On Service Lands Contaminant Investigation: NJ – Preliminary

FINAL REPORT

ON-REFUGE CONTAMINANT INVESTIGATION

PRELIMINARY ASSESSMENT OF THE EFFECTS OF DELAWARE BAY WATER AND A KNOWN ENDOCRINE DISRUPTING COMPOUND ON HORSESHOE CRABS (LIMULUS POLYPHEMUS) USING PROTEOMICS AND OBSERVATION OF EMBRYONIC DEVELOPMENT AND SURVIVAL.

U.S. Fish and Wildlife Service New Jersey Field Office

June 2007

Preliminary assessment of the effects of Delaware Bay water and a known endocrine disrupting compound on horseshoe crabs (Limulus polyphemus) using proteomics and observation of embryonic development and survival

Cape May National Wildlife Refuge, Middle Township, Cape May County, New Jersey

2nd Congressional District represented by the Honorable Frank LoBiondo

DEC ID # 200550005.1 FFS # 5N41

Prepared for:

U.S. Fish and Wildlife Service Northeast Region Division of Refuges and Wildlife Hadley, Massachusetts

Prepared by:

Brian Marsh U.S. Fish and Wildlife Service New Jersey Field Office

Dr. David Bushek and Sean Boyd Haskin Shellfish Research Laboratory Rutgers University Port Norris, New Jersey

Dr. Peter Van Veld Department of Environmental Sciences Virginia Institute of Marine Sciences Gloucester Point, Virginia

June 2007

Project Biologist: Brian Marsh

Assistant Supervisor: Timothy J. Kubiak

EXECUTIVE SUMMARY

The eggs, larvae, and juveniles of horseshoe crabs (L. polyphemus) contribute significantly to the forage base of many species in Delaware Bay. The eggs also provide a crucial forage base for at least 11 species of shorebird. Birds foraging on L. polyphemus eggs significantly contribute to the wildlife-watching industry in Delaware and New Jersey. L. polyphemus also has significant economic importance to the bait fishery and the pharmaceutical industry. Estimates suggest the population of L. polyphemus in Delaware Bay have declined. Disease, harvest for bait, and habitat loss may contribute to the apparent population decline. However, environmental contamination also may play a role.

This investigation looked at potential adverse effects to L. polyphemus by the endocrine disrupting pesticide methoprene. Methoprene is an insect growth regulator that acts as a juvenile hormone mimic. Land managers apply methoprene aerially to marshes of Delaware Bay to control mosquitoes. This investigation hypothesized that mosquitoes and L. polyphemus will experience similar adverse effects from a compound like methoprene. Two procedures were used to assess these effects. First, any macroscopic developmental abnormalities were recorded by observing egg and larval development in vitro. Second, a proteomics approach was used to address the multitude of potential pathways that may become altered as a result of methoprene exposure. The proteomics approach involved three phases: 1) separation and visualization of unknown proteins that are expressed differently in controls as compared to treated eggs, embryos, and potentially larvae; 2) visualization of those proteins that are consistently over- or under-expressed in treated individuals compared to controls; and 3) identification of these proteins by de novo amino acid sequencing.

A total of 55 female and 22 male adult crabs were collected from Kimbles Beach at Cape May National Wildlife Refuge for two separate spawns. Phase I of the investigation examined embryo, larval, and juvenile response to a series of exposures to methoprene solutions at 0 µg/l, 1 µg/, 10 µg/l, and 100 µg/l using pore water from Kimbles Beach and artificial sea water. Eggs, embryos, larvae, and juveniles were monitored for fertilization, hatching, and survival and for any macroscopic abnormities. Eggs and larvae all treatments were periodically removed and archived in liquid nitrogen for later proteomic analysis. Phase II of the investigation involved a proteomics approach to observing whether the methoprene may have caused more subtle effects to the embryos, larvae, and juveniles. The results provided no evidence that a treatment effect occurred. We observed no obvious acute effects of environmentally relevant concentrations of the mosquito larvicide methoprene on developing L. polyphemus embryos, larvae, or first molt post hatch juveniles. Through this investigation we found evidence for a No Observed Adverse Effects Level of 100 µg/l for methoprene exposure. Our findings provide evidence that chemical exposure to methoprene may not be a limiting factor to the population of L. polyphemus. This information will be useful to land managers, agencies involved in mosquito control, and the numerous groups interested in finding and examining the limiting factors on the L. polyphemus population.

Keywords: methoprene, Altosid, proteomics, horseshoe crab, Limulus polyphemus, Cape May National Wildlife Refuge, Delaware Bay, mosquito control

TABLE OF CONTENTS

EXECUTIVE SUMMARY ii

I. INTRODUCTION 1 A. L. polyphemus population biology B. L. polyphemus population decline C. Significance of potential Limulus polyphemus population decline D. Methoprene and juvenile hormone E. Rationale for using proteomics F. Scientific objectives for this investigation

II. METHODS 8 A. Project Site B. Treatments C. Spawn 1 D. Spawn 2 E. Group 3 F. Proteomics

III. RESULTS 12 A. Spawn 1 B. Spawn 2 C. Group 3 D. Macroscopic abnormalities E. Proteomics

IV. DISCUSSION 16 A. Treatments B. Proteomics C. No Observed Adverse Effect Level D. Management actions E. Summary of scientific objectives for this investigation

V. LITERATURE CITED 20

VI. FIGURES 28

VII. TABLES 41

VII. APPENDICES 55

iii LIST OF FIGURES Appended at the end of the report

1. Kimbles Beach, Cape May National Wildlife Refuge Middle Township, Cape May County, New Jersey.

2. Kimbles Beach, Cape May National Wildlife Refuge Middle Township, Cape May County, New Jersey.

3. Development success for Spawn 1 treatments.

4. Percent of Spawn 1 eggs reaching juvenile stage for pore water.

5. Development success for Spawn 2 treatments.

6. Percent of Spawn 2 eggs reaching juvenile stage.

7. Percent of Group 3 larvae reaching the juvenile stage.

8. Representative cytosolic proteins from day xx larvae (panel a) and day yy larvae (panel b) stained with Sypro-Ruby (Invitrogen, Inc.) for total protein. No consistent treatment related differences (control, 10ug/l, 100 ug/l) were observed at these or other time points during the study.

9. Representative two dimensional analyses of florescent-tagged proteins. Large differences in expression of individual proteins (red and green spots) are the result of differences between experiments.

10. Representative two dimensional analyses of florescent-tagged proteins in control and methoprene-exposed day 21 free swimming larvae. Small differences in protein expression appear as pale green or pink spots.

11. Representative Western blot of HSP70 in day 21 free swimming larvae.

12. Phosphoproteins in control and methoprene-exposed day 21 free swimming larvae.

iv LIST OF TABLES Appended at the end of the report

1. Dilution series for deriving methoprene solutions using Altosid SR-5® (51.3 g/l).

2. The 16 methoprene treatments used for Spawn 1 exposures.

3. The 32 methoprene treatments used for Spawn 2 exposures.

4. The 8 methoprene treatments used for Group 3 exposures.

5. Development of samples of approximately 50 eggs from each Spawn 1 treatment at day 2 after fertilization. Stages of development (18-20 to 18-26) correspond to figures in Brown and Clapper (1981) and are depicted in a table in Appendix 1.

6. Development of samples of approximately 50 eggs from each Spawn 1 treatment at day 23 after fertilization. Stages of development correspond to those in Brown and Clapper (1981) and are depicted in a table in Appendix 1.

7. Percent of Spawn 1 eggs reaching a late stage of embryonic development.

8. Percent of Spawn 1 eggs reaching juvenile stage and dates removed from the treatments.

9. Development from each Spawn 2 treatment at day 31 after fertilization. Stages of development correspond to those in Brown and Clapper (1981) and are depicted in a table in Appendix 1. Tallies do not include eggs removed on July 1 but do include larvae removed July 8.

10. Percent of Spawn 2 eggs reaching a late stage of embryonic development.

11. Percent of Spawn 2 eggs reaching juvenile stage and dates removed from the treatments.

12. Summary of Group 3 juveniles removed from larvae treatments (from treatments started on 7/20/05 for larvae sorted out from 7/13/05 beach collected, fertilized eggs).

13. Frequency of abnormalities removed from treatments (replicates combined).

v LIST OF APPENDICES

1. Stages of horseshoe crab development (Brown and Clapper, 1981) 2. Treatment data 3. Samples archived under liquid nitrogen 4. Project pictures 5. Temperature probe data from Kimbles Beach 6. Altosid SR-5 Label

LIST OF ACRONYMS AND ABBREVIATIONS

µg/l – micrograms per liter µm – micrometer μM – microMole 2DE - two-dimensional electrophoresis ASW – artificial seawater cm – centimeter Ecy - Ecdysone EDC – Endocrine Disrupting Compound g/l – grams per liter HSP – Heat Shock Protein HSP70 – Heat Shock Protein 70 JH - Juvenile Hormone mg/l – milligrams per liter ml - millimeter nM - nanoMole NOAEL - No Observed Adverse Effect Level ppt – parts per thousand SDS-PAGE – Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

I. INTRODUCTION

A. L. polyphemus population biology

Much of the life history of the American horseshoe crab, Limulus polyphemus (subphylum Chelicerata) has been well documented (e.g., Botton and Haskin, 1984; Shuster, 1996). The beaches of Delaware Bay provide the largest spawning site for L. polyphemus (Atlantic States Marine Fisheries Commission, 1997). Adults migrate into the nearshore areas of Delaware Bay and spawn during May and June spring tides (Botton et al., 1988; Smith et al., 2002). Along the high tide line, a female deposits approximately 4,000 eggs in a nest approximately 20 cm deep as a paired male and often several surrounding satellite males fertilize the eggs. Each female lays up to 88,000 eggs annually (Shuster, 1982; Shuster and Botton, 1985). Incubation time varies according to temperature, salinity, and oxidation of surrounding sediments (Jegla and Costlow, 1982; Botton et al., 1988). Generally, trilobite larvae emerge from the eggs after 2 to 4 weeks and soon become demersal and undergo their first molt (Jegla and Costlow, 1982). The nocturnal juveniles remain in the intertidal areas close to their natal beaches for approximately 2 years before migrating to deeper water (Rudloe, 1979; Rudloe, 1981; Shuster, 1982).

B. L. polyphemus population decline

The size of the L. polyphemus population is poorly understood and estimates are based on incomplete and disjunct data (Atlantic States Marine Fisheries Commission, 1998). However, many estimates do suggest evidence for a decline. Stranded L. polyphemus surveys performed from 1985-1995 (Botton and Loveland, unpublished data, 1995) indicate natural strandings have decreased by 90% on beaches in New Jersey. Egg surveys (Botton, unpublished data, 1997) suggest a 90% decline in the number of eggs deposited on New Jersey beaches from 1990 to 1997. Trawling data in and outside of Delaware Bay also suggest a decline of this species (Atlantic States Marine Fisheries Commission, 1998). Spawning surveys have been conducted on the Delaware and New Jersey sides of Delaware Bay since 1990. The methodology used for these surveys has varied and did not have statistical validity until 1999 (Smith et al., 2002); however, these data also indicate the population has declined.

Disease (Groff and Leibovitz, 1982; Shuster, 1982), harvesting for bait and the medical industry (Novitsky, 1984), and habitat loss (Botton et al., 1988; Botton, 1995; Krupa, 1998) may be contributing to the apparent population decline. Although each of these factors likely contributes to the decline, the evidence does not clearly implicate any one as the sole factor or that these are the only potential stressors of significance on the population.

The presence of L. polyphemus eggs and larvae in intertidal areas can be assumed to make them vulnerable to contaminants associated with those areas (Weis and Ma, 1987). The role of contaminant exposure in the decline of this species is one of the least studied factors in their mortality. The discreet pathways that contaminants can cause effects and the variety of contaminants in the environment have made such investigations less common than examinations of more obvious impacts such as over-harvesting. A plethora of conventional and non- conventional and regulated and unregulated contaminants are introduced into Delaware Bay.

1 Additionally, some contaminants are introduced into the river close to spawning areas. For example, septic systems along the shores of Delaware Bay can introduce a variety of contaminants into the aquatic system as can mosquito control programs and pesticide use that affect water quality.

Past studies do not provide evidence suggesting unusual sensitivity of L. polyphemus to low dissolved oxygen or high turbidity (Botton, 1995). Botton et al. (1998a) found L. polyphemus embryos and larvae exhibit some resistance to copper and zinc. Other studies have found similar degrees of resistance to tributyltin (Botton et al., 1998b), diflubenzuron (Weis and Ma, 1987), polychlorinated biphenyls (Neff and Giam, 1977), and oil (Laughlin and Neff, 1977; Strobel and Brenowitz, 1981; U.S. Fish and Wildlife Service, 1998a). Maghini (1996) examined a variety of contaminants in the L. polyphemus eggs and birds collected from Delaware. For most of the contaminants, the eggs and birds exhibited background or lower concentrations. Developing horseshoe crabs are also very tolerant of wide temperature and salinity fluctuations (Ehlinger and Tankersley, 2004). However, some studies have revealed contaminant effects upon L. polyphemus. Botton et al. (1998b) found acute organotin exposure delayed the first molt. Itow et al. (1998a) found mercury caused segmental defects and tin caused eye defects. Itow et al. (1998b) found tributyltin, mercury, cadmium, chromium, and zinc to all have inhibitive effects on the regeneration of limbs as well as causing a delay in molting. Burger (1997) found selenium, lead, manganese, and chromium concentrations were higher than expected in L. polyphemus eggs and higher than concentrations in the spawning females, demonstrating that the eggs can accumulate pollutants from water.

The role of endocrine disrupting chemicals (EDCs) on L. polyphemus has not been thoroughly investigated. However, these types of contaminants are being found in coastal environments. EDCs often exert their effects on embryos, fetuses, or juveniles (see: Colborn et al., 1993; deFur et al., 1999). The effects of EDCs have been observed on various arthropods. Sex determination, body morphology, and molt timing and success are some of the outcomes affected by EDCs in aquatic crustaceans (see deFur et al., 1999). Daphnia spp. exposure to the EDCs endosulfan and methoprene alters molting and reproduction (Zou and Fingerman, 1997; Olmstead and LeBlanc, 2001a and 2001b). L. polyphemus shares many biological traits with these organisms. Arthropods are very sensitive to EDCs during molting. Juveniles are believed to molt 16 to 17 times before becoming adults, when molting may cease (Atlantic States Marine Fisheries Commission, 1997). At least half of the molts occur during the first 2 years of life while the animals receive and are most vulnerable to high concentrations of EDCs and other contaminants in nearshore waters. Molts also occur in the embryos as the eggs bathe in nearshore pore waters. Disruptions of molting cycles could contribute to L. polyphemus population declines.

Molting in L. polyphemus is controlled by the molt hormone 20-OH ecdysone (Ecy), the active form of which is found in almost all arthropods. Ecy is a steroid that acts during premolt to initiate changes throughout the organism that are required for molting, such as changes in the epidermis, muscle, and various metabolic processes. The chemical structure of Ecy is identical in every arthropod examined, including L. polyphemus. The endocrinology of crustacean molting includes Ecy, juvenile hormones (JH), and inhibiting hormones. JHs have two major effects: they influence the molt timing and they determine the developmental outcome of the

2 molt. Consistent with other investigations of endocrine disruption (see: Kavlock et al., 1996; Ankley et al., 1998; deFur et al., 1999), investigations need to focus on how EDCs affect reproduction, development of the eggs, embryos, larvae, and molting of juvenile animals.

