assumptions that were made in this risk assessment in the absence of data. Filling the data gaps would reduce the uncertainty associated with the assumptions that were made.

• 850.1075 (acute estuarine/marine fish toxicity) – acute freshwater fish toxicity data were used in lieu of estuarine/marine fish data. • 850.1025/850.1035 (acute estuarine/marine invertebrate toxicity) – acute freshwater invertebrate data were used in lieu of estuarine/marine invertebrate data. • 850.1300 (freshwater invertebrate life cycle test) – exposure was presumed to be negligible because the peptide is expected to immediately break down in aquatic environments, thus, long-term exposures are unlikely. • 850.1350 (estuarine/marine invertebrate life cycle test) – exposure was presumed to be negligible because the peptide is expected to immediately break down in aquatic environments, thus, long-term exposures are unlikely. • 850.1400 (freshwater fish early life stage test) – exposure was presumed to be negligible because the peptide is expected to immediately break down in aquatic environments, thus, long-term exposures are unlikely. • 850.1400 (estuarine/marine fish early life stage test) – exposure was presumed to be negligible because the peptide is expected to immediately break down in aquatic environments, thus, long-term exposures are unlikely. • 850.2100 (acute avian oral toxicity with mallard and passerine species) – acute oral data from the bobwhite quail was used as a surrogate for the mallard and passerine species. • 850.2200 (acute avian dietary toxicity) – acute oral data from the bobwhite quail was used as a surrogate. • 850.2300 (avian reproduction toxicity) – information regarding the lack of toxicity to vertebrates was used to support the assessment. • 870.3800 (mammalian reproduction toxicity) – information regarding the lack of toxicity to vertebrates was used to support the assessment. • 850.4100 (seedling emergence) – reliable information was not available for soybean. Data from the other tested dicots were used as surrogates. • 850.4150 (vegetative vigor) – reliable information was not available for soybean. Data from the other tested dicots were used as surrogates. • 850.4400 (aquatic plant growth – vascular plants) – lack of effects in terrestrial plants and presumed negligible exposure because the peptide is expected to immediately break down in aquatic environments were used to support the assessment.

• 850.4500 (aquatic plant growth – non-vascular plants) – lack of effects in terrestrial plants and presumed negligible exposure because the peptide is expected to immediately break down in aquatic environments were used to support the assessment.

In addition, a honeybee oral toxicity study (non-guideline, OECD 213) and larval toxicity (non- guideline) would also be requested to evaluate toxicity to honeybees and non-listed terrestrial invertebrates. In the absence of these data, risks are assumed to honeybees because the pesticide is designated as an insecticide.

Aquatic Organisms Acute data for freshwater fish and invertebrates are available (Table 4). For the freshwater fish study, a limit test (100 mg ai/L) was performed with rainbow trout. The treatment group exhibited 10% mortality; however, this is considered normal background mortality (USEPA 1996a). No sub-lethal effects were observed and the LC50 was determined to be above the 100 mg ai/L concentration.

The acute freshwater invertebrate limit test was performed with the waterflea. Waterfleas were exposed to a single concentration of 1000 mg ai/L. Mortality (3 to 7 %) occurred in both the control and treatment groups, but it was at rates consistent with normal background mortality (USEPA 1996b). No sub-lethal effects were observed and the EC50 was determined to be above the 1000 mg ai/L concentration.

One uncertainty for both the fish and aquatic invertebrate studies is the stability of GS-U-ACTX- Hv1a-SEQ2 in water. Understanding the stability of a compound is important because it directly affects the exposure concentration in the test. The half-life of the peptide in terrestrial habitats is approximately four days. No hydrolysis data have been submitted, thus the concentration of GS- U-ACTX-Hv1a-SEQ2 over the duration of the aquatic tests is unknown; it is possible that the degradation occurred very rapidly. It is assumed that the acute exposures represent real-life exposure scenarios, regardless of the degradation rate. It is unimportant whether the peptide degraded quickly upon entering the aquatic environment or persisted through the experiment. No effects occurred within the test systems; however, the fish and invertebrate studies are classified as “supplemental” because of the chemical stability uncertainty.

Acute and chronic data are not available for estuarine/marine fish or invertebrates. Likewise, chronic data are not available for freshwater and estuarine/marine fish or aquatic invertebrates. Data for aquatic vascular and non-vascular plants also are not available.

Table 4. Acute Toxicity Effects of GS-U-ACTX-Hv1a-SEQ2 on Aquatic Organisms Species Study A.I. Toxicity Endpoint Study and MRID # Guideline Toxicity Classification

Rainbow Trout 850.1075 31.5 % LC50 > 100 mg ai/A Supplemental 48779014 (Oncorhynchus NOAEC = 100 mg ai/L mykiss) Practically No sub-lethal effects non-toxic Waterflea 850.1010 31.5 % EC50 > 1000 mg ai/L Supplemental 48779015 (Daphnia NOAEC = 1000 mg ai/L magna) Practically No sub-lethal effects non-toxic

Terrestrial Organisms Acute oral toxicity data are available for birds and mammals (Table 5). A limit test was conducted by exposing the bobwhite quail via oral gavage to concentrations of 630 mg ai/kg-bw. Ten control birds were tested and ten birds were exposed to GS-U-ACTX-Hv1a-SEQ2. No mortalities or sub-lethal effects were observed, thus, the LD50 is > 630 mg ai/kg-bw. Acute dietary data are not available for birds. Chronic/reproduction data also are not available.

Similarly, an acute oral toxicity study (up/down procedure) was conducted on three rats. Initially, one female rat was administered an oral dose of 5000 mg ai/kg-bw. When mortality did not occur and no sub-lethal effects (including changes to behavior and decreases in body weight) were observed, an additional two rats were dosed with the same concentration. The experiment was terminated when none of the three rats showed any signs of toxicity over a two week period. The resulting LD50 is > 5000 mg ai/kg-bw. A chronic two-generation reproduction study with rats is not available.

An acute honeybee contact toxicity test was available for GS-U-ACTX-Hv1a-SEQ2 (Table 5). Sixty bees were exposed to a concentration of 25 µg ai/bee and 60 bees were exposed to water (controls) via a droplet of solution to the abdomen (contact exposure). Mortality rates for each group were at or below 8% and no sub-lethal effects were observed. Consequently, the contact LD50 for honeybees is > 25 µg ai/bee. Acute oral and larval data are not available for honeybees.

