University of Florida Thesis Or Dissertation

IMPACTS OF A NON-NATIVE PISCIVORE, THE PIKE KILLIFISH, ON JUVENILE COMMON SNOOK

GEOFFREY HENRY SMITH JR.

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2019

To my loving and understanding wife, Amanda, who not only helped me trudge through the mud to collect fish, but also put up with me throughout this sometimes-stressful process and provided thoughtful conversation, moral support, and much needed breaks from all the work. I couldn’t have done it without you.

ACKNOWLEDGMENTS

I would like to thank my dissertation committee members including Daryl Parkyn,

Jeff Hill, Colette St. Mary, Ed Matheson, Ron Taylor, and especially my advisor Debra

Murie for all their advice, encouragement, and thoughtful discussions on snook biology, non-native species, and aquatic ecology throughout my doctoral studies. I would like to thank Marin Greenwood for all of his knowledge and preliminary investigations on Pike

Killifish in the estuarine waters of Tampa Bay. I would like to thank Chuck Cichra for the use of his boat and seine nets for my field work, the FWRI Marine Fisheries

Independent Monitoring Program for the use of a hand-held coded-wire detector, and the UF Tropical Aquaculture lab for occasional use of vehicles, storage of samples, and on-site lodging. Field work for this study would not have been possible without the assistance of my wife and fellow doctoral student, Amanda Croteau. I would also like to thank the PADI Foundation (Grant #6379), the UF Graduate School Award awarded through the Program of Fisheries and Aquatic Sciences and School of Forest

Resources and Conservation, Steven Berkeley Marine Conservation Fellowship, Guy

Harvey Scholarship from Florida Sea Grant, and the Longboat Key Garden Club

Scholarship who all provided funding to make this study possible. Collection of organisms from the field was conducted under FWC SAL 12-1400-A; sampling within

Terra Ceia Preserve State Park was conducted under FDEP Florida Park Service

Research/Collection Permit 04011324; and sampling within Cockroach Bay Preserve was conducted under a Hillsborough County Letter of Authorization. All field and lab studies were conducted under appropriate protocols including UF IFAS Non-regulatory

Animal Research Protocol 006-12FAS and UF IACUC Protocol 201207451.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 9

ABSTRACT ...... 13

CHAPTER

1 GENERAL INTRODUCTION ...... 15

2 PREDATION OF JUVENILE COMMON SNOOK BY PIKE KILLIFISH ...... 37

Introduction ...... 37 Methods ...... 39 Predation Trials ...... 39 Fish Collection for Diet Analysis ...... 43 Stomach Content Analysis ...... 45 Results ...... 45 Predation Trials ...... 45 Pike Killifish Attacks on Prey ...... 46 Pike Killifish Consumption of Prey ...... 48 Stomach Content Analysis ...... 50 Discussion ...... 51

3 DIET ANALYSIS AND POTENTIAL FOOD RESOURCE COMPETITION BETWEEN EARLY-JUVENILE COMMON SNOOK AND PIKE KILLIFISH ...... 68

Introduction ...... 68 Methods ...... 71 Fish Collection for Diet Analysis ...... 71 Collection of Prey for Abundance Estimates ...... 73 Diet Analysis ...... 74 Prey Abundance ...... 80 Results ...... 81 Prey Regression Curves...... 81 Early-juvenile Common Snook Diet Analysis ...... 82 Pike Killifish Diet Analysis ...... 85 Dietary Overlap ...... 86 Prey Abundance ...... 87 Discussion ...... 88

4 POTENTIAL INTERFERENCE COMPETITION AND SPACE RESOURCE COMPETITION BETWEEN PIKE KILLIFISH AND EARLY-JUVENILE COMMON SNOOK ...... 141

Introduction ...... 141 Methods ...... 143 Results ...... 145 Discussion ...... 147

5 POTENTIAL IMPACTS OF Non-Native PIKE KILLIFISH ON EARLY-JUVENILE COMMON SNOOK ABUNDANCE, GROWTH, AND CONDITION ...... 163