Crabs begin to molt when exposed to insect molting hormones and their synthetic analogs suggesting close similarities in the molting processes of these arthropods (Jegla et al., 1972). For example, the insecticide diflubenzuron, which is a chitin synthesis inhibitor, was tested on L. polyphemus larvae (Weis and Ma, 1987), and 50 µg/l was found to produce severe mortality immediately after ecdysis. Those that survived were smaller than controls. For this investigation, methoprene was used as a typical endocrine disruptor that could adversely affect L. polyphemus since it is used in the near-shore areas of Delaware Bay as a mosquito larvicide.

C. Significance of potential Limulus polyphemus population decline

A potentially depressed or declining L. polyphemus population is highly significant due to several factors. The annual L. polyphemus bait industry servicing eel and conch fisheries is worth and estimated $1.5 million. Biomedical use of L. polyphemus is an estimated $50 million per year industry. The eggs, larvae, and juveniles contribute significantly to the forage base of many fish species in the Delaware Bay including striped bass (Morone saxatilis), white perch (Morone americana), American eel (Anguilla rostrata), killifish (Fundulus spp.), silver perch (Bairdiella chrysoura), weakfish (Cynoscion regalis), kingfish (Menticirrhus saxatilis), silversides (Menidia menidia), summer flounder (Paralichthys dentatus), and winter flounder (Pseudopleuronectes americanus) (Shuster, 1982). All crab species and several gastropods, including conchs (Busycon spp.), also feed on the eggs and larvae. L. polyphemus eggs, juveniles, or adults are also common prey federally threatened loggerhead sea turtles (Caretta caretta) (Musick et al., 1983) and sharks (Squaliformes spp.) (Shuster, 1982).

Delaware Bay has the second highest concentration of shorebirds in the Western Hemisphere during the spring. Between 300,000 to 600,000 shorebirds use Delaware Bay as a staging area on their northward migration route (Wander and Dunne, 1982; Senner and Howe, 1984; Myers, 1986; Clark et al., 1993). A single day count of 426,162 birds was made in 1986 (Clarke et al., 1993). The northward migration of at least 20 species of shorebirds coincides with the L. polyphemus spawning (Wander and Dunne, 1981; Dunne et al., 1982) and at least 11 of these federal trust species depend on L. polyphemus eggs for food (Myers, 1986). Migrant shorebirds have extremely high energy requirements (Kersten and Piersma, 1987). Approximately 320 tons of food is supplied to these birds through L. polyphemus eggs (Delaware Department of Natural Resources, 1987). The birds forage almost entirely on L. polyphemus eggs during their 2 to 3 week stopover eating as many as 9,000 to 10,080 eggs per day and increasing their body weight by 40 to 80% (Myers, 1986; American Bird Conservancy, 1997; Tsipoura and Burger, 1998). Despite this heavy use of the L. polyphemus eggs, the impact upon the L. polyphemus population by birds is probably minimal (Botton et al., 1994). With further declines in the population of L. polyphemus, shorebird species such as ruddy turnstones (Arenaria interpres), semipalmated sandpipers (Calidris pusilla), redknots (Calidris canutus), sanderlings (Calidris alba), dowitcher (Limnodromus spp.), and dunlin (Calidris alpina) would likely not have adequate resources to complete their migrations and successfully breed. More resident types of bird species, such as gulls (Laridae), also rely heavily on L. polyphemus eggs (Burger and Galli, 1987).

3 Birds foraging on L. polyphemus eggs significantly contribute to the wildlife watching industry in Delaware and New Jersey that supports 15,127 jobs and generates a total household-income of $399 million (U.S. Fish and Wildlife Service, 1998b). As the crab population has declined there has been a commensurate and significant decline in shorebird populations (Baker et al., 2004; Clark et al., 1993; Clark and Niles, unpublished data, 1997; Morrison et al., 2004).

D. Methoprene and juvenile hormone

Land managers apply methoprene aerially to marshes of Delaware Bay as a mosquito larvicide. Methoprene was registered in 1975 as a mosquito growth regulator (Ware, 1982) and goes under several trade names such as Altosid®, Precor®, Kabat®, Pharorid®, Dianex®, Apex®, Fleatrol®, Ovitrol®, Extinguish®, and Diacon®.

Methoprene has low persistence in the soil with a reported half-life of 10 days (U.S. Environmental Protection Agency, 1982) and is tightly sorbed to most soils (U.S. Environmental Protection Agency, 1982). Methoprene will tend to stay in the top few inches of soil even after repeated washings (U.S. Environmental Protection Agency, 1982; Zoecon Corporation, 1974). Methoprene rapidly undergoes microbial and photo degradation (U.S. Environmental Protection Agency, 1982; U.S. Environmental Protection Agency, 1991). Methoprene is only slightly soluble in water (1.4 mg/l @ 25°C) but is miscible in organic solvents (Kidd and James, 1991). The water half-life is less than 1 day in light and over 4 weeks in dark and on plants the half-life is 1 to 2 days (U.S. Environmental Protection Agency, 1982; 1991). Methoprene has been shown to have half-lives in pond water of about 30 to 40 hours (Menzie, 1980). Evidence has not indicated that methoprene acts as a teratogen, mutagen, or carcinogen and has indicated only minimal toxicity to fish, mammals, or birds (World Health Organization, 2004). Limited evidence has indicated moderate to high toxicity to some invertebrates (Zoecon Corporation, 1974).

Methoprene is designed to specifically mimic JH. JHs are a class of signaling molecules that play critically important roles in regulation of insect growth and development (Jones, 1995). As an insect develops, higher levels of these hormones prolong juvenile stages and lesser amounts promote progression to more adult stages. In many insects, JHs regulate the production of eggs in the female's ovaries as well. In other species, these hormones appear to play a role in reproductive and social behaviors (Pearce et al., 2001; Scott et al., 2001).

JH’s action is mediated mainly by the intracellular ecdysteroid receptor (Spindler et al., 2001). In insects, species-, tissue- and developmental stage-specific activities of the signal mediated by JH and ecdysteroid receptor interaction coordinate the activity of the diverse signaling pathways. The precise nature of the pathways involved and protein components are not well understood (Wilson, 2004). However, their further study could be revealing to understand the effects of EDCs. The results of a project like this, in addition to potentially revealing a relationship between methoprene and L. polyphemus, can more generally help improve understanding of relationships between altered protein expression and higher order effects on reproduction, growth, and development. In addition, identification of new proteins via proteomics can enhance the understanding of pathways that are altered as a result of exposures to EDCs.

4 JH is a necessary molecule at specific times in insect development but becomes toxic when present during metamorphosis. Methoprene and other synthetic JH mimics are designed to interfere with the timing of metamorphosis in a broad spectrum of insects during embryonic, larval and reproductive stages. Methoprene likely activates JH protein signaling pathways at the wrong times during development. However, the nature of these pathways and therefore the mechanism of action of these compounds is poorly understood (Wilson, 2004) as well as their importance to other arthropods such as L. polyphemus. Crustaceans, like insects, have JH-like compounds involved in the control of their larval development, molting, and reproduction. L. polyphemus are closely related to spiders, scorpions and mites. Whether or not Chelicerata use JH-like compounds for control of molting and development has not been determined and few studies have addressed the toxicity of methoprene or other synthetic JH analogs to this class of organism (El-Banhawy, 1977; Belozerov, 2001).

While methoprene was developed as a JH agonist for the control of insect pests, evidence suggests that this compound is toxic to crustaceans as well (Olmstead and LeBlanc, 2001a and 2001b; Walker et al., 2005; Ghekiere et al., 2006). Olmstead and LeBlanc (2001a and 2001b) found significant methoprene toxicity to endocrine related processes in the crustacean Daphnia magna at the 5 to 50 nM range. McKenney and Celestial (1996) found methoprene may interfere with endocrine controlled systems in Mysidopsis bahia as demonstrated by a variety of effects varying from total lethality to juvenile mysids at 125 μg/l for four days to significant reductions in brood size at 2 μg/l. Horst and Walker (1999) found exposure to methoprene at environmentally relevant concentrations (2-10 μM) produced morbidity and mortality in blue crabs (Callinectes sapidus) as demonstrated by an overall reduction in successful hatching and by lethargic behavior exhibited by the surviving zoeae (first stage larvae). Horst and Walker (1999) also found a methoprene concentration of 3.3 μM delayed the molt from the megalopae (post larval stage) to the first crab form, resulting in death of 80% of the megalopae after 10 days.

E. Rationale for using proteomics

This investigation provided a unique opportunity to examine treatment effects of a putative EDC using a proteomics approach. Through proteomics and the examination of subtle changes in protein expression, subtle treatment effects can be revealed. In a recent review of molecular markers of endocrine disruption Rotchell and Ostrander (2003) suggest that proteomics offers “enormous potential” to highlight “critical candidate proteins” that are up- and down-regulated following exposure to EDCs. Proteomics is particularly relevant for identification of cellular alterations involving multiple proteins, as is likely the case with endocrine-mediated effects. Protein mediated pathways regulate reproduction, molting, growth, and development through hormones, growth factor receptors, and other means. Exposure to EDCs has the potential to alter these pathways especially during reproduction and early development. Processes that may be influenced include, but are not limited to the following: 1) Ecy-mediated pathways; 2) molt inhibiting hormone-mediated pathways; 3) JH receptor-mediated pathways; 4) inductive protein signaling networks that induce developing embryos through various stages of growth and differentiation; 5) numerous proteins involved in hormone synthesis and degradation; and 6) pathways that allow cross-talk between 1-5 above. EDCs have the potential to alter a variety of these pathways.

The biochemical pathways affected by methoprene in insects are only partially understood (Spindler et al., 2001; Wilson, 2004). To our knowledge, information on the toxicity of this compound to Limulus or other Chelicerata does not exist. For this investigation, we chose to include a proteomics approach to address the possibility of diverse alterations resulting from methoprene exposure in L. polyphemus. Our rationale was that two-dimensional electrophoresis (2DE) coupled with sequence information would provide a method for visualization and identification of diverse target proteins whose expression may be altered as a result of methoprene. In addition to proteomics, we chose two other candidate biomarkers (heat shock protein 70 and protein phosphorylation patterns) previously reported to be altered by methoprene exposure in larval lobster (Homarus americanus) (Walker et al., 2005).

F. Scientific objectives for this investigation

1) Assess embryonic molting success in treatments of control water and pore water from a Delaware Bay beach spiked with a known EDC (i.e., methoprene). Positive biological findings, if found, will provide a more rational focus for pursuing endocrine active chemicals that are possibly involved in the decline of L. polyphemus.

Hypothesis 1: L. polyphemus molting success will be reduced in embryos developed in treatment solutions compared to control solution as observed by macroscopic characteristics in embryos or lethality.

Null hypothesis: No difference will occur in L. polyphemus molting success between embryos developed in treatment solutions compared to control solution.

2) Determine if evidence exists for exposure to environmental contaminants using proteomics. Positive biological findings, if found, will provide evidence that environmental contaminants do have an effect upon L. polyphemus.

Hypothesis 2: There will be a protein specific pattern difference between treatment and control embryos as demonstrated by two dimensional gel electrophoresis and subsequent de novo sequencing.

Null hypothesis: No difference will occur between treatment and control individuals.

3) Determine if a known EDC (i.e., methoprene) causes adverse impacts to L. polyphemus embryos and larvae and develop a dose-response curve for this compound. If there are no demonstrable biological or biochemical effects, a No Observable Adverse Effects Level (NOAEL) will be established.

Hypothesis 3: A known EDC will produce adverse impacts to L. polyphemus embryos and larvae as shown by characteristics in embryos or lethality and proteomics, demonstrating L. polyphemus is susceptible to environmental contaminants.

6 Null hypothesis: A known EDC will not produce adverse impacts L. polyphemus embryos and larvae.

4) Address a complex and poorly understood chemical effects scenario on a historically prolific species which is preyed upon by a large number of aquatic species and avian predators. As such, we are addressing a keystone species at the epicenter of its range. We are employing biological and biochemical analyses that should provide a more rigorous and convincing battery of information to either discount or affirm physical habitat constraints or harvesting as the principal stressors associated with crab population declines or whether environmental contaminants may also be a limiting factor to the population.

Hypothesis 4: An examination of L. polyphemus embryos, through biological and biochemical investigation, will provide evidence that environmental contamination is a potential limiting factor on the L. polyphemus population by providing data that statistically support one or more of the above three hypotheses.

Null hypothesis: An examination of L. polyphemus embryos, through biological and biochemical investigation, will provide evidence that environmental contamination is not a potential limiting factor on the L. polyphemus population and other stressors such as over-harvesting warrant more attention by providing data that statistically support the above three null hypotheses.

7 II. METHODS

A. Project Site

L. polyphemus adults and beach pore water were collected from Kimbles Beach in Middle Township, Cape May County, New Jersey (Figure 1 and 2). The beach is part of Cape May NWR, historically well represented in L. polyphemus surveys, and accessible. The beach is characterized by a narrow band (~ 10 m) of sand bordering high salt marsh. The northern and southern end of the 800 meter beach have broader expanses (~ 15 m) of sand and are bordered by tidal inlets. Spawning, cultures, and exposures were completed at the Rutgers University Haskin Shellfish Research Laboratory (HSRL) in Port Norris, Cumberland County, New Jersey. Proteomics analysis was completed at the Virginia Institute of Marine Sciences (VIMS) in Gloucester Point, Gloucester County, Virginia.

B. Treatments

Two spawns were completed (hereafter referred to as Spawn 1 and Spawn 2) which produced two series of treatments at the HSRL that exposed fertilized eggs to various concentrations of methoprene in the proprietary mixture Altosid SR-5® (5% methoprene). The gonopore regions of the crabs were electrically stimulated with a 6 V 800 mA charge, to cause release of gametes; however, this method resulted in little success. Manual pressure applied to the gonopore region was more effective in causing gamete release. All adults were released after spawning. An additional series of treatments (Group 3) were run with larvae collected from samples of fertilized eggs collected directly from Kimbles Beach and allowed to hatch in the lab.

Series of methoprene treatments included those with pore water from Kimbles Beach and artificial seawater (Artificial Ocean® at 25 ppt)(ASW). The beach pore water was collected from pits approximately 40 cm deep dug into the beach just above the tide line. The water was collected weekly throughout the investigation, filtered to 300 µm at the beach, filtered to 1 µm at the lab, and stored at 6-8°C. The water was warmed to room temperature prior to being mixed into solutions. For reference, temperature probes were buried in the north end of the beach in the intertidal zone on May 5 and removed on July 27, 2006. The mean temperature over this time for probes at 10, 20, and 30 cm was 23.75, 23.53, and 23.47°C, respectively. Appendix 5 shows the temperature readings over this time. The salinity of pore water 40 cm deep in the beach immediately above the tide was 21, 20, 20, 20, and 22 ppt NaCl for May 5, 23, 27, 30, and 7, respectively.

Field exposure of L. polyphemus to methoprene that may naturally occur was assumed to be at most approximately 1 µg/l. The recommended application rates for Altosid SR-5® is 219 to 293 ml/hectare. Altosid SR-5® has 5% methoprene by volume (51.3 g/l), making the application rate approximately 10.95 to 14.65 g/hectare of methoprene. If applied over open water 1-cm deep, this application would result in a temporary water concentration of up to 14.65 g of methoprene for 100,000 liters of water or 0.1465 µg/l.

8 Treatment solutions were made at four methoprene concentrations: 0, 1, 10, and 100 µg/l using pore water and ASW. Treatment mixtures were made using a series of dilutions as depicted in Table 1.