Table 5. Acute Toxicity Effects of GS-U-ACTX-Hv1a-SEQ2 on Terrestrial Animals Species Study A.I. Toxicity Endpoint Study and MRID # Guideline Toxicity Classification

Bobwhite quail 850.2100 31.5% LD50 > 630 mg ai/kg-bw Supplemental 48779013 (Colinus NOAEC = 630 mg ai/kg-bw virginianus) Slightly toxic No effects Rat (Rattus 870.1100 31.5% LD50 > 5000 mg ai/kg-bw Acceptable 48779007 norvegicus) NOAEC = 5000 mg ai/kg-bw Practically non-toxic Honeybee (Apis 850.3020 31.5% Contact LD50 > 25 µg ai/bee Supplemental 48779018 mellifera) NOAEC = 25 µg ai/bee Practically No sub-lethal effects non-toxic

Terrestrial plant data were available for GS-U-ACTX-Hv1a-SEQ2 (Table 6). Seedling emergence and vegetative vigor tests were conducted at application rates of 0.8 lb ai/A. In both studies, the emergence rate for soybean was below 70%, indicating a potential problem with the seed stock. Consequently, data for this species are not considered reliable and are not included in the risk assessment. In the seedling emergence test, none of the species in the treatment group showed reductions in emergence, diameter, length, or dry weight that were greater than 25%. Therefore, the EC25 is > 0.8 lb ai/A for seedling emergence. In the vegetative vigor study, onion and lettuce showed significant decreases (greater than 25% in root weight); however these differences did not translate into above-ground biomass. Root weight is generally considered an unreliable endpoint because of the difficulty in obtaining accurate measurements. Effects of 25% or more were not seen for plant height, diameter or dry length; thus, the EC25 is > 0.8 lb ai/A for vegetative vigor.

Table 6. Toxicity Effects of GS-U-ACTX-Hv1a-SEQ2 on Terrestrial Plants Species Study A.I. Toxicity Endpoint Study MRID # Guideline Classification

Monocots Seedling 31.5 % EC25 > 0.8 lb ai/A Supplemental 48779016 Oat (Avena sativa) emergence NOAEC = 0.8 lb ai/A Ryegrass (Lolium perenne) Onion (Allium cepa) 850.4100 No effects Corn (Zea mays)

Dicots Tomato (Lycopersicon esculentum) Cucumber (Cucumis sativus) Lettuce (Lactuca sativa) Soybean (Glycine max) Cabbage (Brassica oleracea) Carrot (Daucus carota) Monocots Vegetative 31.5 % EC25 > 0.8 lb ai/A Supplemental 48779017 Oat (Avena sativa) vigor NOAEC = 0.8 lb ai/A Ryegrass (Lolium perenne) Onion (Allium cepa) 850.4150 >25% root weight Corn (Zea mays) decrease in onion and lettuce that was not Dicots considered reliable Tomato (Lycopersicon esculentum) Cucumber (Cucumis sativus) Lettuce (Lactuca sativa) Soybean (Glycine max) Cabbage (Brassica oleracea) Carrot (Daucus carota)

Incident Database Review

Searches of the Ecological Incident Information System (EIIS v. 2.1.1), Avian Incident Monitoring System (AIMS), and the Incident Database System (IDS) were performed on May 2,

2013. No ecological incidents were reported in any of the databases. This is expected given that GS-U-ACTX-Hv1a-SEQ2 is a new chemical.

5. RISK DESCRIPTION

Results of the exposure modeling and toxicity studies are used to evaluate the likelihood of adverse ecological effects on non-target species. For GS-U-ACTX-Hv1a-SEQ2, this will be a qualitative assessment because of the limited fate and toxicity data.

5.1.1 Risk to Aquatic Organisms

A peak concentration of GS-U-ACTX-Hv1a-SEQ2 was generated by calculating the amount of active ingredient distributed per liter within the standard PRZM/EXAMS pond. This EEC (0.044 mg ai/L) was compared directly to the toxicity values generated in the registrant- submitted studies.

Acute Risks to Fish and Invertebrates The LC50 for freshwater fish is > 100 mg ai/L. Compared to the peak EEC of 0.044 mg ai/L, the LC50 is at least four orders of magnitude higher. This indicates that risk concerns to freshwater fish on an acute exposure basis are unlikely. Acute toxicity data were not available for estuarine/marine fish. In lieu of this, freshwater fish are used as a surrogate. This adds some uncertainty to the analysis; however, given that no effects (including sub-lethal) were seen in freshwater fish and the extremely large difference between the LC50 value and the peak EEC, it is unlikely that estuarine/marine fish would be markedly different in their sensitivity to GS-U- ACTX-Hv1a-SEQ2. Consequently, estuarine/marine fish are not expected to be at risk on an acute exposure basis.

The EC50 for freshwater invertebrates is >1000 mg ai/L. Compared to the peak EEC of 0.044 mg ai/L, the EC50 is at least five orders of magnitude larger. Therefore, acute risk concerns to freshwater invertebrates are unlikely. Acute toxicity data were not available for estuarine/marine invertebrates, thus, the freshwater invertebrate data were used as a surrogate. For the same reasons identified for fish, the freshwater invertebrate data are considered a reasonably protective surrogate for estuarine/marine invertebrates. Based on this, acute risk concerns are not expected for estuarine/marine invertebrates.

Chronic Risks to Fish and Invertebrates Toxicity data were not available for chronic exposures to fish or invertebrates. One of the uncertainties in the acute aquatic studies was the concern that GS-U-ACTX-Hv1a-SEQ2 degraded very quickly and that the actual exposure experienced by the test organism was far lower than the nominal concentration of active ingredient. Hydrolysis data for the peptide are not available, but it is likely that degradation rapidly occurs in water. The quaternary structure (protein folding) of GS-U-ACTX-Hv1a-SEQ2 is very complex; conditions that even slightly alter the protein’s configuration (e.g., exposure to water) will interfere with its receptor binding. Terrestrial data indicate that the half-life of GS-U-ACTX-Hv1a-SEQ2 is only 4 days. If a similar or smaller half-life occurs in water, aquatic organisms would not reasonably be expected to experience chronic exposure conditions. Therefore, risk concerns from chronic exposures are considered unlikely.

Aquatic Plants Toxicity data were not available for aquatic vascular and non-vascular plants. At an application rate of 0.8 lb ai/A, terrestrial plants showed no effects. As with the aquatic invertebrate and fish analysis, if the 0.8 lb ai/A were sprayed directly onto a 1 ha 20,000,000 L pond, the concentration is expected to be 0.044 mg ai/L. Exposures to fish (100 mg ai/L) and aquatic invertebrates (1000 mg ai/L) at much higher rates that 0.044 mg ai/L also showed no effects. Given the specificity of GS-U-ACTX-Hv1a-SEQ2 to insects, the low probability that aquatic plants have this same receptor, and the peptide’s predicted instability in water, risk concerns for aquatic plants are not expected. To confirm this conclusion, toxicity data for aquatic vascular and non-vascular plants could be requested (850.4400 and 850.4500).

5.1.2 Risk to Terrestrial Organisms For terrestrial animals, EECs were generated using T-REX. These were then compared to the non-definitive toxicity values to characterize risks. EECs for GS-U-ACTX-Hv1a-SEQ2 exposure to terrestrial plants could not be generated because solubility data for the protein were not available. Consequently, the discussion of risks to terrestrial plants is based strictly on the toxicity information from the seedling emergence and vegetative vigor studies.

In addition to the Agency guideline toxicity studies, the registrant submitted several studies with detailed information about the specificity of the GS-U-ACTX-Hv1a-SEQ2 to insect receptors versus bird/mammal receptors. This information is useful to further characterize the absence of effects in the acute avian and mammal studies as well as to define potential chronic effects.