Treatments were placed in 300 ml glass dishes filled to 200 ml with solution. All the dishes were kept in a uniform, covered, water bath maintained at a temperature of 23°C and 14 hours light/10 hours dark cycle. Treatments were not aerated. Each dish was drained and filled with fresh solution every Monday, Wednesday, and Friday. Periodically, eggs, larvae, and/or juveniles were removed from the treatments and archived in liquid nitrogen for later proteomics analysis (see Appendix 3 for a list of samples archived). Egg envelopes and debris were periodically removed from the treatments.

C. Spawn 1

On May 23, 2005, sixteen adult females and eight adult males were collected from Kimbles Beach. The adults were transported to the HSRL and placed in aerated holding tanks (22°C, 16 ppt NaCl). Sperm was collected from five of the males and pooled together in 30 ml of ASW. Eggs from each of six females were placed separately into flat bottomed glass dishes (300 ml) with 200 ml of ASW and mixed with 1 ml of the sperm solution for 5 to 50 minutes. A sample of unfertilized eggs from one female was archived.

Fertilized eggs from each female were then placed together into each of 16 treatments using plastic transfer pipettes. Eggs from a single female were divided approximately equally among the 16 treatments. However, each of the 16 treatments had unequal amounts of fertilized eggs from the different females due to variation in the amount of eggs collected from each individual. The 16 treatments consisted of two replicates for each of eight separate treatments. Table 2 lists the treatments.

The sixteen dishes were examined after 2, 18, 25, 30, 65, and 72 days. Examination of the treatments included scanning the dishes of eggs, larvae, and juveniles using a dissecting microscope for signs of development or abnormalities and tallying undeveloped eggs, free swimming larvae, and juveniles. The stages of development outlined in Brown and Clapper (1981) were used as a reference. Periodically, juveniles were removed from the 16 treatments and placed into separate parallel treatments with replicates pooled. Juveniles received the same exposures and protocol, except for the addition of freshly hatched brine shrimp nauplii after each water change. The original 16 treatments were concluded on August 3 and any unhatched eggs or remaining larvae were archived. At the end of the parallel juvenile treatments on August 17, all juveniles from Spawn 1 were archived.

D. Spawn 2

On June 7, 2005, 39 adult females and 14 adult males were collected from Kimbles Beach. The crabs were taken to the HSRL, separated by sex, and placed in aerated holding tanks. On June 8, 13 males and 36 females were spawned. The spawning was completed in four batches with each involving 3 to 4 males and 6 to 13 females. The eggs from each batch were combined with a 3 ml suspension of sperm from the same batch and left to fertilize in 200 ml of ASW for 27 to 52

9 minutes, then rinsed with ASW and placed into new treatments. Each batch was considered a separate replicate of treatments. A sample of eggs from two batches was archived before fertilization and eggs from the other two batches were archived after fertilization. The number of eggs gathered from each female varied greatly and thus the number entering the treatments from each female varied. Table 2 lists the treatments.

The 32 dishes were examined after 30, 56, 63, and 70 days. As with Spawn 1, periodically, juveniles were removed from the treatments and placed into separate parallel treatments with replicates pooled. Juveniles received the same treatments and protocol, except for addition of freshly hatched brine shrimp nauplii during each thrice weekly solution change. The original 16 treatments were concluded on August 3 and August 10 and any unhatched eggs or remaining larvae were archived. At the end of the parallel juvenile treatments on August 17, all juveniles from Spawn 2 were archived.

E. Group 3

On July 13, eggs were collected directly from Kimbles Beach, rinsed with ASW, and placed in glass dishes in the water bath described above. Most of the eggs, when collected, appeared to be in late stages of development with the egg envelope breaking away as outlined as stages 20 and 21 in Brown and Clapper (1981). Once hatched, free swimming larvae were removed and placed into a series of ASW methoprene treatments with two replicates each. As they were found during thrice weekly solution changes, juveniles were removed and placed into separate parallel treatments with replicates grouped. On August 17 the treatments were ended, the contents of the dishes tallied, and all the remains archived separately by treatment replicate.

F. Proteomics

Two-Dimensional Electrophoresis (2DE) was used as a method for visualization and isolation of abundantly expressed proteins including those that may have been altered as a result of environmental contaminants and specifically methoprene acting as an EDC. A three phase approach was used: 1) separation of unknown proteins that are differentially expressed in control versus treated eggs, embryos, and larvae; 2) visualization of those proteins that are consistently over or under expressed in control verse treated individuals; and 3) identification of these proteins by de novo amino acid sequencing.

1. Sample processing. Samples of eggs, larvae, or juveniles that had been archived during the treatments were homogenized in 1X Tris-Buffered saline (pH 7.4) containing 20 percent glycerol, 1.0 mM dithiothreiotol, 0.1 mM EDTA, and 1 mM phenyl methyl sulfonyl fluoride with a Tissue–Tearor (Biospec Products Inc, Bartlesville, Oklahoma) homogenizer followed by ultracentrifugation. The cytosolic fraction (100,000 xg supernanant) was obtained by high speed centrifugation. Cytosolic protein content in each sample was estimated by the method of Bradford (1976) and cytosols were frozen in liquid nitrogen.

2. One dimensional gel electrophoresis and imaging. Cytosolic protein (1-5 μg ) were separated by Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) in discontinuous, pre-electrophoresed, 12% polyacrylamide gels using a Mini-Protean II apparatus

10 (Bio-Rad Laboratories, Hercules, California) according to manufacturer’s instructions. Gels were stained with either Sypro-Ruby (Invitrogen, Carlesbad, California) for total protein or Pro- Q Diamond (Invitrogen, Carlesbad California) for phosphoproteins according to manufacturer’s instructions. Fluorescent images were collected and quantified with a Fluorchem SP Imaging System (Alpha Innotech Corp, San Leandro, California) using excitation at 365 nm.

3. Western blotting (HSP70). Proteins were resolved by SDS-polyacrylamide gel electrophoresis on 10% gels and were transferred to nitrocellulose membranes (Amersham Biosciences). The membranes were probed with monoclonal antibody mouse anti-heat shock protein 70 (#MA3-006 Affinity BioReagents ,Golden, Colorado). Western blots were probed with goat anti-mouse conjugated to Alexa Fluor 680 (Invitrogen, Inc). Blotted proteins were visualized using an Odyssey infrared imaging system (LI-COR).

4. Two dimensional electrophoresis (2DE). Cytsolic proteins from day 21 free swimming larvae control, 10 µg/l, 100 µg/l) were labeled with one of three fluorescent CY dyes (Amersham Biosciences) as described previously (Tonge et al., 2001). Differentially labeled proteins from each of three different treatments (0, 10 µg/l methoprene, 100 µg/l methoprene) were then electrophoresed on the same gel. Proteins were separated by denaturing isoelectric focusing (1st dimension) using 24 cm Immoboline (Amersham Biosciences) dry strips (pH 3-7) with an IPGphore (Amersham Biosciences). For second dimension separation, the strips were transferred to large format (Ettan Dalt II Large Vertical System, Amersham Biosciences) and proteins were electrophoresed through gradient (8-15%) SDS-polyacrylamide gels. Images were collected and analyzed with a Typhoon 9400 imaging system (Amersham Biosciences) and DeCyder software (Amersham Biosciences).

11 III. RESULTS

A. Spawn 1

At day 2 after fertilization, approximately 50 Spawn 1 eggs from each treatment were examined under a dissecting microscope for signs of development or abnormalities. Accurately distinguishing the developmental stages proved impractical. Distinct stages are interspersed with indistinct opaque stages. Eggs were often coated with a fine film of organic matter that further made accurately deciphering developmental stages difficult, especially in the pore water samples. Moreover, signs of development can only be seen at certain angles. However, some degree of differentiation within most of the eggs across all treatments was visible approximately corresponding to stages 1 through 3. Table 5 displays these results. The pore water treatments appeared to have more eggs showing no differentiation (possibly infertile) but this may be due to the inability to view the development due to the organic film on the eggs.

On day 9 after fertilization, the dishes were scanned under a dissecting microscope for the furthest stage of development observable. The ASW treatments all revealed some eggs to at least stage 12 with a distinct body forming and some to stage 14 with limbbuds beginning to form. Only one (B100-1) of the 8 pore water treatments had eggs showing any sign of development and they appeared to still be in stage 1. Eggs in 5 of the pore water treatments appeared to have a red fungus-like film on them.

Twenty three days after fertilization, approximately 50 eggs from each Spawn 1 treatment were randomly scanned to determine the stages of development. Table 6 displays these results. Differentiation was difficult to see in the eggs for all stages except stage 20. However, most of the eggs in all of the treatments appeared to be in stage 20 with the egg envelope breaking away and the clearly visible larvae starting to move freely within the extraembryonic shell. The pore water samples consistently had more eggs showing no signs of development but irregardless of methoprene concentration. Many of the pore water treatment eggs showing no differentiation continued to be covered with the red fungus.

Remaining undeveloped eggs from each treatment were counted and archived at day 30; however, any abnormal eggs were counted and left in the treatments for photographing and further monitoring. On day 46 after fertilization, juveniles began to appear in the Spawn 1 treatments. Four of the 16 treatments contained juveniles (B0-1 had 3, B1-2 had 2, B10-1 had 1, and A1-1 had 21). On day 51 after fertilization, we began to remove juveniles from the Spawn 1 treatments and place them into the separate parallel juvenile treatments but with replicates combined. Any remaining eggs and embryos were removed from the Spawn 1 treatments and archived. At day 65 after fertilization, more juveniles were removed from the Spawn 1 treatments and placed into the parallel juvenile treatments. Dead larvae were removed from the Spawn 1 treatments and archived. At day 72 after fertilization, all remaining eggs and larvae were tallied and removed from the Spawn 1 treatments and archived. Any abnormal eggs or larvae were noted and some were photographed (see pictures in Appendix 4).

12 On day 79 after fertilization, all the juveniles in the Spawn 1 pore water treatments were found dead. Remains were tallied and archived. It was not possible to entirely separate the brine shrimp from the remains being archived for these and other samples.

At day 86 after fertilization, all the treatments were concluded and the remains were tallied and archived. Juvenile survivorship was poor across all the treatments. All but 50 juveniles (49 in ASW 1 µg/l and 1 in ASW 0 µg/l) were dead by day 86. No juveniles were recorded reaching their first juvenile to juvenile molt. All the juveniles were archived. Table 7 and Figure 3 summarize the results for egg development for Spawn 1. Table 8 and Figure 4 summarize the results for reaching juvenile stage for Spawn 1. Appendix 3 lists samples archived throughout the treatments.

B. Spawn 2

At day 24 after fertilization, 15 stage 20 eggs were removed from all the 16 ASW Spawn 2 treatments and archived. Too few eggs were developed in the pore water treatments to provide archive samples. On day 31 after fertilization, all the Spawn 2 pore water treatments were discarded. The treatments were filled with a white fungus and had a putrid, anoxic smell. No sign of development was discernable.

On day 31 after fertilization, eggs showing no sign of development were counted and archived, except for a few abnormal eggs left in the treatments for photographing and further observation. The remaining dish contents were tallied and the treatments continued. Table 9 lists a summary of results by treatment for day 31. The data do not appear to suggest any trend associated with methoprene concentration. Eggs in the 10 µg/l ASW treatment showed some signs of the red fungus found in pore water Spawn 1 treatments but only on undeveloped eggs.

At day 50 after fertilization, we began removing juveniles from the Spawn 2 treatments and placed them into parallel treatments with the replicates combined. On day 57 after fertilization, additional juveniles were removed from the Spawn 2 treatments and placed into the parallel treatments. Eight of the 32 treatments were stopped and the contents tallied and archived due to there being too little remaining in the treatments to warrant their maintenance. On day 64 after fertilization, additional juveniles were removed from the Spawn 2 treatments and placed into the parallel treatments and any remaining eggs and larvae in the Spawn 2 treatments were tallied and archived. Any abnormal eggs or larvae were noted and some were photographed. Table 10 summarizes the results for egg development for Spawn 2 and Figure 5 depicts the success by treatments. There does not appear to be evidence that methoprene concentration is correlated in any way with the percent of the eggs reaching a late stage of development.

Table 11 summarizes the results for reaching juvenile stage for Spawn 2 and Figure 6 depicts the percent success to juvenile stage by treatment. There does not appear to be a trend for how many juveniles were produced in a treatment or when they were found and removed from a treatment relative to methoprene concentration.

13 C. Group 3

No treatment effect was apparent with the Group 3 results. Table 12 shows the results of the Group 3 treatments. The larvae from each treatment appeared to have similar success reaching the larva-juvenile molt.

D. Macroscopic abnormalities

Several of the treatments produced embryos and larvae that were abnormal in appearance. Appendix 4 includes pictures of some of these abnormalities. Abnormal eggs or larvae were observed and removed from the treatments as depicted in Table 13. Note that the table only lists those abnormalities that were ultimately removed. For example abnormal eggs or embryos may have been observed but were left in the treatments and thus may be ultimately recorded as abnormal larvae.

Abnormal eggs were generally asymmetrical, included two fused eggs, expanded but with no development inside, or were yellowed. The yellowed eggs were the most common and may have been simply infertile. Abnormal embryos generally had a bell shaped prosoma or some other disfigurement or there was an opaque partly developed embryo in a clear egg. Abnormal larvae had a bell shaped prosoma or were asymmetrical. No abnormal juveniles were observed. No trends were observed that indicated the abnormalities were correlated with any component of the treatments.

E. Proteomics

Results from proteomics and biomarkers components of this study indicated no significant treatment-related responses resulting from methoprene exposure. Prior to two dimensional analyses, samples from all treatments and time points were screened by SDS-PAGE with Sypro- Ruby stain. Our intention was to identify obvious large differences in protein expression as a result of exposure in specific life stages following exposures. These samples would then become candidates for more exhaustive and revealing 2DE. No such differences were revealed by one dimensional electrophoresis by theses analyses (Figure 8). In the absence of obvious treatment- related differences revealed by this approach, we chose day 21 free swimming larvae from two separate experiments (i.e., Spawn 1 and 2) for subsequent 2DE for the three treatment concentrations and the control. 2DE of differentially tagged proteins was performed in order to identify specific changes in protein expression resulting from methoprene exposure. For 2DE, we combined day 21 free swimming larvae from two separate experiments (Spawn 1 and Spawn 2) in a randomized block design. Using different fluorescent labels for proteins in each treatment, large differences in protein expression would be expected to appear as intense red or green spots in gel overlays; slight differences (≤ two- fold) as faint pink or light green spots; no difference as yellow spots (Tonge et al., 2001). Although we observed several differences in protein expression between experiments (Figure 9), few differences appear to have resulted from methoprene exposure itself (Figure 10). For example, no treatment related differences were observed in levels of HSP70 during our study (Figure 11). We did not observe consistent treatment-related differences in patterns of expression of phosphoproteins in eggs, larvae, or juvenile horseshoe crabs (Figure 12).

IV. DISCUSSION

A. Treatments

Accurately examining early signs of development in the eggs was not possible. However, later stages of development in the eggs and free swimming larvae and juveniles were easily distinguished. No sign of treatment effects was observed by looking at development success.

The results of this investigation were surprising due to their apparent contrast to previous studies on other taxa. For example, Olmstead and Leblanc (2001a and 2001b) found methoprene concentrations as low as 6.4 nM altered the ratio of sexual verse parthenogenetic reproduction in Daphnia magna. However, others have found results of arthropods not being sensitive to methoprene. For example, Zulkosky et al. (2005) found methoprene showed no toxicity to American lobsters at the highest concentration (10 µg/l) they tested.

The methoprene exposure route may not have been entirely relevant to field conditions. However, the experimental design was probably more likely to allow for higher exposures due to a lack of sediment in the treatments. Methoprene will quickly adsorb to sediment. On July 1, one larva was removed from all Spawn 1 pore water treatments and placed into a dish with sand and ASW. All these larvae had buried themselves in the sand within 3 hours and remained that way through the conclusion of the treatments. We performed the treatments without a bottom substrate to allow easy monitoring and collection of the eggs, larvae, and juveniles as well as to be able to change the treatment solutions.

Carriers (e.g., ethanol or acetone) were not considered necessary in the treatments. However, a solvent/carrier mixed into the treatments could have increased exposures and produced different results. However, the toxicity of a carrier like ethanol or acetone would be an added variable. The Altosid SR-5® appeared to be sufficiently soluble even when making 10 percent solutions during the dilutions series. Additionally, solvents are not part of the Altosid SR-5® formulation and would not be part of an actual environmental exposure. The Altosid SR-5® should have remained effective during the 1 to 2 day periods between solution changes for the treatments. Altosid SR-5® is applied every 7 to 10 days during actual field use (Wellmark International, 2000).

We did not reach a point in any of the treatments to see the first juvenile to juvenile molt. This molt would be expected to occur approximately 4½ months after hatching (Brown and Clapper, 1981). Perfecting the rearing of the juveniles and more thoroughly examining the effects of an endocrine disruptor on this second molt rather than the egg, larval, and first juvenile stages may prove more revealing. The effects of the methoprene could have been mitigated for by the egg barrier and the fact that the larvae do not feed. Generally the number of juveniles removed from the original treatments did not equal the number in the final juvenile samples. This discrepancy could be due to several factors such as the remains of the juveniles being hard to separate and count.

Treatments in pore water from Kimbles Beach clearly had lower development success than those in ASW for Spawn 1 and did not develop to a stage to allow any observations for Spawn 2. The

16 likely explanation of the poor success in the pore water was bacterial or fungal growth as indicated by the film covering the eggs and the putrid smells of the treatments. This effect may have been removable with water treatment albeit by introducing an additional chemical variable. Additionally, a red growth was common on many of the egg surfaces, particularly those in pore water. The fine particles in the pore water may have also created higher biological oxygen demand, reduced dissolved oxygen, and lower development success. Another less likely explanation for the lower success in the pore water treatments could be an additional contaminant in the pore water that acts adversely but independently of the methoprene. The numerous contaminant sources to Delaware Bay, such as industrial and municipal effluent and urban runoff, introduce a mix of chemicals with unknown impact upon L. polyphemus. This same investigation could be performed with a different putative EDC found in beach sediments or Delaware Bay water. Chemical analysis of beach sediments and near-shore Delaware Bay water would be necessary. Methoprene was used for this investigation because it is known to be used in the study area and is specifically designed to be an endocrine disruptor to arthropods.

B. Proteomics

Results from proteomics and biomarker components of this study indicated no significant treatment-related responses resulting from methoprene exposure. Combining day 21 free swimming larvae from two separate experiments (Spawn 1 and Spawn 2) may have confounded visualization of results but not likely the overall conclusions. We are in the process of performing similar trials (for publication purposes) where individual samples from the two different exposures are separated in all gels. However, based on results thus far, it is unlikely that major changes in protein expression resulting from methoprene exposure will be revealed.

Cells have developed numerous adaptive and protective mechanisms to various forms of environmental stress. Among these, heat shock proteins (HSP) are molecular chaperones, involved in many cellular functions such as protein folding, transport, maturation and degradation (Mallouk et al., 1999). Altered levels of HSP70 have been observed in diverse organisms exposed to a variety of environmental stressors including anthropogenic chemicals (Werner and Nagel, 1997; Ait-Aissa et al., 2000; Staempfli et al., 2002). Walker et al. (2005) reported large differences in expression of HSP70 in methoprene–exposed lobster larvae. Therefore, the lack of any treatment effects observed in this investigation was surprising.

Within the cell, protein kinases and protein phosphatases act as molecular “on” and “off” switches in diverse signaling pathways mediated by hormone-receptor interaction (Katzenellenbogen, 1996; Driggers and Segars, 2002; Freeman and Gordon, 2002). By phosphorylating signal proteins, specific kinases turn specific proteins and pathways “on” while dephosphorylations catalyzed by protein phosphatase deactivate these proteins and pathways they regulate. These mechanisms are highly conserved in nature and have been identified in all classes of organisms from bacteria, yeast to mammals (Sears and Nevins, 2002). Walker et al. (2005) reported that juvenile lobster exhibited altered pattern of protein phosphorylation following methoprene exposures suggesting that methoprene may interfere with specific cell signaling pathways in this crustacean. In that study, no attempt was made to identify the proteins or pathways affected. In our study, we did not observe consistent treatment-related differences

17 in patterns of expression of phosphoproteins in eggs, larvae, or juvenile horseshoe crabs suggesting that similar pathways were unaffected in this species.

Results from the biomarker component of this study are in agreement in the apparent absence of toxicity (hatching success, survival, growth, development) observed during horseshoe crab exposures. Differences observed between our study and that of Walker et al. (2005) with lobster likely involve differences in signaling molecules used by crustaceans and Chelicerata. Crustaceans, like insects, have JH-like compounds that are involved in the control of their larval development and reproduction. Numerous studies indicate that methoprene adversely affects growth and molting in several crustacean species (DeFur et al., 1999; Olmstead and LeBlanc, 2001a and 2001b; Ghekiere et al., 2006).

Hormone-receptor interaction requires specific recognition of the hormone by its receptor. In the absence of such recognition, hormone-receptor mediated activation of specific pathways cannot occur. It is possible that pathways regulating growth, molting, and development of Chelicerata are similar to that of crustaceans and insects. However, the specific structure of the JH counterpart in Chelicerata may be substantially different from that of crustaceans and insects. Thus, JH hormone analogs such as methoprene may not mimic any physiological hormones in L. polyphemus and may not be recognized by a specific (ecdysteroid) receptor in this organism. If this is the case, then there would be no specific pathways to trick into receiving the wrong set of signals.

Belozerov (2001) reported results of several studies that indicated effects of methoprene on molting and limb regeneration in ticks and mites. Because these processes are normally under the control of JH-like compounds, the investigator concluded that JH- or methyl farnesoate-like compounds may also be present in these organisms. To our knowledge, JH or methyl farnesoate have not been identified in any of the Chelicerata. This area of research appears to be controversial. At least one investigator suggests that spiders lack JH entirely (Craig, 2003).

C. No Observed Adverse Effect Level

This study provides evidence that 100 µg/l of methoprene is a No Observed Adverse Effect Level (i.e., NOAEL) on L. polyphemus. The publication of this number will provide useful information to managers of L. polyphemus. The data may ultimately be applicable in criteria development for methoprene or other novel EDCs. Development of methodology for examining endocrine disruption in L. polyphemus builds a foundation from which more refined and ambitious studies with broader management implications can be pursued.

D. Management actions

The study addressed two of six priorities identified by the Delaware River/Delmarva Coastal Watershed Ecosystem Team (U.S. Fish and Wildlife Service, 1996). Resource Priority 1 addresses the need to “protect, restore and enhance migratory bird habitat and populations...” and Strategy 2, Action E addresses the need to “document the cumulative environmental impacts and develop protection strategies for waterfowl, colonial nesting waterbirds and shorebirds.” The second priority addresses Resource Priority 2, which is to “protect, restore, and enhance wetland

18 values, with emphasis on areas of exceptional value.” Strategy 7 directs Service programs to “identify, and prioritize environmental contaminant impacts to natural resources and coordinate Service activities.”

The principal reason for conducting this investigation was to examine a potential contributing factor to the relationship between L. polyphemus and migrating bird populations. We had hypothesized that population decline in L. polyphemus and the correlated decline in avian predators could be associated with contaminant exposure to early developmental crab stages.

Developing an understanding of limiting factors on L. polyphemus equates to developing an understanding of limiting factors on shorebird species such as the red knot (Calidris canutus rufa). This investigation did not provide information suggesting methoprene is a limiting factor on the L. polyphemus population. Evidence was not found that an alteration of mosquito control pesticide spraying practices is necessary for the protection of L. polyphemus. Groups such as the Atlantic States Marine Fisheries Commission, Delaware River Basin Commission, fisheries interests, the New Jersey Department of Environmental Protection, the Delaware Department of Natural Resources and Environmental Control, the medical industry, and passive recreational natural resource industry will benefit from understanding what role contaminants may play in L. polyphemus biology.

E. Summary of scientific objectives for this investigation

For all of the four objectives of this study, evidence was found supportive of the null hypotheses.

Null hypothesis: No difference will occur in L. polyphemus molting success between embryos developed in treatment solutions compared to control solution.

Hypothesis 2: There will be a protein specific pattern difference between treatment and control embryos as demonstrated by two dimensional gel electrophoresis and subsequent de novo sequencing.

Null hypothesis: No difference will occur between treatment and control individuals.

Null hypothesis: A known EDC will not produce adverse impacts L. polyphemus embryos and larvae.

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27 VI. FIGURES

Figure 3. Development success for Spawn 1 treatments.

ASW Pore water 0.95

0.9

0.85

0.8

0.75

0.7

0.65 Percent reaching at least stage 20 stage least reaching at Percent

0.6 0 20 40 60 80 100 Methoprene concentration (µg/l)

31 Figure 4. Percent of Spawn 1 eggs reaching juvenile stage.

ASW Pore water 0.65

0.6

0.55

0.5

0.45

0.4

0.35 Percent to juvenilePercent stage

0.3

0.25 0 20 40 60 80 100 Methoprene concentration (µg/l)

Figure 5. Development success for Spawn 2 treatments.

ASW 0.66

0.64

0.62

0.6

0.58

0.56

0.54 Percent reaching at least stage 20 stage least reaching at Percent

0.52 0 20 40 60 80 100 Methoprene concentration (µg/l)

33 Figure 6. Percent of Spawn 2 eggs reaching juvenile stage.

ASW 0.35

0.3

0.25

0.2

0.15 Percent to juvenilePercent stage 0.1

0.05 0 20 40 60 80 100 Methoprene concentration (µg/l)

34 Figure 7. Percent of Group 3 larvae reaching the juvenile stage.

ASW 0.91

0.89

0.87

0.85

0.83

0.81

0.79 Percent larvae to juvenilelarvae Percent molt 0.77

0.75 0 20 40 60 80 100 Methoprene concentration (µg/l)

35 Figure 8. Representative cytosolic proteins from day xx larvae (panel a) and day yy larvae (panel b) stained with Sypro-Ruby (Invitrogen, Inc.) for total protein. No consistent treatment related differences (control, 10ug/l, 100 ug/l) were observed at these or other time points during the study.

a) b)

75 kD

25 kD control 10 ug/l 100 ug/l control 10 ug/l 100 ug/l

36 Figure 9. Representative gel 211 two dimensional analyses of florescent-tagged proteins. Large differences in expression of individual proteins (red and green spots) are the result of differences between experiments.

37 gel 212 Figure 10. Representative two dimensional analyses of florescent-tagged proteins in control and methoprene-exposed day 21 free swimming larvae. Small differences in protein expression appear as pale green or pink spots

Figure 11. Representative Western blot of HSP70 in day 21 free swimming larvae.

control 10 ug/l 100 ug/l

39 Figure 12. Phosphoproteins in control and methoprene-exposed day 21 free swimming larvae.

150

110

25 control 0 ug/l 10 ug/l 100 ug l

40 VII. TABLES

Table 1. Dilution series for deriving methoprene solutions using Altosid SR-5® (51.3 g/l).

Dilution Altosid Water Methoprene (µg/l) 51,300,000 Dilution 1 100x 1 ml 99 ml 513,000 Dilution 2 500x 1 ml 499 ml 1,026 Dilution 3 10x 50 ml 450 ml 102.6 Dilution 4 10x 50 ml 450 ml 10.26 Dilution 5 10x 50 ml 450 ml 1.026

42 Table 2. The 16 methoprene treatments used for Spawn 1 exposures.

Artificial sea water treatments Beach pore water treatments replicate 1 replicate 2 replicate 1 replicate 2 0 µg / l 0 µg / l 0 µg / l 0 µg / l 1 µg / l 1 µg / l 1 µg / l 1 µg / l 10 µg / l 10 µg / l 10 µg / l 10 µg / l 100 µg / l 100 µg / l 100 µg / l 100 µg / l

43 Table 3. The 32 methoprene treatments used for Spawn 2 exposures.

Artificial sea water treatments replicate 1 replicate 2 replicate 3 replicate 4 0 µg / l 0 µg / l 0 µg / l 0 µg / l 1 µg / l 1 µg / l 1 µg / l 1 µg / l 10 µg / l 10 µg / l 10 µg / l 10 µg / l 100 µg / l 100 µg / l 100 µg / l 100 µg / l

Beach pore water treatments replicate 1 replicate 2 replicate 3 replicate 4 0 µg / l 0 µg / l 0 µg / l 0 µg / l 1 µg / l 1 µg / l 1 µg / l 1 µg / l 10 µg / l 10 µg / l 10 µg / l 10 µg / l 100 µg / l 100 µg / l 100 µg / l 100 µg / l

44 Table 4. The 8 methoprene treatments used for Group 3 exposures.

Artificial sea water treatments replicate 1 replicate 2 0 µg / l 0 µg / l 1 µg / l 1 µg / l 10 µg / l 10 µg / l 100 µg / l 100 µg / l

45 Table 5. Development of samples of approximately 50 eggs from each Spawn 1 treatment at day 2 after fertilization. Stages of development (18-20 to 18-26) correspond to figures in Brown and Clapper (1981) and are listed in Appendix 1.

Treatment Estimated stage of development

18 18 18 18 18 No

- - - - - Water Methoprene Replicate Abnormal different 20 21 22 25 26 -tiation

ASW 0 µg/l 1 0 0 8 0 15 16 6 ASW 0 µg/l 2 0 1 19 0 18 17 2 ASW 1 µg/l 1 0 0 20 0 25 10 0 ASW 1 µg/l 2 0 1 18 0 20 10 0 ASW 10 µg/l 1 0 0 6 0 25 24 4 ASW 10 µg/l 2 1 0 3 0 18 23 6 ASW 100 µg/l 1 0 19 14 0 10 8 0 ASW 100 µg/l 2 2 0 9 0 31 7 1 Pore 0 µg/l 1 0 24 8 0 7 11 0 Pore 0 µg/l 2 1 6 31 0 3 10 0 Pore 1 µg/l 1 0 10 4 0 13 28 0 Pore 1 µg/l 2 1 4 18 1 19 13 0 Pore 10 µg/l 1 0 23 1 0 17 11 0 Pore 10 µg/l 2 0 10 2 0 30 10 0 Pore 100 µg/l 1 1 18 15 0 6 10 0 Pore 100 µg/l 2 2 29 10 0 5 4 0

46 Table 6. Development of samples of approximately 50 eggs from each Spawn 1 treatment at day 23 after fertilization. Stages of development correspond to those in Brown and Clapper (1981) and are depicted in a table in Appendix 1.