The GS-U-ACTX-Hv1a-SEQ2 is closely related to the omega peptide of the same form (50% homology in structure) and exhibits a similar folding pattern (quaternary structure); it also shares homology with the kappa peptide (κ-ACTX-Hv1c). It has been documented to share activity on a molecular level with these two other peptides and toxicity information from the omega and kappa peptides are considered appropriate surrogates (MRID 48779019). In vitro studies indicate that the omega peptide blocks insect calcium channels and affects the ganglionic neurotransmission, but does not inhibit mammalian neuronal calcium channels (Fletcher et al., 1997). A homolog of the omega peptide with similar insect calcium channel activity was also tested in vertebrates using rat and chick smooth and skeletal nerve-muscle, respectively. No effects were observed (Chong et al., 2007). The kappa group of peptides, which shares the calcium activated potassium channel inhibition mode of action with GS-U-ACTX-Hv1a-SEQ2, also is insect specific and is believed to exhibit little to no activity at rat dorsal root ganglion neuronal calcium-activated potassium channels (Gunning et al., 2008). Sollod (2006) demonstrated insect specificity with hybrid peptide compounds – similar to GS-U-ACTX-Hv1a- SEQ2.

Acute Risks to Birds and Mammals The highest acute dose-based EEC generated for birds is 219 mg ai/kg-bw (20 g bird eating short grass). The LD50 of > 630 mg ai/kg-bw, when compared with the EEC, is three times higher. Thus, acute risks to birds are not expected. Similarly, the acute dose-based EEC for mammals is 183 mg ai/kg-bw (15 g mammal eating short grass). The corresponding LD50 is > 5000 mg

ai/kg-bw, which is one order of magnitude higher than the EEC. Therefore, acute risk concerns for mammals are not expected.

Chronic Risks to Birds and Mammals Toxicity data were not available for chronic exposures to birds and mammals. The bioavailability of GS-U-ACTX-Hv1a-SEQ2 is expected to be low, based on the physical structure of the peptide. Spider venoms are designed to be injected into their prey. Exposure through oral means is likely to drastically decrease the toxicity of the compound or eliminate it altogether; peptides are rapidly digested in the gut. As an example, peptide drugs that have been developed for human oral administration are generally limited to a bioavailability of 1-2 % because of their molecular size (large), hydrophillicity, and physical stability in the digestive tract (Hamman and Steenekamp, 2011). Consequently, absorption enhancing agents and chemical and physical modifications to the peptide molecules are made to increase the absorption. Drugs derived from the cone shell (Conus fulmen) are based on a peptide with the similar cystine knot of 3 disulfide bonds that is found in GS-U-ACTX-Hv1a-SEQ2; they also share similar physical chemical characteristics. The drug is delivered intrathecally because of a lack of bioavailability through the oral route (Lee, 2010). The limited bioavailability and similarity of the cone shell peptide and GS-U-ACTX-Hv1a-SEQ2 further supports that premise that the oral exposure route poses little chance of toxicity to vertebrates. Consequently, chronic risk concerns are not expected for birds and mammals.

Acute Risks to Honeybees EECs were generated for both the oral and contact exposure pathways for honeybees. Toxicological data were available for the contact route; the LD50 is > 25 µg ai/bee for adult honeybees. The contact EEC (converted to a dose) is 9.6 µg ai/bee, which is lower than the LD50. Consequently, direct risks to honeybees via the contact exposure route are not considered likely. Data were not available for the dietary exposure route to adult honeybees nor were toxicity data provided for larvae. Given that GS-U-ACTX-Hv1a-SEQ2 is an insecticide and is highly specific to insects, risk cannot be precluded. The insecticide is intended to be delivered to its targets through the oral route; therefore, risk concerns (via ingestion) are identified for honeybees (adult and larval) in the absence of toxicity data. To reduce the uncertainty associated with this assumption, an acute oral toxicity study on adult honeybees (OECD 213) and a larval toxicity study (non-guideline study) could be performed. In addition, conducting a honeybee toxicity of residues on foliage study (850.3030) would confirm the conclusion of low risk from honeybee exposure to aged residues.

Terrestrial Plants Toxicity data for terrestrial plants were available; however the solubility information for GS-U- ACTX-Hv1a-SEQ2 needed to run the TerrPlant model was not. The plant toxicity studies did not exhibit reductions in seedling emergence and vegetative vigor endpoints that were greater than 25%. Given that they were conducted at the highest application rate, effects to plants are probably unlikely for the proposed uses. Consequently, no risk concerns are identified for plants from the proposed application rate and uses.

6. THREATENED AND ENDANGERED SPECIES CONCERN

Terrestrial insects were the only group of organisms identified to be potentially at risk from the proposed uses of GS-U-ACTX-Hv1a-SEQ2. To determine whether GS-U-ACTX-Hv1a-SEQ2 use sites are geographically associated with known locations of listed species, a screening-level search of the LOCATES (version 2.2.5) database was conducted (query performed on 05/07/2013). The database compares county-level location data for listed species with county- level crop production data (as available in the 2007 agricultural census) to identify any coarse overlaps of listed species with the proposed labeled uses of GS-UACTX-Hv1a-SEQ2. See Appendix A for a complete list of the crops used in the analysis.

Based on the endangered species analysis, risk concerns for direct effects to terrestrial invertebrates have been identified (Table 7). There is a concern for indirect effects to terrestrial plants that have obligate pollinator requirements or other dependencies on terrestrial invertebrates. Species that depend on terrestrial invertebrates for food (amphibians, birds, fish, mammals, reptiles, terrestrial invertebrates) may also be at indirect risk. Appendix A lists the federally listed species (123 species) on which direct effects could potentially occur because of the co-occurrence with crops included in the proposed new uses of GS-U-ACTX-Hv1a-SEQ2.

Table 7. Listed Species Risks Associated with the GS-U-ACTX-Hv1a-SEQ2 Proposed New Uses Listed Taxa Direct Effects Indirect Effects

Terrestrial and semi-aquatic plants No Yes (monocots and dicots) Birds No Yes Terrestrial phase amphibians No Yes Reptiles No Yes Mammals No Yes Terrestrial invertebrates Yes (oral) Yes Aquatic vascular plants No No Aquatic non-vascular plants No No Freshwater fish No Yes Aquatic phase amphibians No Yes Freshwater invertebrates No No Marine/estuarine fish No Yes Marine/estuarine invertebrates No No

7. LITERATURE CITED

Burns, L.A. 2000. Exposure Analysis Modeling System (EXAMS II) User Manual and System Documentation. EPA/600/R-00/081. http://www.epa.gov/ceampubl/

Carousel, R.F., J.C. Imhoff, P.R., Hummel, Cheplick, J.M., A.S. Donigian, and L.A. Suárez. 1997. PRZM-3, A Model for Predicting Pesticide and Nitrogen Fate in the Crop Root and Unsaturated Soil Zones: Users Manual for Release 3.12.2. http://www.epa.gov/ceampubl/

Chong Y, Hayes JL, Sollod B, Wen S, Wilson DT, Hains PG, Hodgson WC, Broady KW, King GF, Nicholson GM. 2007. The ω-atracotoxins: Selective blockers of insect M-LVA and HVA calcium channels. Biochemical Pharmacology 74:623-638.