Treatment Estimated stage of development

No differen- Water Methoprene Replicate Abnormal tiation 18 19 20

ASW 0 µg/l 1 0 1 0 4 45 ASW 0 µg/l 2 0 3 8 5 37 ASW 1 µg/l 1 0 5 1 0 44 ASW 1 µg/l 2 0 3 5 0 42 ASW 10 µg/l 1 0 6 0 15 34 ASW 10 µg/l 2 0 3 12 3 35 ASW 100 µg/l 1 0 8 16 8 19 ASW 100 µg/l 2 0 1 0 1 48 Pore 0 µg/l 1 0 19 0 0 33 Pore 0 µg/l 2 0 24 0 0 28 Pore 1 µg/l 1 0 12 2 0 35 Pore 1 µg/l 2 0 22 0 0 29 Pore 10 µg/l 1 0 16 1 0 46 Pore 10 µg/l 2 0 24 28 0 15 Pore 100 µg/l 1 0 12 0 0 38 Pore 100 µg/l 2 1 20 2 0 27

47 Table 7. Percent of Spawn 1 eggs reaching a late stage of embryonic development.

Treatment Total Total eggs Total eggs Percent Replicate undeveloped reaching at originally in reaching at Water Methoprene eggs least stage 20 treatment least stage 20 ASW 0 µg/l 1 13 167 180 0.928 ASW 0 µg/l 2 22 179 201 0.891 ASW 1 µg/l 1 13 143 156 0.917 ASW 1 µg/l 2 16 155 171 0.906 ASW 10 µg/l 1 22 157 179 0.877 ASW 10 µg/l 2 26 161 187 0.861 ASW 100 µg/l 1 22 162 184 0.880 ASW 100 µg/l 2 19 182 201 0.905 Pore 0 µg/l 1 53 98 151 0.649 Pore 0 µg/l 2 43 125 168 0.744 Pore 1 µg/l 1 64 150 214 0.701 Pore 1 µg/l 2 44 128 172 0.744 Pore 10 µg/l 1 44 147 191 0.770 Pore 10 µg/l 2 53 132 185 0.714 Pore 100 µg/l 1 41 132 173 0.763 Pore 100 µg/l 2 46 91 137 0.664

48 Table 8. Percent of Spawn 1 eggs reaching juvenile stage and dates removed from the treatments.

Total molts recorded molts Total

Total eggs originally originally eggs Total

Percent success to success to Percent

Treatment stage juvenile

in treatmentin

7/13/2005 7/14/2005 7/15/2005 7/18/2005 7/20/2005 7/22/2005 7/25/2 7/27/2005 7/29/2005

Replicate

8/1/2005 8/3/2005

Methoprene

005

Water

ASW 0 µg/l 1 25 2 9 35 6 9 8 2 0 1 1 98 180 0.544 ASW 0 µg/l 2 1 0 6 10 27 17 18 5 13 9 0 106 201 0.527 ASW 1 µg/l 1 57 9 2 2 1 4 6 5 1 1 0 88 156 0.564 ASW 1 µg/l 2 11 1 0 64 17 0 5 2 3 0 1 104 171 0.608 ASW 10 µg/l 1 4 0 2 27 3 20 18 5 9 5 1 94 179 0.525 ASW 10 µg/l 2 1 0 0 21 45 13 10 3 3 3 0 99 187 0.529 ASW 100 µg/l 1 0 0 0 0 4 5 26 10 20 18 4 87 184 0.473 ASW 100 µg/l 2 2 0 2 9 34 16 16 9 7 4 3 102 201 0.507 Pore 0 µg/l 1 25 2 1 2 0 6 5 0 0 0 1 42 151 0.278 Pore 0 µg/l 2 20 1 5 9 1 8 7 2 3 1 5 62 168 0.369 Pore 1 µg/l 1 36 2 1 10 2 6 1 0 1 1 0 60 214 0.280 Pore 1 µg/l 2 42 3 2 3 0 18 4 1 6 1 0 80 172 0.465 Pore 10 µg/l 1 34 2 3 10 3 13 8 4 1 1 0 79 191 0.414 Pore 10 µg/l 2 0 3 0 6 2 21 30 2 3 1 0 68 185 0.368 Pore 100 µg/l 1 8 3 1 16 7 5 11 2 5 4 0 62 173 0.358 Pore 100 µg/l 2 1 0 2 3 5 10 6 3 1 2 2 35 137 0.255

49 Table 9. Development from each Spawn 2 treatment at day 31 after fertilization. Stages of development correspond to those in Brown and Clapper (1981) and are depicted in a table in Appendix 1. Tallies do not include eggs removed on July 1 but do include larvae removed July 8.

Treatment Estimated stage of development

No Water Methoprene Replicate Abnormal differen- 20-21 Larvae tiation

ASW 0 µg/l 1 1 360 3 92 ASW 0 µg/l 2 4 140 30 37 ASW 0 µg/l 3 3 169 41 19 ASW 0 µg/l 4 14 106 19 110 ASW 1 µg/l 1 4 304 21 42 ASW 1 µg/l 2 1 151 1 84 ASW 1 µg/l 3 6 153 3 51 ASW 1 µg/l 4 14 126 68 45 ASW 10 µg/l 1 0 370 13 58 ASW 10 µg/l 2 0 187 3 60 ASW 10 µg/l 3 4 139 31 33 ASW 10 µg/l 4 15 114 49 59 ASW 100 µg/l 1 0 385 0 92 ASW 100 µg/l 2 1 154 4 58 ASW 100 µg/l 3 1 156 0 58 ASW 100 µg/l 4 0 144 112 37

Table 10. Percent of Spawn 2 eggs reaching a late stage of embryonic development.

Treatment Total Total eggs Total eggs Percent Replicate undeveloped reaching at originally in reaching at Water Methoprene eggs least stage 20 treatment least stage 20 ASW 0 µg/l 1 361 404 765 0.528 ASW 0 µg/l 2 147 213 360 0.592 ASW 0 µg/l 3 173 211 384 0.549 ASW 0 µg/l 4 120 178 298 0.597 ASW 1 µg/l 1 310 348 658 0.529 ASW 1 µg/l 2 152 188 340 0.553 ASW 1 µg/l 3 159 193 352 0.548 ASW 1 µg/l 4 140 268 408 0.657 ASW 10 µg/l 1 371 423 794 0.533 ASW 10 µg/l 2 187 225 412 0.546 ASW 10 µg/l 3 144 178 322 0.553 ASW 10 µg/l 4 126 194 320 0.606 ASW 100 µg/l 1 387 425 812 0.523 ASW 100 µg/l 2 155 191 346 0.552 ASW 100 µg/l 3 157 187 344 0.544 ASW 100 µg/l 4 144 198 342 0.579

Table 11. Percent of Spawn 2 eggs reaching juvenile stage and dates removed from the treatments.

Total molts recorded molts Total

Total eggs originally originally eggs Total

Percent success to success to Percent

Treatment stage juvenile

in treatmentin

7/22/2005 7/25/2005 7/27/2005 7/29/2005 8/10/2005

Replicate

8/1/2005 8/3/2005 8/5/2005 8/8/2005

Methoprene

Water

ASW 0 µg/l 1 14 4 20 17 5 2 0 0 0 62 765 0.081 ASW 0 µg/l 2 0 1 9 10 11 3 0 NA NA 34 360 0.094 ASW 0 µg/l 3 0 0 7 30 2 0 0 NA NA 39 384 0.102 ASW 0 µg/l 4 0 23 34 20 9 2 3 4 1 96 298 0.322 ASW 1 µg/l 1 1 3 13 12 7 2 3 0 1 42 658 0.064 ASW 1 µg/l 2 2 26 15 7 14 2 0 NA NA 66 340 0.194 ASW 1 µg/l 3 1 28 2 3 2 0 0 NA NA 36 352 0.102 ASW 1 µg/l 4 0 0 6 9 15 16 2 1 1 50 408 0.123 ASW 10 µg/l 1 0 13 11 12 5 0 0 3 0 44 794 0.055 ASW 10 µg/l 2 0 15 13 7 6 4 0 NA NA 45 412 0.109 ASW 10 µg/l 3 2 12 2 5 1 0 0 NA NA 22 322 0.068 ASW 10 µg/l 4 0 2 6 18 16 14 4 9 8 77 320 0.241 ASW 100 µg/l 1 10 19 13 25 2 0 0 3 0 72 812 0.089 ASW 100 µg/l 2 1 20 8 9 4 0 0 NA NA 42 346 0.121 ASW 100 µg/l 3 2 31 5 2 2 0 0 NA NA 42 344 0.122 ASW 100 µg/l 4 0 1 3 10 59 9 1 14 5 102 342 0.298

52 Table 12. Summary of Group 3 juveniles removed from larvae treatments (from treatments started on 7/20/05 for larvae sorted out from 7/13/05 beach collected, fertilized eggs).

8/1/2005 8/3/2005 8/5/2005 8/8/2005 8/10/2005 8/17/2005 removed juveniles total 8/17/05 larvae remaining in treatment put total molt to success percent

Treatment

Water Methop Replicate

rene

ASW 0 µg/l 1 5 15 14 29 16 4 83 16 99 0.838 ASW 0 µg/l 2 6 26 14 41 7 8 102 26 128 0.797 ASW 1 µg/l 1 9 23 12 16 1 12 73 12 85 0.859 ASW 1 µg/l 2 15 24 12 20 11 5 87 9 96 0.906 ASW 10 µg/l 1 8 30 20 30 6 6 100 12 112 0.893 ASW 10 µg/l 2 4 9 12 33 1 2 61 20 81 0.753 ASW 100 µg/l 1 4 14 14 29 10 10 81 14 95 0.853 ASW 100 µg/l 2 3 6 17 29 11 13 79 18 97 0.814

Table 13. Frequency of abnormalities removed from treatments (replicates combined).

Water Methoprene Eggs Embryos Larvae Juveniles Spawn 1 ASW 0 µg/l 1 0 1 0 ASW 1 µg/l 2 0 0 0 ASW 10 µg/l 3 0 0 0 ASW 100 µg/l 1 2 0 0 Pore 0 µg/l 6 1 3 0 Pore 1 µg/l 5 2 2 0 Pore 10 µg/l 1 1 0 0 Pore 100 µg/l 0 0 0 0

Spawn 2 ASW 0 µg/l 19 0 0 0 ASW 1 µg/l 0 2 0 0 ASW 10 µg/l 4 2 1 0 ASW 100 µg/l 1 0 0 0

Group 3 ASW 0 µg/l NA NA NA 0 ASW 1 µg/l NA NA NA 0 ASW 10 µg/l NA NA NA 0 ASW 100 µg/l NA NA NA 0

VIII. APPENDICES

Appendix 1

Stages of horseshoe crab development (Brown and Clapper, 1981)

Time from Stage Description fertilization Unfertilized egg. The surface is smooth. Indentations are caused by 1 0 oviducal packing and will eventually disappear. The appearance, coalescence and disappearance of pits represent the egg 1 30 min cortical reaction. Pit appearance can usually be observed 10 min after insemination and the pits appear uniformly over the surface. After disappearance of the pits, the surface becomes smooth. The 1 45 min duration of the cortical reaction is variable but generally is completed by this time. The first granulation of the surface begins to appear about 2 ½ hr after fertilization and end 1 ½ hr later when surface becomes smooth. A 1 3 ½ hr second granulation cycle will occur between 5 hr and 11 hr after fertilization. 1 4 Smooth surface embryo between the first two granulation cycles. On the surface, 8-16 large nuclei are equally distributed. They frequently appear in pairs as if in mitosis. Often, but not always, each nucleus 2 21-28 hr appears to be enclosed in a cell. A third granulation cycle often makes viewing of the nuclei difficult. Segmentation or yolk block formation, possibly representing the beginning of blastulation. The stage begins with deep grooves and the appearance of 4-8 large blocks and continues to about 150 blastomeres on 3 28-46 hr surface. Another granulation cycle occurs on the surfaced of each blastomere near end of stage. Nuclei are rarely obvious, although some are observed between the large blocks. Immediately after the previously mentioned granulation cycle is completed, the nucleus in each blastomere is obvious. Blastomeres vary 4 45-58 hr greatly in shape and size and the surface of the embryo is quite irregular. Patches of smaller blastomeres are frequently observed in the latter half of this stage. Due to rapid cell division, blastomeres are now smaller in size and more uniform in shape. They are loosely attached to one another and give a 5 58-73 hr jumbled appearance to surface of embryo. However, as this stage progresses, surface becomes smoother. Unlike previous stage, difficult to see margins of blastomeres and nuclei 6 3 ½ days appear equally distributed. Embryo appears very smooth with a few cells sticking above the surface. Individual blastomeres can only be observed at high magnifications. The 7 4 days appearance of the irregularly shaped indentation represents the area which will become the germ disc.

Time from Stage Description fertilization Area around previous indentation is now uplifting and is approximately 8 5 days 1/16 the diameter of the embryo. This area is the developing germ disc. Germ disc increases in size to approximate ¼ diameter of embryo. The disc is still an uplifted area and has equal margins on all sides. 9 6 days Unfortunately, stages 9, 10, and 11 are very difficult to reproduce photographically, as the germ disc is difficult to observe. Margin is not obvious in light micrograph. Germ disc is now approximately ½ diameter of embryo and can be observed as an uplifted area if embryo is viewed laterally. This stage is 10 7 days unfortunately variable since germ disc does not always appear as an uplifted area but sometimes quite smooth. The germ disc definitely has a marginal groove which is much more 11 8 days pronounced along one edge. If viewed laterally, this particular edge appears as a deep groove. Usually multiple grooves become obvious around germ disc. In addition, 12 9 days extensive wrinkles and ridges are observed in other regions of embryo and continue to be present throughout stage 12 and into stage 13. Germ disc continues to differentiate with multiple grooves around margin 13 10 days becoming obvious. Characteristic is the presence of an apparent groove transecting the germ disc. Six pairs of elongated blocks representing the limbbuds are observed. In 14 11 days addition the abdominal region shows some differentiation. The first five pairs of limbbuds are becoming round. The stomodaeum 15 12 days appears and is anterior to the first pair of limbbuds. The other regions of embryo are smooth. As limbbuds elongate into appendages, stomodaeum moves posteriorly to 16 13 days a position between first pair of appendages. Round lateral organs appear laterally to fourth pair of appendages. Final stage before first embryonic molt. If carefully examined the loosening of the exuvia can eventually be observed in the region between the fourth-sixth pairs of appendages. Appendages are becoming articulated and frequently move. Anterior end continues to differentiate 17 14 days and stomodaeum is located at the end of a ridge-like structure. Opisthosomal segmentation is obvious. By careful focusing, polygonal plates of extra-embryonic shell (located beneath egg envelope) can be observed. Material released during first embryonic molt is very cloudy and obscures many features of this stage. However, initial segmentation of the green 18 15 days hepatopancreas (occupying most of the embryo’s internal space) can be observed.

Time from Stage Description fertilization After second embryonic molt, body segmentation of hepatopancreas has become more pronounced and extends over the entire dorsal surface of 19 17 days this organ. Although medium surrounding embryo is clearer than before, the extra-embryonic shell is more opaque and causes difficulties in observing embryo. Lateral organs are oval. After third embryonic molt, the egg envelope breaks away. The process is slow with a crack occurring in the egg envelope and the extra- 20 21 days embryonic shell eventually swelling to maximum size. Infrequently, this swelling can occur earlier in stage 19. The egg envelope in time falls away. The larva freely moves around within the extra-embryonic shell. If no obstructions are present, larval movements cause rolling around of shell 20 22 days in the Petri dish. As a result, behavioral responses to various tropisms can be studied. The fourth embryonic molt produces the trilobite larva, which generally 21 26 days hatches from extra-embryonic shell in 1-1 ½ days. The opisthosomal appendages are greatly flattened. Hatched trilobite larva swims freely and frequently in a dorsal down 21 27 days position. Posterior margin of opisthosoma is serrated and sensory filaments can be observed on prosoma. Larva does not feed. 59 days First posthatched juvenile. Begins feeding. 4 ½ months second posthatched juvenile 6 months third posthatched juvenile 7 ½ months fourth posthatched juvenile 9 months fifth posthatched juvenile

3 Appendix 2

Treatment data

SPAWN 1 EGGS AND LARVAE

5/23/2005 Spawn 1 completed and 8 treatments x 2 replicates started (sample of unfertilized eggs from one female placed in cryovial and archived)

5/25/2005 Brown and Clapper (1981) stage of approximately 50 randomly selected eggs (value of counts questionable because seeing and distinguishing stages very difficult)

no sign of total eggs 18-20 18-21 18-22 18-25 18-26 abnormal? development counted A0.1 0 8 0 15 16 6 0 45 A0.2 1 19 0 18 17 2 0 57 A1.1 0 20 0 25 10 0 0 55 A1.2 1 18 0 20 10 0 0 49 A10.1 0 6 0 25 24 4 0 59 A10.2 0 3 0 18 23 6 1 51 A100.1 13 14 0 10 9 0 0 46 A100.2 0 9 0 31 7 1 2 50 B0.1 24 8 0 7 11 0 0 50 B0.2 6 31 0 3 10 0 1 51 B1.1 10 4 0 13 23 0 0 50 B1.2 4 18 1 19 13 0 1 56 B10.1 23 1 0 17 11 0 0 52 B10.2 10 2 0 30 10 0 0 52 B100.1 18 15 0 6 10 0 1 50 B100.2 28 10 0 5 4 0 2 49 (fine organic film on bay water eggs further conceals development)

6/10/2005 archived eggs

vials marked as no. of eggs follows (replicates combined) A0.1 15 A0 610 A0.2 15 A1.1 15 A1 610 A1.2 15 A10.1 15 A10 610 A10.2 15 A100.1 15 A100 610 A100.2 15 B0.1 15 B0 610 B0.2 15 B1.1 15 B1 610

1 B1.2 15 B10.1 15 B10 610 B10.2 15 B100.1 15 B100 610 B100.2 15

6/15/2005 Brown and Clapper (1981) stage of approximately 50 randomly selected eggs (value of counts questionable because seeing and distinguishing stages very difficult)

smooth no smooth no total eggs Stage 18-19 Stage 20 differentiation irregular differentiation counted with red A0.1 4 45 1 0 0 50 A0.2 13 32 3 0 0 48 A1.1 1 44 5 0 0 50 A1.2 5 42 3 0 0 50 A10.1 15 34 6 0 0 55 A10.2 15 35 3 0 0 53 A100.1 24 19 8 0 0 51 A100.2 1 48 1 0 0 50 B0.1 0 33 19 0 0 50 B0.2 0 28 23 1 0 48 B1.1 2 35 7 6 0 50 B1.2 0 29 8 14 0 50 B10.1 1 46 16 0 0 55 B10.2 28 15 23 1 0 53 B100.1 0 38 9 4 0 51 B100.2 2 27 9 12 1 50 (fine organic film on bay water eggs further conceals development)

6/17/2005 archived stage 20 eggs

vial grease pencil no. of eggs marked as vial marking A0-1 15 6-17 A0-1 A A0-2 15 6-17 A0-2 B A1-1 15 6-17 A1-1 C A1-2 15 6-17 A1-2 D 6-17 A10- A10-1 15 E 1 6-17 A10- A10-2 15 F 2 6-17 A100-1 15 G A100-1 6-17 A100-2 15 H A100-2 B0-1 15 6-17 B0-1 I B0-2 15 6-17 B0-2 J

2 B1-1 15 6-17 B1-1 K B1-2 15 6-17 B1-2 L 6-17 B10- B10-1 15 M 1 6-17 B10- B10-2 15 N 2 6-17 B100-1 15 O B100-1 6-17 B100-2 15 P B100-2

6/22/2005 archived all Spawn 1 eggs not showing signs of development (a few potentially abnormal eggs were left in treatments to monitor for signs of development, to photograph, etc.)

vial no. of eggs marked as A0-1 6-22 A0-1 12 E A0-2 6-22 A0-2 22 E A1-1 6-22 A1-1 13 E A1-2 6-22 A1-2 13 E A10-1 6- A10-1 21 22 E A10-2 6- A10-2 24 22 E A100-1 6- A100-1 22 22 E A100-2 6- A100-2 19 22 E B0-1 6-22 B0-1 53 E B0-2 6-22 B0-2 43 E B1-1 6-22 B1-1 64 E B1-2 6-22 B1-2 44 E B10-1 6- B10-1 44 22 E B10-2 6- B10-2 53 22 E B100-1 6- B100-1 41 22 E B100-2 6- B100-2 46 22 LE

3 6/22/2005 archived samples of Spawn 1 free swimming larvae

vial no. of larvae marked as A0-1 6-22 A0-1 15 L A0-2 6-22 A0-2 15 L A1-1 6-22 A1-1 15 L A1-2 6-22 A1-2 15 L A10-1 6- A10-1 15 22 L A10-2 6- A10-2 15 22 L A100-1 6- A100-1 15 22 L A100-2 6- A100-2 15 22 L B0-1 6-22 B0-1 15 L B0-2 6-22 B0-2 15 L B1-1 6-22 B1-1 15 L B1-2 6-22 B1-2 15 L B10-1 6- B10-1 15 22 L no larvae B10-2 0 hatched B100-1 6- B100-1 15 22 L B100-2 6- B100-2 15 22 L

6/22/2005 dish contents at end of 6-22-05

free late stage eggs in early potentially potentially swimming 20 or stages of abnormal abnormal larvae stage 21 development eggs embryos A0.1 62 60 0 0 0 A0.2 2 137 0 1 3 A1.1 113 12 0 0 0 A1.2 59 62 0 2 1 A10.1 3 121 0 5* 0 A10.2 5 114 0 1 0 A100.1 16 113 0 3 1 A100.2 105 39 1 0 2

4 B0.1 28 32 0 1 2 B0.2 4 86 0 0 5 B1.1 27 82 0 0 6 B1.2 55 35 0 0 2 B10.1 11 96 0 1 1 B10.2 0 104 0 0 9 B100.1 23 69 0 0 0 B100.2 24 39 0 0 0 *the 5 eggs include one egg with red blotches left in dish - only egg in artificial treatments to have red blotches

7/27/2005 Dead larvae removed from bay water Spawn 1 treatments and live larvae left in dishes

in cryovial live larvae left dead marked as in dish B0-1 0 x 13 B0-2 0 x 28 B1-1 41 B1-1 7-27 5 B1-2 0 9 B10-1 7- B10-1 6 19 27 B10-2 7- B10-2 11 27 27 B00-1 7- B100-1 2 31 27 B100-2 0 x 16

8/3/2005 removed all remaining eggs or larvae in the original Spawn 1 treatments

cryovial dead larvae live larvae marked as A0-1 0 24 A0-1 8-3 A0-2 0 28 A0-2 8-3 1 egg removed and discarded, 1 bell shaped larvae - picture taken A1-1 0 10 A1-1 8-3 A1-2 0 6 A1-2 8-3 3 eggs removed and discarded A10-1 0 18 A10-1 8-3 1 egg removed and discarded A10-2 0 17 A10-2 8-3 2 eggs removed and discarded A100-1 0 30 A100-1 8-3 A100-2 0 35 A100-2 8-3 B0-1 1 10 B0-1 8-3 one of the dead molts appears incomplete, one side molted the other not - picture taken B0-2 2 16 B0-2 8-3 B1-1 4 0 B1-1 8-3 B1-2 0 3 B1-2 8-3 B10-1 17 0 B10-1 8-3 B10-2 23 0 B10-2 8-3 B100-1 3 20 B100-1 8-3 B100-2 0 11 B100-2 8-3

8/3/2005 summary of eggs by dish

total archived archived 6-22 total archived 6- archived 6-22 archived 7- archived 8-3 irregular total developed total in % to late 6-17 eggs juveniles 10 stage 20 larvae 27 larvae larvae eggs undeveloped to late treatment stage stage 20 (undeveloped) removed stages A0.1 15 15 15 12 0 24 98 1 13 167 180 0.928 A0.2 15 15 15 22 0 28 106 0 22 179 201 0.891 A1.1 15 15 15 13 0 10 88 0 13 143 156 0.917 A1.2 15 15 15 13 0 6 104 3 16 155 171 0.906 A10.1 15 15 15 21 0 18 94 1 22 157 179 0.877 A10.2 15 15 15 24 0 17 99 2 26 161 187 0.861 A100.1 15 15 15 22 0 30 87 0 22 162 184 0.880 A100.2 15 15 15 19 0 35 102 0 19 182 201 0.905 B0.1 15 15 15 53 0 11 42 0 53 98 151 0.649 B0.2 15 15 15 43 0 18 62 0 43 125 168 0.744 B1.1 15 15 15 64 41 4 60 0 64 150 214 0.701 B1.2 15 15 15 44 0 3 80 0 44 128 172 0.744 B10.1 15 15 15 44 6 17 79 0 44 147 191 0.770 B10.2 15 15 0 53 11 23 68 0 53 132 185 0.714 B100.1 15 15 15 41 2 23 62 0 41 132 173 0.763 B100.2 15 15 15 46 0 11 35 0 46 91 137 0.664

SPAWN 1 JUVENILES

7/1/2005 a larva was removed from each of the bay water treatments and placed in dish with sand

8/3/2005 Spawn 1 counts of juveniles removed from egg/larvae treatments (juveniles then placed into separate culture dishes for continuation of treatment- with replicates pooled)

percent total success total in 7/13/2005 7/14/2005 7/15/2005 7/18/2005 7/20/2005 7/22/2005 7/25/2005 7/27/2005 7/29/2005 8/1/2005 8/3/2005 molts to treatment recorded juvenile stage A0-1 25 2 9 35 6 9 8 2 0 1 1 98 180 0.544 A0-2 1 0 6 10 27 17 18 5 13 9 0 106 201 0.527 A1-1 57 9 2 2 1 4 6 5 1 1 0 88 156 0.564 A1-2 11 1 0 64 17 0 5 2 3 0 1 104 171 0.608 A10-1 4 0 2 27 3 20 18 5 9 5 1 94 179 0.525 A10-2 1 0 0 21 45 13 10 3 3 3 0 99 187 0.529 A100-1 0 0 0 0 4 5 26 10 20 18 4 87 184 0.473 A100-2 2 0 2 9 34 16 16 9 7 4 3 102 201 0.507 B0-1 25 2 1 2 0 6 5 0 0 0 1 42 151 0.278 B0-2 20 1 5 9 1 8 7 2 3 1 5 62 168 0.369

6 B1-1 36 2 1 10 2 6 1 0 1 1 0 60 214 0.280 B1-2 42 3 2 3 0 18 4 1 6 1 0 80 172 0.465 B10-1 34 2 3 10 3 13 8 4 1 1 0 79 191 0.414 B10-2 0 3 0 6 2 21 30 2 3 1 0 68 185 0.368 B100-1 8 3 1 16 7 5 11 2 5 4 0 62 173 0.358 B100-2 1 0 2 3 5 10 6 3 1 2 2 35 137 0.255

7/13/2005 Spawn 1 juveniles archived (included in the juveniles removed counts from 7-13)

number cryovial

removed marked as A0J 18 A0J 7-13 A1J 18 A1J 7-13 B0J 18 B0J 7-13 B1J 18 B1J 7-13 B10J 18 B10J 7-13 B100J 7- B100J 9 13

8/3/2005 All B10 Spawn 1 juveniles removed from treatment (all were dead) and placed in vial marked B10J Juv 8-3 (number archived = 129)

8/10/2005 removed all the Spawn 1 juveniles in the remaining three bay water spawn 1 treatments (all were dead). Placed in vials marked as follows.

vial marked number

as archived B0J B0J 1 8-10 86 B1J B1J 1 8-10 122 B100J B100J 1 8-10 88

8/17/2005 ended Spawn 1 juvenile treatments vials vials marked live dead marked as as A0J 1 x 190 A0J 8-17D1 A1J 8- A1J 49 47 A1J 8-17D1 (much of the remains uncountable) 17L1 A10J 0 x 194 A10J 8-17 D1 A100J 8- A100J 1 81 A100J 8-17D1 (much of the remains uncountable) 17L1

7 8/17/2005 Summary of Spawn 1 juveniles percent total total in 8/17/2005 7/13/2005 8/3/2005 8/10/2005 success to juveniles treatments molt A0J 191 18 0 0 209 381 0.549 A1J 96 18 0 0 114 327 0.349 A10J 194 0 0 0 194 366 0.530 A100J 82 0 0 0 82 385 0.213 B0J 0 18 0 86 104 319 0.326 B1J 0 18 0 122 140 386 0.363 B10J 0 18 129 0 147 376 0.391 B100J 0 9 0 88 97 310 0.313

SPAWN 2 EGGS AND LARVAE

7/1/2005 archived stage 20 eggs (not enough eggs developed in bay water treatments to sample) vial marked no. of eggs as A0-1 15 7-1 A0-1 A0-2 15 7-1 A0-2 A0-3 15 7-1 A0-3 A0-4 15 7-1 A0-4 A1-1 15 7-1 A1-1 A1-2 15 7-1 A1-2 A1-3 15 7-1 A1-3 A1-4 15 7-1 A1-4 A10-1 15 7-1 A10-1 A10-2 15 7-1 A10-2 A10-3 15 7-1 A10-3 A10-4 15 7-1 A10-4 A100-1 15 7-1 A100-1 A100-2 15 7-1 A100-2 A100-3 15 7-1 A100-3 A100-4 15 7-1 A100-4

7/8/2005 archived free swimming Spawn 2 larvae (no larvae hatched in bay water treatments) vial marked no. of larvae as A0-1 15 A0-1 7-8 L A0-2 15 A0-2 7-8 L

8 A0-3 15 A0-3 7-8 L A0-4 15 A0-4 7-8 L A1-1 15 A1-1 7-8 L A1-2 15 A1-2 7-8 L A1-3 15 A1-3 7-8 L A1-4 15 A1-4 7-8 L A10-1 15 A10-1 7-8 L A10-2 15 A10-2 7-8 L A10-3 15 A10-3 7-8 L A10-4 15 A10-4 7-8 L A100-1 15 A100-1 7-8 L A100-2 15 A100-2 7-8 L A100-3 15 A100-3 7-8 L A100-4 15 A100-4 7-8 L

7/8/2005 archived all Spawn 2 eggs not showing signs of development (a few potentially abnormal eggs were left in treatments to monitor for signs of development, to photograph, etc.) vial marked no. of eggs as A0-1 361 A0-1 7-8 E A0-2 144 A0-2 7-8 E A0-3 172 A0-3 7-8 E A0-4 120 A0-4 7-8 E A1-1 308 A1-1 7-8 E A1-2 152 A1-2 7-8 E A1-3 155 A1-3 7-8 E A1-4 140 A1-4 7-8 E A10-1 370 A10-1 7-8 E A10-2 187 A10-2 7-8 E A10-3 139 A10-3 7-8 E A10-4 125 A10-4 7-8 E A100-1 7-8 A100-1 385 E A100-2 7-8 A100-2 154 E A100-3 7-8 A100-3 156 E A100-4 7-8 A100-4 144 E

7/8/2005 all eggs from bay water treatments disposed of and bay water Spawn 2 treatments stopped

7/8/2005 dish contents at end of 7-8-05 free eggs in early potentially potentially swimming late stage 20 stages of abnormal abnormal larvae or stage 21 development eggs embryos A0.1 77 3 0 0 0 A0.2 7 30 0 1 0 15 larvae were spilled and disposed of from A0-2 A0.3 4 41 0 0 0 A0.4 95 19 0 0 1 A1.1 27 21 0 0 1 one of the larvae put back in A1.1 was clearly dead A1.2 69 1 0 0 0 A1.3 36 3 0 0 4 A1.4 30 68 0 0 0 A10.1 43 13 0 0 0 A10.2 45 3 0 0 0 A10.3 18 31 0 4 0 A10.4 44 49 0 0 4 one of the larvae put back in A10.4 was clearly dead A100.1 77 0 0 0 0 A100.2 43 4 0 1 0 A100.3 43 0 0 0 1 A100.4 22 97 0 0 0 one of the larvae put back in A100.4 was clearly dead