Fletcher JI, Smith R, O’Donoghue SI, Nilges M. Connor M, Howden MEH, Christie MJ, King GF. 1997. The structure of a novel insecticidal neurotoxin, ω-atracotoxin-HV1, from the venom of an Australian funnel web spider. Nature Structural Biology, 4(7)559-566.

Gunning, S. J., F. Maggio, M. J. Windley, S. M. Valenzuela, G. F. King, and G. M. Nicholson. 2008. The Janus-faced atracotoxins are specific blockers of invertebrate KCa channels. The FEBS Journal, 275: 4045-4059.

Hamman JH, and Steenekamp JH. 2011. Oral peptide drug delivery: Strategies to overcome challenges. In: Peptide Drug Discovery and Development Ed, MC Castanho and NC Santos. Wiley-VCH, Verlag GmbH& Co KGaA.

Lee S, Kim Y, B SK, Choi HW, Lee JY, Jung HH, Ryu JH, Suh HW, Na HS, Kim HJ, Rhim H, Kim JI. 2010. Analgesic effect of highly reversible ω-conotoxin FIVA on N type Ca2+ channels. Molecular Pain. 6;97.

Sollod, B.L. 2006. From venoms to insecticides: exploring the structure, function, and evolution of peptide toxins found in the venom of Australian funnel-web spiders. Ph.D. Thesis, University of Connecticut.

USEPA. 1996a. Ecological effects test guidelines: OPPTS 850.1075 fish acute toxicity test, freshwater and marine. Office of Chemical Safety and Pollution Prevention, United States Environmental Protection Agency.

USEPA. 1996b. Ecological effects test guidelines: OPPTS 850.1010 aquatic invertebrate acute toxicity test, freshwater daphnids. Office of Chemical Safety and Pollution Prevention, United States Environmental Protection Agency.

USEPA. 2012. White paper in support of the proposed risk assessment process for bees. Environmental Protection Agency, Health Canada, and California Department of Pesticide Regulation

APPENDIX A: LOCATES Endangered Species Reporting Crops modeled include: alfalfa hay, almonds, apples, apricots, artichokes-excluding Jerusalem, asparagus, avocados, bananas, barley for gain, beans-all other, beans-dry edible excluding limas, beans-dry lima, beans-green lima, beans-snap (green), beans-yard long, beets, blackberries and dewberries, blueberries-tame, blueberries-wild, boysenberries, broccoli, Brussels sprouts, buckwheat, cabbage-chinese, cabbage-head, cabbage-mustard, canola, cantaloupes, carrots, cassava, cauliflower, celery, cherries-sweet, cherries-tart, chestnuts, chicory, Christmas trees, Christmas trees cut, citrus fruit-all, coffee, collards, corn for grain, corn for silage of greenchop, corn-popcorn and sweet corn, corn-sweet for seed, cotton-all, cotton-pima, cotton-upland, cranberries, cropland used only for pasture or grazing, cucumbers and pickles, currents, dill for oil, dry beans, eggplant, escarole and endive, field and grass seed crops-all, figs, floriculture crops, forage-land used for all hay/haylage grass silage and greenchop, garlic, ginger root, ginseng, grapefruit, grapes, guavas, hay-all hay including alfalfa other tame small grain and wild, haylage (grass silage and greenchop), haylage or greenchop from alfalfa or alfalfa mixtures, hay-other tame dry, hay-wild dry, hazelnuts (filberts), herb and spice plants harvested, herbs-dried, herbs-freshcut, honeydew melons, hops, horseradish, jojoba harvested, kale, kiwifruit, lemons, lemons and limes, lentils, lettuce-all, lettuce-head, lettuce- leaf, lettuce-romaine, limes, loganberries, macadamia nuts, mangoes, mangos, mint for oil-all, mint for oil- peppermint, mint for oil-spearmint, muskmelons and pepinos, mustard greens, nectarines, oats for grain, okra, olives, onions-dry, onions-green, oranges-all, oranges-valencia, orchards, other berries, other haylage grass silage and greenchop, other oranges, other tame hay, papayas, parsley, passion fruit, peaches-all, peaches-clingstone, peaches-freestone, peanuts for nuts, pears-all, pears-bartlett, pears-other, peas-austrian winter, peas-chinese (sugar and snow), peas-dry edible, peas-dry southern (cowpeas), peas-pigeon, peas- green (excluding southern), peas-green southern (cowpeas) – blackeyed, crowder, etc., pecans-all, pecans-improved, pecans-native and seedling, peppers harvested, peppers other than bell (including chile), peppers-bell (excluding pimientos), permanent pasture and rangeland, other than cropland and woodland pastured, persimmons, pineapples harvested, pineapples not harvested, pistachios, plums, plums and prunes, pomegranates, potatoes, proso millet, prunes, pumpkins radishes, rapeseed, raspberries-all, raspberries-black, raspberries-red, rhubarb, rice, rice-wild, rye for grain, safflower, small grain hay, sod harvested (in the open), sorghum for grain, sorghum for silage or greenchop, sorghum for syrup, soursop, soybeans, spinach, squash-all, squash-summer, squash-winter, starfruit, strawberries, sugarbeets for seed, sugarbeets for sugar, sunflower seed-all, sunflower seed-non-oil varieties, sunflower seed-oil varieties, sweet potatoes, tangerines, tobacco, tomatoes in the open, triticale, turnip greens, turnips, walnuts-english, watercress, watermelons, wheat-all, wheat-durum, wheatgrass seed, wheat-spring (other), wheat-winter, yam (lesser), yams

Common Name Scientific Name Medium STATE NAME County Taxon Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Alabama Bibb Insect Snail, Tulotoma Tulotoma magnifica Terrestrial Alabama Clay Gastropod Snail, Tulotoma Tulotoma magnifica Terrestrial Alabama Coosa Gastropod Snail, Tulotoma Tulotoma magnifica Terrestrial Alabama Elmore Gastropod Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Alabama Greene Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Alabama Jackson Insect Snail, Tulotoma Tulotoma magnifica Terrestrial Alabama Monroe Gastropod Snail, Tulotoma Tulotoma magnifica Terrestrial Alabama Shelby Gastropod Snail, Tulotoma Tulotoma magnifica Terrestrial Alabama St. Clair Gastropod Snail, Tulotoma Tulotoma magnifica Terrestrial Alabama Talladega Gastropod Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Alabama Tuscaloosa Insect Springsnail, San Pyrgulopsis Terrestrial, Bernardino bernardina Freshwater Arizona Cochise Gastropod Oxyloma haydeni Terrestrial, Ambersnail, Kanab kanabensis Freshwater Arizona Coconino Gastropod Beetle, American Nicrophorus Burying americanus Terrestrial Arkansas Franklin Insect