8/3/2005 removed all remaining eggs or larvae in eight of the Spawn 2 treatments vial marked dead larvae live larvae as A0-2 0 3 A0-2 2- 8-3 3 abnormal eggs, included in vial A0-3 1 3 A0-3 2 8-3 1 abnormal egg, included in vial A1-2 0 3 A1-2 2 8-3 A1-3 1 1 A1-3 2 8-3 4 abnormal eggs, included in vial A10-2 0 4 A10-2 2 8-3 A10-3 0 2 A10-3 2 8-3 5 abnormal eggs, included in vial A100-2 0 3 A100-2 2 8-3 1 abnormal egg, included in vial A100-3 0 0 x 1 abnormal egg, discarded

8/10/2005 ended the remaining larvae treatments from Spawn 2 vial marked as live larva dead larva eggs A0-1 A0-1 2 8-10 7 0 0 A0-4 A0-4 2 8-10 11 3 0 A1-1 A1-1 2 8-10 2 1 1 plus 1 abnormal egg 2 A1-4 A1-4 2 8-10 (discarded) 47 0 A10-1 2 8- A10-1 10 11 0 1 A10-4 2 8- A10-4 10 18 0 0 plus 1 abnormal egg and 1 abnormal larva A100-1 2 8- A100-1 10 4 0 2 A100-4 2 8- A100-4 10 11 1 0

8/17/2005 summary of eggs by dish at end of 8-10-05 for Spawn 2 total developed archived 7-8 8-3 and 8- other total to late percent archived 7-1 archived 7-8 eggs archived 8-3 archived 8- 10 eggs larvae juveniles total stages or total in success to embryos larvae (undeveloped) larvae 10 larvae archived removed removed undeveloped hatched treatment late stages A0.1 15 15 361 0 7 0 0 368 361 404 765 0.528 A0.2 15 15 144 3 0 3 15 165 147 213 360 0.592 A0.3 15 15 172 4 0 1 0 177 173 211 384 0.549 A0.4 15 15 120 0 14 0 0 134 120 178 298 0.597 A1.1 15 15 308 0 4 2 0 314 310 348 658 0.529 A1.2 15 15 152 3 0 0 0 155 152 188 340 0.553 A1.3 15 15 155 2 0 4 0 161 159 193 352 0.548 A1.4 15 15 140 0 49 0 0 189 140 268 408 0.657 A10.1 15 15 370 0 11 1 0 382 371 423 794 0.533 A10.2 15 15 187 4 0 0 0 191 187 225 412 0.546 A10.3 15 15 139 2 0 5 0 146 144 178 322 0.553 A10.4 15 15 125 0 18 1 1 145 126 194 320 0.606 A100.1 15 15 385 0 4 2 0 391 387 425 812 0.523 A100.2 15 15 154 3 0 1 0 158 155 191 346 0.552 A100.3 15 15 156 0 0 1 0 157 157 187 344 0.544 A100.4 15 15 144 0 12 0 0 156 144 198 342 0.579

11 SPAWN 2 JUVENILES

Spawn 2 juveniles removed from egg/larvae treatments

percent success to total molts total in juvenile 7/22/2005 7/25/2005 7/27/2005 7/29/2005 8/1/2005 8/3/2005 8/5/2005 8/8/2005 8/10/2005 recorded treatment stage A0-1 14 4 20 17 5 2 0 0 0 62 765 0.081 A0-2 0 1 9 10 11 3 0 0 0 34 360 0.094 A0-3 0 0 7 30 2 0 0 0 0 39 384 0.102 A0-4 0 23 34 20 9 2 3 4 1 96 298 0.322 A1-1 1 3 13 12 7 2 3 0 1 42 658 0.064 A1-2 2 26 15 7 14 2 0 0 0 66 340 0.194 A1-3 1 28 2 3 2 0 0 0 0 36 352 0.102 A1-4 0 0 6 9 15 16 2 1 1 50 408 0.123 A10-1 0 13 11 12 5 0 0 3 0 44 794 0.055 A10-2 0 15 13 7 6 4 0 0 0 45 412 0.109 A10-3 2 12 2 5 1 0 0 0 0 22 322 0.068 A10-4 0 2 6 18 16 14 4 9 8 77 320 0.241 A100-1 10 19 13 25 2 0 0 3 0 72 812 0.089 A100-2 1 20 8 9 4 0 0 0 0 42 346 0.121 A100-3 2 31 5 2 2 0 0 0 0 42 344 0.122 A100-4 0 1 3 10 59 9 1 14 5 102 342 0.298

8/17/2005 ended Spawn 2 juvenile treatments (remains hard to accurately count) percent vials marked vials marked juveniles in juvenile live as dead as treatment survival A0J 17 A0J 8-17L 235 A0J 8-17D 252 0.06746032 A1J 4 A1J 8-17L 195 A1J 8-17D 199 0.0201005 A10J 1 x 138 A10J 8-17D 139 0.00719424 A100J 25 A100J 8-17L 107 A100J 8-17D 132 0.18939394

GROUP 3 LARVAE TO JUVENILES

Summary of juveniles removed from larvae treatments (from treatments started on 7/20/05 for larvae sorted out from 7/13/05 beach collected, fertilized eggs)

total larvae percent total put in 8/1/2005 8/3/2005 8/5/2005 8/8/2005 8/10/2005 8/17/2005 juveniles remaining success to treatment removed 8/17/05 molt W0-1 5 15 14 29 16 4 83 16 99 0.838 W0-2 6 26 14 41 7 8 102 26 128 0.797 W1-1 9 23 12 16 1 12 73 12 85 0.859 W1-2 15 24 12 20 11 5 87 9 96 0.906 W10-1 8 30 20 30 6 6 100 12 112 0.893 W10-2 4 9 12 33 1 2 61 20 81 0.753 W100-1 4 14 14 29 10 10 81 14 95 0.853 W100-2 3 6 17 29 11 13 79 18 97 0.814

8/17/2005 ended all Group 3 treatments

vials dead vials vials live larvae juveniles marked as larvae marked as marked as W0-1 4 0-1 8-17L 12 0-1 8-17D 4 x W0-2 0 x 26 0-2 8-17D 8 dead 0-2 8-17La W1-1 9 1-1 8-17L 3 1-1 8-17D 12 live 1-1 8-17La W1-2 8 1-2 8-17L 1 x 5 x 5 live 1 10-1 8- W10-1 9 10-1 8-17L 3 x dead 17La W10-2 0 x 20 10-2 8-17D 2 x 100-1 8- 100-1 8- 100-1 8- W100-1 8 6 7 dead plus 3 live juveniles not archived 17L 17D 17La 100-2 8- 100-2 8- 100-2 8- W100-2 14 4 11 live plus 2 dead juveniles not archived 17L 17D 17La

8/17/2005 ended Group 3 juvenile treatment

vials vials live dead marked as marked as W0 9 W0 8-17L 158 W0 8-17D W1 136 W1 8-17L 9 W1 8-17D W10 82 W10 8-17L 82 W10 8-17D W100 8- W100 8- W100 131 3 17L 17D

13 Appendix 3

Samples archived under liquid nitrogen

Vial ID Group Water Treatment Replicate Date archived Contents A0 610 Spawn 1 ASW 0 1 + 2 6/10/2005 30 fertilized eggs A1 610 Spawn 1 ASW 1 1 + 2 6/10/2005 30 fertilized eggs A10 610 Spawn 1 ASW 10 1 + 2 6/10/2005 30 fertilized eggs A100 610 Spawn 1 ASW 100 1 + 2 6/10/2005 30 fertilized eggs B0 610 Spawn 1 Pore 0 1 + 2 6/10/2005 30 fertilized eggs B1 610 Spawn 1 Pore 1 1 + 2 6/10/2005 30 fertilized eggs B10 610 Spawn 1 Pore 10 1 + 2 6/10/2005 30 fertilized eggs B100 610 Spawn 1 Pore 100 1 + 2 6/10/2005 30 fertilized eggs 6-17 A0-1 Spawn 1 ASW 0 1 617/2005 15 stage 20 eggs 6-17 A0-2 Spawn 1 ASW 0 2 617/2005 15 stage 20 eggs 6-17 A1-1 Spawn 1 ASW 1 1 617/2005 15 stage 20 eggs 6-17 A1-2 Spawn 1 ASW 1 2 617/2005 15 stage 20 eggs 6-17 A10-1 Spawn 1 ASW 10 1 617/2005 15 stage 20 eggs 6-17 A10-2 Spawn 1 ASW 10 2 617/2005 15 stage 20 eggs 6-17 A100-1 Spawn 1 ASW 100 1 617/2005 15 stage 20 eggs 6-17 A100-2 Spawn 1 ASW 100 2 617/2005 15 stage 20 eggs 6-17 B0-1 Spawn 1 Pore 0 1 617/2005 15 stage 20 eggs 6-17 B0-2 Spawn 1 Pore 0 2 617/2005 15 stage 20 eggs 6-17 B1-1 Spawn 1 Pore 1 1 617/2005 15 stage 20 eggs 6-17 B1-2 Spawn 1 Pore 1 2 617/2005 15 stage 20 eggs 6-17 B10-1 Spawn 1 Pore 10 1 617/2005 15 stage 20 eggs 6-17 B10-2 Spawn 1 Pore 10 2 617/2005 15 stage 20 eggs 6-17 B100-1 Spawn 1 Pore 100 1 617/2005 15 stage 20 eggs 6-17 B100-2 Spawn 1 Pore 100 2 617/2005 15 stage 20 eggs A0-1 6-22 E Spawn 1 ASW 0 1 6/22/2005 12 undeveloped eggs A0-2 6-22 E Spawn 1 ASW 0 2 6/22/2005 22 undeveloped eggs A1-1 6-22 E Spawn 1 ASW 1 1 6/22/2005 13 undeveloped eggs A1-2 6-22 E Spawn 1 ASW 1 2 6/22/2005 13 undeveloped eggs A10-1 6-22 E Spawn 1 ASW 10 1 6/22/2005 21 undeveloped eggs A10-2 6-22 E Spawn 1 ASW 10 2 6/22/2005 24 undeveloped eggs A100-1 6-22 E Spawn 1 ASW 100 1 6/22/2005 22 undeveloped eggs A100-2 6-22 E Spawn 1 ASW 100 2 6/22/2005 19 undeveloped eggs B0-1 6-22 E Spawn 1 Pore 0 1 6/22/2005 53 undeveloped eggs B0-2 6-22 E Spawn 1 Pore 0 2 6/22/2005 43 undeveloped eggs B1-1 6-22 E Spawn 1 Pore 1 1 6/22/2005 64 undeveloped eggs B1-2 6-22 E Spawn 1 Pore 1 2 6/22/2005 44 undeveloped eggs B10-1 6-22 E Spawn 1 Pore 10 1 6/22/2005 44 undeveloped eggs B10-2 6-22 E Spawn 1 Pore 10 2 6/22/2005 53 undeveloped eggs B100-1 6-22 E Spawn 1 Pore 100 1 6/22/2005 41 undeveloped eggs B100-2 6-22 LE Spawn 1 Pore 100 2 6/22/2005 46 undeveloped eggs A0-1 6-22 L Spawn 1 ASW 0 1 6/22/2005 15 free swimming larvae A0-2 6-22 L Spawn 1 ASW 0 2 6/22/2005 15 free swimming larvae A1-1 6-22 L Spawn 1 ASW 1 1 6/22/2005 15 free swimming larvae A1-2 6-22 L Spawn 1 ASW 1 2 6/22/2005 15 free swimming larvae A10-1 6-22 L Spawn 1 ASW 10 1 6/22/2005 15 free swimming larvae A10-2 6-22 L Spawn 1 ASW 10 2 6/22/2005 15 free swimming larvae A100-1 6-22 L Spawn 1 ASW 100 1 6/22/2005 15 free swimming larvae A100-2 6-22 L Spawn 1 ASW 100 2 6/22/2005 15 free swimming larvae B0-1 6-22 L Spawn 1 Pore 0 1 6/22/2005 15 free swimming larvae B0-2 6-22 L Spawn 1 Pore 0 2 6/22/2005 15 free swimming larvae

1 Vial ID Group Water Treatment Replicate Date archived Contents B1-1 6-22 L Spawn 1 Pore 1 1 6/22/2005 15 free swimming larvae B1-2 6-22 L Spawn 1 Pore 1 2 6/22/2005 15 free swimming larvae B10-1 6-22 L Spawn 1 Pore 10 1 6/22/2005 15 free swimming larvae B100-1 6-22 L Spawn 1 Pore 100 1 6/22/2005 15 free swimming larvae B100-2 6-22 L Spawn 1 Pore 100 2 6/22/2005 15 free swimming larvae B1-1 7-27 Spawn 1 Pore 1 1 7/27/2005 41 dead free swimming larvae B10-1 7-27 Spawn 1 Pore 10 1 7/27/2005 6 dead free swimming larvae B10-2 7-27 Spawn 1 Pore 10 2 7/27/2005 11 dead free swimming larvae B100-1 7-27 Spawn 1 Pore 100 1 7/27/2005 2 dead free swimming larvae A0-1 8-3 Spawn 1 ASW 0 1 8/3/2005 24 live free swimming larvae A0-2 8-3 Spawn 1 ASW 0 2 8/3/2005 28 live free swimming larvae A1-1 8-3 Spawn 1 ASW 1 1 8/3/2005 10 live free swimming larvae A1-2 8-3 Spawn 1 ASW 1 2 8/3/2005 6 live free swimming larvae A10-1 8-3 Spawn 1 ASW 10 1 8/3/2005 18 live free swimming larvae A10-2 8-3 Spawn 1 ASW 10 2 8/3/2005 17 live free swimming larvae A100-1 8-3 Spawn 1 ASW 100 1 8/3/2005 30 live free swimming larvae A100-2 8-3 Spawn 1 ASW 100 2 8/3/2005 35 live free swimming larvae B0-1 8-3 Spawn 1 Pore 0 1 8/3/2005 1 dead 10 live free swimming larvae B0-2 8-3 Spawn 1 Pore 0 2 8/3/2005 2 dead 16 live free swimming larvae B1-1 8-3 Spawn 1 Pore 1 1 8/3/2005 4 dead free swimming larvae B1-2 8-3 Spawn 1 Pore 1 2 8/3/2005 3 live free swimming larvae B10-1 8-3 Spawn 1 Pore 10 1 8/3/2005 17 dead free swimming larvae B10-2 8-3 Spawn 1 Pore 10 2 8/3/2005 23 dead free swimming larvae B100-1 8-3 Spawn 1 Pore 100 1 8/3/2005 3 dead 20 live free swimming larvae B100-2 8-3 Spawn 1 Pore 100 2 8/3/2005 11 live free swimming larvae A0J 7-13 Spawn 1- juv ASW 0 - 7/13/2005 18 live juveniles A1J 7-13 Spawn 1- juv ASW 1 - 7/13/2005 18 live juveniles B0J 7-13 Spawn 1- juv Pore 0 - 7/13/2005 18 live juveniles B1J 7-13 Spawn 1- juv Pore 1 - 7/13/2005 18 live juveniles B10J 7-13 Spawn 1- juv Pore 10 - 7/13/2005 18 live juveniles B100J 7-13 Spawn 1- juv Pore 100 - 7/13/2005 9 live juveniles B10J Juv 8-3 Spawn 1- juv Pore 10 - 8/3/2005 129 dead juveniles B0J 1 8-10 Spawn 1- juv Pore 0 - 8/10/2005 86 dead juveniles B1J 1 8-10 Spawn 1- juv Pore 1 - 8/10/2005 122 dead juveniles B100J 1 8-10 Spawn 1- juv Pore 100 - 8/10/2005 88 dead juveniles A0J 8-17D1 Spawn 1- juv ASW 0 - 8/17/2005 190 dead juveniles A1J 8-17L1 Spawn 1- juv ASW 1 - 8/17/2005 49 live juveniles A1J 8-17D1 Spawn 1- juv ASW 1 - 8/17/2005 47 dead juveniles A10J 8-17 D1 Spawn 1- juv ASW 10 - 8/17/2005 194 dead juveniles A100J 8-17L1 Spawn 1- juv ASW 100 - 8/17/2005 1 live juvenile A100J 8-17D1 Spawn 1- juv ASW 100 - 8/17/2005 81 dead juveniles 7-1 A0-1 Spawn 2 ASW 0 1 7/1/2005 15 stage 20 eggs 7-1 A0-2 Spawn 2 ASW 0 2 7/1/2005 15 stage 20 eggs 7-1 A0-3 Spawn 2 ASW 0 3 7/1/2005 15 stage 20 eggs 7-1 A0-4 Spawn 2 ASW 0 4 7/1/2005 15 stage 20 eggs 7-1 A1-1 Spawn 2 ASW 1 1 7/1/2005 15 stage 20 eggs 7-1 A1-2 Spawn 2 ASW 1 2 7/1/2005 15 stage 20 eggs 7-1 A1-3 Spawn 2 ASW 1 3 7/1/2005 15 stage 20 eggs 7-1 A1-4 Spawn 2 ASW 1 4 7/1/2005 15 stage 20 eggs 7-1 A10-1 Spawn 2 ASW 10 1 7/1/2005 15 stage 20 eggs