Beetle, American Nicrophorus Burying americanus Terrestrial Arkansas Little River Insect Beetle, American Nicrophorus Burying americanus Terrestrial Arkansas Logan Insect Shagreen, Magazine Mesodon Mountain magazinensis Terrestrial Arkansas Logan Gastropod Beetle, American Nicrophorus Burying americanus Terrestrial Arkansas Scott Insect Beetle, American Nicrophorus Burying americanus Terrestrial Arkansas Sebastian Insect Butterfly, Bay Checkerspot (Wright's Euphydryas editha euphydryas) bayensis Terrestrial California Alameda Insect Butterfly, Callippe Speyeria callippe Silverspot callippe Terrestrial California Alameda Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Amador Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Butte Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Calaveras Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Colusa Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Contra Costa Insect Butterfly, Callippe Speyeria callippe Silverspot callippe Terrestrial California Contra Costa Insect Butterfly, Lange's Apodemia mormo Metalmark langei Terrestrial California Contra Costa Insect Butterfly, Oregon Speyeria zerene Silverspot hippolyta Terrestrial California Del Norte Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California El Dorado Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Fresno Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Glenn Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Kern Insect Moth, Kern Primrose Euproserpinus Sphinx euterpe Terrestrial California Kern Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Kings Insect Beetle, Valley Desmocerus Terrestrial California Lake Insect

Elderberry Longhorn californicus dimorphus Skipper, Carson Pseudocopaeodes Wandering eunus obscurus Terrestrial California Lassen Insect Butterfly, El Euphilotes battoides Segundo Blue allyni Terrestrial California Los Angeles Insect Glaucopsyche Butterfly, Palos lygdamus Verdes Blue palosverdesensis Terrestrial California Los Angeles Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Madera Insect Butterfly, Mission Icaricia icarioides Blue missionensis Terrestrial California Marin Insect Butterfly, Myrtle's Speyeria zerene Silverspot myrtleae Terrestrial California Marin Insect Butterfly, San Bruno Callophrys mossii Elfin bayensis Terrestrial California Marin Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Mariposa Insect Butterfly, Behren's Speyeria zerene Silverspot behrensii Terrestrial California Mendocino Insect Lycaeides Butterfly, Lotis Blue argyrognomon lotis Terrestrial California Mendocino Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Merced Insect Butterfly, Smith's Euphilotes enoptes Blue smithi Terrestrial California Monterey Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Napa Insect Butterfly, Callippe Speyeria callippe Silverspot callippe Terrestrial California Napa Insect Butterfly, Myrtle's Speyeria zerene Silverspot myrtleae Terrestrial California Napa Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Nevada Insect Euphydryas editha Butterfly, Quino quino (=E. e. Checkerspot wrighti) Terrestrial California Orange Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Placer Insect Beetle, Casey's June Dinacoma caseyi Terrestrial California Riverside Insect Euphydryas editha Butterfly, Quino quino (=E. e. Checkerspot wrighti) Terrestrial California Riverside Insect Rhaphiomidas Fly, Delhi Sands terminatus Flower-loving abdominalis Terrestrial California Riverside Insect Beetle, Delta Green Terrestrial, Ground Elaphrus viridis Vernal pool California Sacramento Insect

Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Sacramento Insect Euphydryas editha Butterfly, Quino quino (=E. e. Checkerspot wrighti) Terrestrial California San Bernardino Insect Rhaphiomidas Fly, Delhi Sands terminatus Flower-loving abdominalis Terrestrial California San Bernardino Insect Euphydryas editha Butterfly, Quino quino (=E. e. Checkerspot wrighti) Terrestrial California San Diego Insect Skipper, Laguna Pyrgus ruralis Mountain lagunae Terrestrial California San Diego Insect Butterfly, Mission Icaricia icarioides Blue missionensis Terrestrial California San Francisco Insect Butterfly, San Bruno Callophrys mossii Elfin bayensis Terrestrial California San Francisco Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California San Joaquin Insect Desmocerus Beetle, Valley californicus San Luis Elderberry Longhorn dimorphus Terrestrial California Obispo Insect Butterfly, Smith's Euphilotes enoptes San Luis Blue smithi Terrestrial California Obispo Insect Snail, Morro Helminthoglypta San Luis Shoulderband walkeriana Terrestrial California Obispo Gastropod Butterfly, Bay Checkerspot (Wright's Euphydryas editha euphydryas) bayensis Terrestrial California San Mateo Insect Butterfly, Callippe Speyeria callippe Silverspot callippe Terrestrial California San Mateo Insect Butterfly, Mission Icaricia icarioides Blue missionensis Terrestrial California San Mateo Insect Butterfly, Myrtle's Speyeria zerene Silverspot myrtleae Terrestrial California San Mateo Insect Butterfly, San Bruno Callophrys mossii Elfin bayensis Terrestrial California San Mateo Insect Butterfly, Bay Checkerspot (Wright's Euphydryas editha euphydryas) bayensis Terrestrial California Santa Clara Insect Beetle, Mount Terrestrial, Hermon June Polyphylla barbata Subterraneous California Santa Cruz Insect Beetle, Ohlone Tiger Cicindela ohlone Terrestrial California Santa Cruz Insect Grasshopper, Zayante Band- Trimerotropis winged infantilis Terrestrial California Santa Cruz Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Shasta Insect Beetle, Delta Green Terrestrial, Ground Elaphrus viridis Vernal pool California Solano Insect

Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Solano Insect Butterfly, Callippe Speyeria callippe Silverspot callippe Terrestrial California Solano Insect Butterfly, Behren's Speyeria zerene Silverspot behrensii Terrestrial California Sonoma Insect Butterfly, Myrtle's Speyeria zerene Silverspot myrtleae Terrestrial California Sonoma Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Stanislaus Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Sutter Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Tehama Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Tulare Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Tuolumne Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Yolo Insect Desmocerus Beetle, Valley californicus Elderberry Longhorn dimorphus Terrestrial California Yuba Insect Butterfly, Uncompahgre Fritillary Boloria acrocnema Terrestrial Colorado Chaffee Insect Butterfly, Uncompahgre Fritillary Boloria acrocnema Terrestrial Colorado Dolores Insect Skipper, Pawnee Hesperia leonardus Montane montana Terrestrial Colorado Douglas Insect Butterfly, Uncompahgre Fritillary Boloria acrocnema Terrestrial Colorado Eagle Insect Butterfly, Uncompahgre Fritillary Boloria acrocnema Terrestrial Colorado Gunnison Insect Butterfly, Uncompahgre Fritillary Boloria acrocnema Terrestrial Colorado Hinsdale Insect Skipper, Pawnee Hesperia leonardus Montane montana Terrestrial Colorado Jefferson Insect Butterfly, Uncompahgre Fritillary Boloria acrocnema Terrestrial Colorado La Plata Insect Butterfly, Uncompahgre Boloria acrocnema Terrestrial Colorado Lake Insect Fritillary

Butterfly, Uncompahgre Fritillary Boloria acrocnema Terrestrial Colorado Mineral Insect Butterfly, Uncompahgre Fritillary Boloria acrocnema Terrestrial Colorado Ouray Insect Butterfly, Uncompahgre Fritillary Boloria acrocnema Terrestrial Colorado Park Insect Skipper, Pawnee Hesperia leonardus Montane montana Terrestrial Colorado Park Insect Butterfly, Uncompahgre Fritillary Boloria acrocnema Terrestrial Colorado Pitkin Insect Butterfly, Uncompahgre Fritillary Boloria acrocnema Terrestrial Colorado Rio Grande Insect Butterfly, Uncompahgre Fritillary Boloria acrocnema Terrestrial Colorado Saguache Insect Butterfly, Uncompahgre Fritillary Boloria acrocnema Terrestrial Colorado San Miguel Insect Butterfly, Uncompahgre Fritillary Boloria acrocnema Terrestrial Colorado Summit Insect Skipper, Pawnee Hesperia leonardus Montane montana Terrestrial Colorado Teller Insect Terrestrial, Beetle, Puritan Tiger Cicindela puritana Coastal Connecticut Hartford Insect Terrestrial, Beetle, Puritan Tiger Cicindela puritana Coastal Connecticut Middlesex Insect Butterfly [Cassius Leptotes and Blue, Ceraunus Blue, Hemiargus and Nickerbean Blue] Cyclargus genus Terrestrial Florida Miami-Dade Insect Butterfly, Ceranus Hemiargus ceraunus Blue antibubastus Terrestrial Florida Miami-Dade Insect Butterfly, Miami Cyclargus thomasi Blue bethunebakeri Terrestrial Florida Miami-Dade Insect Butterfly, Nickerbean Blue Cyclargus ammon Terrestrial Florida Miami-Dade Insect Butterfly [Cassius Leptotes and Blue, Ceraunus Blue, Hemiargus and Nickerbean Blue] Cyclargus genus Terrestrial Florida Monroe Insect Butterfly, Ceranus Hemiargus ceraunus Blue antibubastus Terrestrial Florida Monroe Insect Butterfly, Miami Cyclargus thomasi Blue bethunebakeri Terrestrial Florida Monroe Insect Butterfly, Nickerbean Blue Cyclargus ammon Terrestrial Florida Monroe Insect Heraclides Butterfly, Schaus aristodemus Swallowtail ponceanus Terrestrial Florida Monroe Insect Snail, Stock Island Orthalicus reses (not Tree incl. nesodryas) Terrestrial Florida Monroe Gastropod

Damselfly, Pacific Megalagrion Hawaiian pacificum Terrestrial Hawaii Hawaii Insect Fly, Hawaiian Drosophila picture-wing heteroneura Terrestrial Hawaii Hawaii Insect Fly, Hawaiian picture-wing Drosophila mulli Terrestrial Hawaii Hawaii Insect Fly, Hawaiian Drosophila picture-wing ochrobasis Terrestrial Hawaii Hawaii Insect Moth, Blackburn's Sphinx Manduca blackburni Terrestrial Hawaii Hawaii Insect Damselfly, Pacific Megalagrion Hawaiian pacificum Terrestrial Hawaii Kauai Insect Fly, Hawaiian Drosophila picture-wing musaphilia Terrestrial Hawaii Kauai Insect Hawaiian picture- wing Fly Drosophila sharpi Terrestrial Hawaii Kauai Insect Moth, Blackburn's Sphinx Manduca blackburni Terrestrial Hawaii Kauai Insect Spider, Kauai Cave Terrestrial, Wolf Adelocosa anops Subterraneous Hawaii Kauai Arachnid Damselfly, Flying Megalagrion Earwig Hawaiian nesiotes Terrestrial Hawaii Maui Insect Damselfly, Pacific Megalagrion Hawaiian pacificum Terrestrial Hawaii Maui Insect Fly, Hawaiian Drosophila picture-wing neoclavisetae Terrestrial Hawaii Maui Insect Moth, Blackburn's Sphinx Manduca blackburni Terrestrial Hawaii Maui Insect Megalagrion blackline Hawaiian nigrohamatum damselfly nigrolineatum Terrestrial Hawaii Oahu Insect Crimson Hawaiian Megalagrion damselfly leptodemas Terrestrial Hawaii Oahu Insect Fly, Hawaiian picture-wing Drosophila aglaia Terrestrial Hawaii Oahu Insect Fly, Hawaiian Drosophila picture-wing montgomeryi Terrestrial Hawaii Oahu Insect Fly, Hawaiian picture-wing Drosophila obatai Terrestrial Hawaii Oahu Insect Fly, Hawaiian Drosophila picture-wing substenoptera Terrestrial Hawaii Oahu Insect Fly, Hawaiian Drosophila picture-wing tarphytrichia Terrestrial Hawaii Oahu Insect Fly, Hawaiian Drosophila picture-wing hemipeza Terrestrial Hawaii Oahu Insect Moth, Blackburn's Sphinx Manduca blackburni Terrestrial Hawaii Oahu Insect Oceanic Hawaiian Megalagrion damselfly oceanicum Terrestrial Hawaii Oahu Insect Snail, O'ahu Tree (Achatinella Achatinella abbreviata) abbreviata Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree Achatinella (Achatinella apexfulva Terrestrial Hawaii Oahu Gastropod

23 apexfulva) Snail, O'ahu Tree (Achatinella bellula) Achatinella bellula Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella buddii) Achatinella buddii Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella bulimoides) bulimoides Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella byronii) Achatinella byronii Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella caesia) Achatinella caesia Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella casta) Achatinella casta Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella cestus) Achatinella cestus Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella concavospira) concavospira Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella curta) Achatinella curta Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella decipiens) decipiens Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella decora) Achatinella decora Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella dimorpha) dimorpha Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella elegans) Achatinella elegans Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella fulgens) Achatinella fulgens Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella fuscobasis) fuscobasis Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella juddii) Achatinella juddii Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella juncea) Achatinella juncea Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella lehuiensis) lehuiensis Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella leucorraphe) leucorraphe Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella lila) Achatinella lila Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella livida) Achatinella livida Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella lorata) Achatinella lorata Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella mustelina) mustelina Terrestrial Hawaii Oahu Gastropod

Snail, O'ahu Tree (Achatinella Achatinella papyracea) papyracea Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella phaeozona) phaeozona Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella pulcherrima) pulcherrima Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella pupukanioe) pupukanioe Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella rosea) Achatinella rosea Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella sowerbyana) sowerbyana Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella spaldingi) spaldingi Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella stewartii) Achatinella stewartii Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella swiftii) Achatinella swiftii Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella taeniolata) taeniolata Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella Achatinella thaanumi) thaahumi Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella turgida) Achatinella turgida Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella valida) Achatinella valida Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella viridans) Achatinella viridans Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella vittata) Achatinella vittata Terrestrial Hawaii Oahu Gastropod Snail, O'ahu Tree (Achatinella vulpina) Achatinella vulpina Terrestrial Hawaii Oahu Gastropod Snail, Snake River Physa Physa natricina Terrestrial Idaho Cassia Gastropod Snail, Snake River Physa Physa natricina Terrestrial Idaho Elmore Gastropod Snail, Snake River Physa Physa natricina Terrestrial Idaho Gooding Gastropod Snail, Snake River Physa Physa natricina Terrestrial Idaho Jerome Gastropod Snail, Snake River Physa Physa natricina Terrestrial Idaho Minidoka Gastropod Snail, Snake River Physa Physa natricina Terrestrial Idaho Owyhee Gastropod Snail, Snake River Physa natricina Terrestrial Idaho Twin Falls Gastropod