2 Vial ID Group Water Treatment Replicate Date archived Contents 7-1 A10-2 Spawn 2 ASW 10 2 7/1/2005 15 stage 20 eggs 7-1 A10-3 Spawn 2 ASW 10 3 7/1/2005 15 stage 20 eggs 7-1 A10-4 Spawn 2 ASW 10 4 7/1/2005 15 stage 20 eggs 7-1 A100-1 Spawn 2 ASW 100 1 7/1/2005 15 stage 20 eggs 7-1 A100-2 Spawn 2 ASW 100 2 7/1/2005 15 stage 20 eggs 7-1 A100-3 Spawn 2 ASW 100 3 7/1/2005 15 stage 20 eggs 7-1 A100-4 Spawn 2 ASW 100 4 7/1/2005 15 stage 20 eggs A0-1 7-8 L Spawn 2 ASW 0 1 7/8/2005 15 free swimming larvae A0-2 7-8 L Spawn 2 ASW 0 2 7/8/2005 15 free swimming larvae A0-3 7-8 L Spawn 2 ASW 0 3 7/8/2005 15 free swimming larvae A0-4 7-8 L Spawn 2 ASW 0 4 7/8/2005 15 free swimming larvae A1-1 7-8 L Spawn 2 ASW 1 1 7/8/2005 15 free swimming larvae A1-2 7-8 L Spawn 2 ASW 1 2 7/8/2005 15 free swimming larvae A1-3 7-8 L Spawn 2 ASW 1 3 7/8/2005 15 free swimming larvae A1-4 7-8 L Spawn 2 ASW 1 4 7/8/2005 15 free swimming larvae A10-1 7-8 L Spawn 2 ASW 10 1 7/8/2005 15 free swimming larvae A10-2 7-8 L Spawn 2 ASW 10 2 7/8/2005 15 free swimming larvae A10-3 7-8 L Spawn 2 ASW 10 3 7/8/2005 15 free swimming larvae A10-4 7-8 L Spawn 2 ASW 10 4 7/8/2005 15 free swimming larvae A100-1 7-8 L Spawn 2 ASW 100 1 7/8/2005 15 free swimming larvae A100-2 7-8 L Spawn 2 ASW 100 2 7/8/2005 15 free swimming larvae A100-3 7-8 L Spawn 2 ASW 100 3 7/8/2005 15 free swimming larvae A100-4 7-8 L Spawn 2 ASW 100 4 7/8/2005 15 free swimming larvae A0-1 7-8 E Spawn 2 ASW 0 1 7/8/2005 361 undeveloped eggs A0-2 7-8 E Spawn 2 ASW 0 2 7/8/2005 144 undeveloped eggs A0-3 7-8 E Spawn 2 ASW 0 3 7/8/2005 172 undeveloped eggs A0-4 7-8 E Spawn 2 ASW 0 4 7/8/2005 120 undeveloped eggs A1-1 7-8 E Spawn 2 ASW 1 1 7/8/2005 308 undeveloped eggs A1-2 7-8 E Spawn 2 ASW 1 2 7/8/2005 152 undeveloped eggs A1-3 7-8 E Spawn 2 ASW 1 3 7/8/2005 155 undeveloped eggs A1-4 7-8 E Spawn 2 ASW 1 4 7/8/2005 140 undeveloped eggs A10-1 7-8 E Spawn 2 ASW 10 1 7/8/2005 370 undeveloped eggs A10-2 7-8 E Spawn 2 ASW 10 2 7/8/2005 187 undeveloped eggs A10-3 7-8 E Spawn 2 ASW 10 3 7/8/2005 139 undeveloped eggs A10-4 7-8 E Spawn 2 ASW 10 4 7/8/2005 125 undeveloped eggs A100-1 7-8 E Spawn 2 ASW 100 1 7/8/2005 385 undeveloped eggs A100-2 7-8 E Spawn 2 ASW 100 2 7/8/2005 154 undeveloped eggs A100-3 7-8 E Spawn 2 ASW 100 3 7/8/2005 156 undeveloped eggs A100-4 7-8 E Spawn 2 ASW 100 4 7/8/2005 144 undeveloped eggs A0-2 2- 8-3 Spawn 2 ASW 0 2 8/3/2005 3 live larvae, 3 abnormal eggs A0-3 2 8-3 Spawn 2 ASW 0 3 8/3/2005 1 dead larva, 3 live larvae, 1 abnormal egg A1-2 2 8-3 Spawn 2 ASW 1 2 8/3/2005 3 live larvae A1-3 2 8-3 Spawn 2 ASW 1 3 8/3/2005 1 dead and 1 live larvae, 4 abnormal eggs A10-2 2 8-3 Spawn 2 ASW 10 2 8/3/2005 4 live larvae A10-3 2 8-3 Spawn 2 ASW 10 3 8/3/2005 2 live larvae and 5 abnormal eggs A100-2 2 8-3 Spawn 2 ASW 100 2 8/3/2005 3 live larvae, 1 abnormal egg A0-1 2 8-10 Spawn 2 ASW 0 1 8/10/2005 7 live larvae, A0-4 2 8-10 Spawn 2 ASW 0 4 8/10/2005 11 live larvae, 3 dead larvae A1-1 2 8-10 Spawn 2 ASW 1 1 8/10/2005 2 live larvae, 1 dead larvae, 1 egg, 1 abnormal egg A1-4 2 8-10 Spawn 2 ASW 1 4 8/10/2005 47 dead larvae

3 Vial ID Group Water Treatment Replicate Date archived Contents A10-1 2 8-10 Spawn 2 ASW 10 1 8/10/2005 11 live larvae, 1 egg A10-4 2 8-10 Spawn 2 ASW 10 4 8/10/2005 18 live larvae, 1 abnormal egg, 1 abnormal larva A100-1 2 8-10 Spawn 2 ASW 100 1 8/10/2005 4 live larvae, 2 eggs A100-4 2 8-10 Spawn 2 ASW 100 4 8/10/2005 11 live larvae, 1 dead larvae A0J 8-17L Spawn 2 - juv ASW 0 - 8/17/2005 17 live juveniles A0J 8-17D Spawn 2 - juv ASW 0 - 8/17/2005 235 dead juveniles A1J 8-17L Spawn 2 - juv ASW 1 - 8/17/2005 4 live juveniles A1J 8-17D Spawn 2 - juv ASW 1 - 8/17/2005 195 dead juveniles A10J 8-17D Spawn 2 - juv ASW 10 - 8/17/2005 138 dead juveniles A100J 8-17L Spawn 2 - juv ASW 100 - 8/17/2005 25 live juveniles A100J 8-17D Spawn 2 - juv ASW 100 - 8/17/2005 107 dead juveniles 0-1 8-17L Group 3 ASW 0 1 8/17/2005 4 live larvae 0-1 8-17D Group 3 ASW 0 1 8/17/2005 12 dead larvae 0-2 8-17D Group 3 ASW 0 2 8/17/2005 26 dead larvae 0-2 8-17La Group 3 ASW 0 2 8/17/2005 8 dead juveniles 1-1 8-17L Group 3 ASW 1 1 8/17/2005 9 live larvae 1-1 8-17D Group 3 ASW 1 1 8/17/2005 3 dead larvae 1-1 8-17La Group 3 ASW 1 1 8/17/2005 12 live juveniles 1-2 8-17L Group 3 ASW 1 2 8/17/2005 8 live larvae 10-1 8-17L Group 3 ASW 10 1 8/17/2005 9 live larvae 10-1 8-17La Group 3 ASW 10 1 8/17/2005 5 live and 1 dead juvenile 10-2 8-17D Group 3 ASW 10 2 8/17/2005 20 dead larvae 100-1 8-17L Group 3 ASW 100 1 8/17/2005 8 live larvae 100-1 8-17D Group 3 ASW 100 1 8/17/2005 6 dead larvae 100-1 8-17La Group 3 ASW 100 1 8/17/2005 7 dead juveniles 100-2 8-17L Group 3 ASW 100 2 8/17/2005 14 live larvae 100-2 8-17D Group 3 ASW 100 2 8/17/2005 4 dead larvae 100-2 8-17La Group 3 ASW 100 2 8/17/2005 11 live juveniles W0 8-17L Group 3 - juv ASW 0 - 8/17/2005 9 live juveniles W0 8-17D Group 3 - juv ASW 0 - 8/17/2005 158 dead juveniles W1 8-17L Group 3 - juv ASW 1 - 8/17/2005 136 live juveniles W1 8-17D Group 3 - juv ASW 1 - 8/17/2005 9 dead juveniles W10 8-17L Group 3 - juv ASW 10 - 8/17/2005 82 live juveniles W10 8-17D Group 3 - juv ASW 10 - 8/17/2005 82 dead juveniles W100 8-17L Group 3 - juv ASW 100 - 8/17/2005 131 live juveniles W100 8-17D Group 3 - juv ASW 100 - 8/17/2005 9 dead juveniles

4 Appendix 4

Project pictures

Kimbles Beach collection area for adult L. polyphemus

Adult L. polyphemus collection from Kimbles Beach

1 Pore water collection from Kimbles Beach

Adult L. polyphemus awaiting spawning

2 Spawning

Treatment mixing

3 Typical treatment

Water bath

4 Covered water bath

Typical embryo 4 days after fertilization

5 Typical embryos 12 days after fertilization

Typical embryos 19 days after fertilization

6 Typical larvae in extraembryonic shell 24 days after fertilization

Abnormal egg

7 Abnormal egg

Abnormal egg or larvae

8 Abnormal embryo

Abnormal embryo

9 Abnormal embryo

Abnormal larvae

10 Abnormal larvae

Abnormal larvae

11 Abnormal larvae

Abnormal larvae

12 Abnormal larvae

Abnormal larvae

13 Abnormal larvae

Abnormal larvae

14 Fungal growth in a pore water treatment during Spawn 2 treatments

Pink fungal growth on eggs in pore water treatment

15 Larvae with pink coloration in treatment

Appendix 5

Temperature probe data from Kimbles Beach

Date 10 cm 20 cm 30 cm 06/18/05 25.15 25.10 24.98 05/05/05 15.23 15.53 16.47 06/19/05 23.85 23.62 23.06 05/06/05 12.41 12.18 11.70 06/20/05 21.90 21.67 21.16 05/07/05 13.59 13.83 14.32 06/21/05 22.77 22.89 23.13 05/08/05 14.96 15.04 15.19 06/22/05 24.23 24.31 24.36 05/09/05 17.01 17.18 17.53 06/23/05 24.17 24.29 24.50 05/10/05 16.55 16.61 16.73 06/24/05 25.27 25.51 25.92 05/11/05 17.93 18.31 19.02 06/25/05 25.98 26.25 26.78 05/12/05 19.23 19.46 19.79 06/26/05 26.17 26.43 26.98 05/13/05 17.60 17.68 17.72 06/27/05 25.01 24.85 24.41 05/14/05 18.73 19.00 19.49 06/28/05 25.36 25.73 26.34 05/15/05 20.25 20.45 20.71 06/29/05 26.22 26.26 26.18 05/16/05 20.20 20.52 21.02 06/30/05 26.36 26.54 26.72 05/17/05 20.17 20.35 20.59 07/01/05 27.46 27.56 27.51 05/18/05 19.89 20.07 20.33 07/02/05 27.00 26.99 26.75 05/19/05 19.59 19.70 19.79 07/03/05 26.08 26.06 25.90 05/20/05 15.45 15.04 14.23 07/04/05 25.71 25.77 25.68 05/21/05 15.73 15.98 16.38 07/05/05 26.64 26.75 26.82 05/22/05 16.91 17.03 17.22 07/06/05 26.54 26.67 26.77 05/23/05 17.38 17.44 17.43 07/07/05 26.37 26.30 26.01 05/24/05 17.32 17.32 17.29 07/08/05 23.50 23.37 23.08 05/25/05 15.06 14.87 14.51 07/09/05 24.58 24.88 25.43 05/26/05 14.44 14.46 14.43 07/10/05 26.61 26.83 27.16 05/27/05 16.97 17.39 18.13 07/11/05 27.72 27.98 28.36 05/28/05 18.78 18.93 19.16 07/12/05 28.70 28.92 29.26 05/29/05 19.18 19.45 20.06 07/13/05 28.28 28.35 28.45 05/30/05 19.99 20.32 20.96 07/14/05 28.26 28.38 28.53 06/01/05 20.75 21.08 21.71 07/15/05 28.89 29.13 29.46 06/01/05 20.50 20.68 20.93 07/16/05 29.12 29.17 29.12 06/02/05 19.31 19.34 19.33 07/17/05 28.09 28.07 27.95 06/03/05 18.10 18.00 17.73 07/18/05 29.49 29.57 29.61 06/04/05 18.47 18.58 18.76 07/19/05 30.68 30.72 30.75 06/05/05 21.20 21.60 22.32 07/20/05 31.30 30.96 31.35 06/06/05 23.77 24.14 24.78 07/21/05 31.24 30.83 31.71 06/07/05 24.53 24.82 25.23 07/22/05 31.06 30.43 31.09 06/08/05 26.08 26.45 27.07 07/23/05 30.06 29.26 29.81 06/09/05 26.39 26.63 27.00 07/24/05 29.11 28.26 28.75 06/10/05 25.88 26.09 26.42 07/25/05 28.38 27.52 28.01 06/11/05 26.63 26.91 27.26 07/26/05 30.18 29.49 30.49 06/12/05 27.14 27.43 27.85 07/27/05 27.24 26.31 27.00 06/13/05 27.87 28.21 28.62 06/14/05 28.69 29.07 29.61 06/15/05 29.24 29.39 29.40 06/16/05 28.58 28.66 28.66 06/17/05 25.45 25.33 25.03

Appendix 6

Altosid SR-5 Label