Physa Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Illinois Cook Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Illinois Du Page Insect Snail, Iowa Pleistocene Discus macclintocki Terrestrial Illinois Jo Daviess Gastropod Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Illinois Lake Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Illinois Will Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Indiana La Porte Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Indiana Lagrange Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Indiana Lake Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Indiana Porter Insect Snail, Iowa Pleistocene Discus macclintocki Terrestrial Iowa Clayton Gastropod Snail, Iowa Pleistocene Discus macclintocki Terrestrial Iowa Clinton Gastropod Snail, Iowa Pleistocene Discus macclintocki Terrestrial Iowa Dubuque Gastropod Snail, Iowa Pleistocene Discus macclintocki Terrestrial Iowa Fayette Gastropod Snail, Iowa Pleistocene Discus macclintocki Terrestrial Iowa Jackson Gastropod Beetle, American Nicrophorus Burying americanus Terrestrial Kansas Chautauqua Insect Beetle, American Nicrophorus Burying americanus Terrestrial Kansas Elk Insect Beetle, American Nicrophorus Burying americanus Terrestrial Kansas Montgomery Insect Beetle, American Nicrophorus Burying americanus Terrestrial Kansas Wilson Insect Beetle, American Nicrophorus Burying americanus Terrestrial Kentucky Henderson Insect Beetle, American Nicrophorus Burying americanus Terrestrial Kentucky Jefferson Insect Beetle, American Nicrophorus Burying americanus Terrestrial Kentucky Trigg Insect Beetle, Northeastern Cicindela dorsalis Beach Tiger dorsalis Terrestrial Maryland Calvert Insect Terrestrial, Beetle, Puritan Tiger Cicindela puritana Coastal Maryland Calvert Insect Terrestrial, Beetle, Puritan Tiger Cicindela puritana Coastal Maryland Cecil Insect Terrestrial, Beetle, Puritan Tiger Cicindela puritana Coastal Maryland Kent Insect Beetle, Northeastern Cicindela dorsalis Beach Tiger dorsalis Terrestrial Maryland Somerset Insect Beetle, Northeastern Cicindela dorsalis Beach Tiger dorsalis Terrestrial Massachusetts Barnstable Insect

Beetle, American Nicrophorus Burying americanus Terrestrial Massachusetts Dukes Insect Beetle, Northeastern Cicindela dorsalis Beach Tiger dorsalis Terrestrial Massachusetts Dukes Insect Terrestrial, Beetle, Puritan Tiger Cicindela puritana Coastal Massachusetts Hampshire Insect Beetle, American Nicrophorus Burying americanus Terrestrial Massachusetts Nantucket Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Michigan Allegan Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Michigan Alpena Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Michigan Barry Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Michigan Berrien Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Michigan Branch Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Michigan Cass Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Michigan Ionia Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Michigan Jackson Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Michigan Kalamazoo Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Michigan Kent Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Michigan Lake Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Michigan Mackinac Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Michigan Mason Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Michigan Mecosta Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Michigan Monroe Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Michigan Montcalm Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Michigan Muskegon Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Michigan Newaygo Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Michigan Oceana Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Michigan Presque Isle Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Michigan St. Joseph Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Michigan Van Buren Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Michigan Washtenaw Insect Butterfly, Karner Lycaeides melissa Terrestrial Minnesota Winona Insect

Blue samuelis Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Missouri Dent Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Missouri Iron Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Missouri Phelps Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Missouri Reynolds Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Missouri Ripley Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Missouri Shannon Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Missouri Wayne Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Blaine Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Boyd Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Brown Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Cherry Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Custer Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Dawson Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Frontier Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Garfield Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Gosper Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Holt Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Keya Paha Insect Beetle, Salt Creek Cicindela nevadica Tiger lincolniana Terrestrial Nebraska Lancaster Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Lincoln Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Loup Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Rock Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Thomas Insect Beetle, American Nicrophorus Burying americanus Terrestrial Nebraska Wheeler Insect Skipper, Carson Pseudocopaeodes Wandering eunus obscurus Terrestrial Nevada Carson City Insect Naucorid, Ash Ambrysus Meadows amargosus Terrestrial Nevada Nye Insect Skipper, Carson Pseudocopaeodes Wandering eunus obscurus Terrestrial Nevada Washoe Insect

Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial New Hampshire Merrimack Insect Beetle, Northeastern Cicindela dorsalis Beach Tiger dorsalis Terrestrial New Jersey Monmouth Insect Springsnail, Koster's Juturnia kosteri Terrestrial New Mexico Chaves Gastropod Springsnail, Pyrgulopsis Terrestrial, Chupadera chupaderae Freshwater New Mexico Socorro Gastropod Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial New York Albany Insect Snail, Chittenango Succinea Terrestrial, Ovate Amber chittenangoensis Freshwater New York Madison Gastropod Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial New York Saratoga Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial New York Schenectady Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial New York Warren Insect Spider, Spruce-fir Microhexura Moss montivaga Terrestrial North Carolina Avery Arachnid Spider, Spruce-fir Microhexura Moss montivaga Terrestrial North Carolina Caldwell Arachnid Butterfly, Saint Neonympha Francis' Satyr mitchellii francisci Terrestrial North Carolina Craven Insect Spider, Spruce-fir Microhexura Moss montivaga Terrestrial North Carolina Haywood Arachnid Butterfly, Saint Neonympha Francis' Satyr mitchellii francisci Terrestrial North Carolina Hoke Insect Spider, Spruce-fir Microhexura Terrestrial North Carolina Mitchell Arachnid Moss montivaga Mesodon clarki Snail, Noonday nantahala Terrestrial North Carolina Swain Gastropod Spider, Spruce-fir Microhexura Moss montivaga Terrestrial North Carolina Swain Arachnid Spider, Spruce-fir Microhexura Moss montivaga Terrestrial North Carolina Watauga Arachnid Spider, Spruce-fir Microhexura Moss montivaga Terrestrial North Carolina Yancey Arachnid Beetle, American Nicrophorus Burying americanus Terrestrial Ohio Athens Insect Beetle, American Nicrophorus Burying americanus Terrestrial Ohio Hocking Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Ohio Lucas Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Ohio Portage Insect Beetle, American Nicrophorus Burying americanus Terrestrial Ohio Vinton Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Adair Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Atoka Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Bryan Insect Beetle, American Nicrophorus Terrestrial Oklahoma Cherokee Insect

Burying americanus Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Choctaw Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Coal Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Craig Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Creek Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Delaware Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Haskell Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Hughes Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Johnston Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Latimer Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Le Flore Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Marshall Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Mayes Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma McCurtain Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma McIntosh Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Muskogee Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Nowata Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Okfuskee Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Okmulgee Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Osage Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Ottawa Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Pawnee Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Pittsburg Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Pontotoc Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Pushmataha Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Rogers Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Seminole Insect Beetle, American Nicrophorus Terrestrial Oklahoma Sequoyah Insect

Burying americanus Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Tulsa Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Wagoner Insect Beetle, American Nicrophorus Burying americanus Terrestrial Oklahoma Washington Insect Butterfly, Fender's Icaricia icarioides Blue fenderi Terrestrial Oregon Benton Insect Butterfly, Oregon Speyeria zerene Silverspot hippolyta Terrestrial Oregon Clatsop Insect Butterfly, Fender's Icaricia icarioides Blue fenderi Terrestrial Oregon Lane Insect Butterfly, Oregon Speyeria zerene Silverspot hippolyta Terrestrial Oregon Lane Insect Butterfly, Oregon Speyeria zerene Silverspot hippolyta Terrestrial Oregon Lincoln Insect Butterfly, Fender's Icaricia icarioides Blue fenderi Terrestrial Oregon Linn Insect Butterfly, Fender's Icaricia icarioides Blue fenderi Terrestrial Oregon Polk Insect Butterfly, Oregon Speyeria zerene Silverspot hippolyta Terrestrial Oregon Tillamook Insect Butterfly, Fender's Icaricia icarioides Blue fenderi Terrestrial Oregon Yamhill Insect Butterfly, Oregon Speyeria zerene Silverspot hippolyta Terrestrial Oregon Yamhill Insect Beetle, American Nicrophorus Burying americanus Terrestrial Rhode Island Washington Insect Beetle, American Nicrophorus Burying americanus Terrestrial South Dakota Bennett Insect Beetle, American Nicrophorus Burying americanus Terrestrial South Dakota Brookings Insect Beetle, American Nicrophorus Burying americanus Terrestrial South Dakota Gregory Insect Beetle, American Nicrophorus Burying americanus Terrestrial South Dakota Haakon Insect Beetle, American Nicrophorus Burying americanus Terrestrial South Dakota Todd Insect Beetle, American Nicrophorus Burying americanus Terrestrial South Dakota Tripp Insect Beetle, American Nicrophorus Burying americanus Terrestrial South Dakota Union Insect Spider, Spruce-fir Microhexura Moss montivaga Terrestrial Tennessee Carter Arachnid Snail, Painted Snake Coiled Forest Anguispira picta Terrestrial Tennessee Franklin Gastropod Marstonia, Royal Pyrgulopsis (=Royal Snail) ogmorhaphe Terrestrial Tennessee Marion Gastropod Spider, Spruce-fir Microhexura Moss montivaga Terrestrial Tennessee Sevier Arachnid Beetle, Helotes Mold Batrisodes venyivi Subterraneous Texas Bexar Insect Harvestman, Texella Terrestrial, Cokendolpher Cave cokendolpheri Subterraneous Texas Bexar Arachnid

Meshweaver, Braken Terrestrial, Bat Cave Cicurina venii Subterraneous Texas Bexar Arachnid Meshweaver, Government Canyon Terrestrial, Bat Cave Cicurina vespera Subterraneous Texas Bexar Arachnid Meshweaver, Terrestrial, Madla's Cave Cicurina madla Subterraneous Texas Bexar Arachnid Meshweaver, Robber Terrestrial, Baron Cave Cicurina baronia Subterraneous Texas Bexar Arachnid Terrestrial, Rhadine exilis (ncn) Rhadine exilis Subterraneous Texas Bexar Insect Rhadine infernalis Terrestrial, (ncn) Rhadine infernalis Subterraneous Texas Bexar Insect Spider, Government Neoleptoneta Terrestrial, Canyon Bat Cave microps Subterraneous Texas Bexar Arachnid Harvestman, Bee Terrestrial, Creek Cave Texella reddelli Subterraneous Texas Burnet Arachnid Beetle, American Nicrophorus Burying americanus Terrestrial Texas Lamar Insect Beetle, American Nicrophorus Burying americanus Terrestrial Texas Red River Insect Beetle, Kretschmarr Texamaurops Cave Mold reddelli Subterraneous Texas Travis Insect Beetle, Tooth Cave Ground Rhadine persephone Subterraneous Texas Travis Insect Harvestman, Bee Terrestrial, Creek Cave Texella reddelli Subterraneous Texas Travis Arachnid Harvestman, Bone Terrestrial, Cave Texella reyesi Subterraneous Texas Travis Arachnid Pseudoscorpion, Tartarocreagris Terrestrial, Tooth Cave texana Subterraneous Texas Travis Arachnid Terrestrial, Spider, Tooth Cave Leptoneta myopica Subterraneous Texas Travis Arachnid Beetle, Coffin Cave Mold Batrisodes texanus Subterraneous Texas Williamson Insect Beetle, Tooth Cave Ground Rhadine persephone Subterraneous Texas Williamson Insect Harvestman, Bone Terrestrial, Cave Texella reyesi Subterraneous Texas Williamson Arachnid Oxyloma haydeni Terrestrial, Ambersnail, Kanab kanabensis Freshwater Utah Kane Gastropod Beetle, Northeastern Cicindela dorsalis Beach Tiger dorsalis Terrestrial Virginia Accomack Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Virginia Carroll Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Virginia Floyd Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Virginia Franklin Insect Spider, Spruce-fir Microhexura Moss montivaga Terrestrial Virginia Grayson Arachnid Beetle, Northeastern Cicindela dorsalis Beach Tiger dorsalis Terrestrial Virginia Lancaster Insect Beetle, Northeastern Cicindela dorsalis Terrestrial Virginia Mathews Insect

Beach Tiger dorsalis Beetle, Northeastern Cicindela dorsalis Beach Tiger dorsalis Terrestrial Virginia Middlesex Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Virginia Montgomery Insect Beetle, Northeastern Cicindela dorsalis Beach Tiger dorsalis Terrestrial Virginia Northampton Insect Beetle, Northeastern Cicindela dorsalis Beach Tiger dorsalis Terrestrial Virginia Northumberland Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Virginia Patrick Insect Butterfly, Mitchell's Neonympha Terrestrial, Satyr mitchellii mitchellii Perm. wetland Virginia Pulaski Insect Snail, Virginia Polygyriscus Fringed Mountain virginianus Terrestrial Virginia Pulaski Gastropod Butterfly, Oregon Speyeria zerene Silverspot hippolyta Terrestrial Washington Grays Harbor Insect Butterfly, Oregon Speyeria zerene Silverspot hippolyta Terrestrial Washington Pacific Insect Snail, Flat-spired Triodopsis Three-toothed platysayoides Terrestrial West Virginia Monongalia Gastropod Snail, Flat-spired Triodopsis Three-toothed platysayoides Terrestrial West Virginia Preston Gastropod Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Adams Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Barron Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Burnett Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Chippewa Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Clark Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Wisconsin Door Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Dunn Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Eau Claire Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Green Lake Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Jackson Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Juneau Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Kenosha Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Wisconsin Kewaunee Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Marquette Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Menominee Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Monroe Insect

Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Oconto Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Outagamie Insect Dragonfly, Hine's Somatochlora Terrestrial, Emerald hineana Freshwater Wisconsin Ozaukee Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Polk Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Portage Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Shawano Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin St. Croix Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Waupaca Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Waushara Insect Butterfly, Karner Lycaeides melissa Blue samuelis Terrestrial Wisconsin Wood Insect