Introduction ...... 163 Methods ...... 165 Assessment of Growth Impacts ...... 165 Assessment of Abundance and Condition Impacts ...... 168 Modelling Theoretical Impacts ...... 170 Results ...... 172 Growth Impacts ...... 172 Abundance Impacts ...... 173 Condition Impacts ...... 175 Theoretical Impacts ...... 175 Discussion ...... 176

6 CONCLUSION ...... 196

APPENDIX: R CODE FOR POPULATION MODEL ...... 201

LIST OF REFERENCES ...... 202

BIOGRAPHICAL SKETCH ...... 216

LIST OF TABLES

Table page

1-1 History of Common Snook regulations in Florida ...... 33

2-1 Comparison of logistic regression curves for Pike Killifish attacks on and consumption of different prey types based on size...... 58

2-2 Comparison of Pike Killifish 48 h attacks on different prey species in 20 mm TL size bins...... 59

2-3 Comparison of Pike Killifish 48 h consumption of different prey species in 20 mm TL size bins ...... 60

2-4 Comparison of Pike Killifish 48 h ratio consumption to attack rates of different prey species in 20 mm TL size bins ...... 61

3-1 Regression equations for various fish and invertebrate prey species used to back-calculate the weight of partially digested prey items based on a variety of measurements ...... 100

3-2 Geometric shapes and equations used to estimate the biomass of small organisms including planktonic crustaceans, some small benthic crustacean, and some aquatic insects ...... 106

3-3 Prey functional groups used in diet and prey availability analysis...... 108

3-4 Diet composition of early-juvenile Common Snook and Pike Killifish collected from Tampa Bay tidal tributaries ...... 109

3-5 Diet composition of early-juvenile Common Snook of different size classes based on prey functional groups ...... 114

3-6 Diet composition of early-juvenile Common Snook with and without Pike Killifish co-occurring ...... 115

3-7 Diet composition, based on prey functional groups, of early-juvenile Common Snook of different size classes from locations without Pike Killifish co- occurring ...... 120

3-8 Diet composition, based on prey functional groups, of early-juvenile Common Snook of different size classes from locations with Pike Killifish co-occurring. . 121

3-9 Levin’s standardized index for early-juvenile Common Snook and Pike Killifish ...... 122

3-10 Ivlev electivity index values for early-juvenile Common Snook and Pike Killifish based on prey collections from both bag seine and minnow seine hauls ...... 123

3-11 Diet composition, based on prey functional groups, of Pike Killifish of different size classes ...... 124

3-12 Morisita’s index of similarity calculated for several pairings of Pike Killifish and early-juvenile Common Snook ...... 125

3-13 Multivariate abundance tests comparing prey abundance, calculated based on total length (TL) size groups, between locations with and without Pike Killifish co-occurring ...... 126

3-14 Multivariate abundance tests comparing prey abundance, calculated based on prey maximum depth (MD) size groups, between locations with and without Pike Killifish co-occurring ...... 127

4-1 Proportion of time spent near the bottom, proportion of time spent in cover, and proportion of prey consumed by Pike Killifish and early-juvenile Common Snook in an experimental aquarium habitats...... 156

5-1 Estimated percent consumption of juvenile Common Snook by Pike Killifish based on standard length (SL) ...... 181

5-2 Mean abundance of early-juvenile Common Snook with 95% confidence intervals from tidal tributaries of Tampa Bay, FL...... 182

5-3 Mean abundance of early-juvenile Common Snook from different locations based on area and shore length sampled in Tampa Bay, FL ...... 183

5-4 Mean abundance of early-juvenile Pike Killifish with 95% confidence intervals from tidal tributaries of Tampa Bay, FL...... 183

5-5 Estimated number of days for Common Snook to exceed 100 mm standard length under varying degrees of reduced daily growth...... 184

LIST OF FIGURES

Figure page

1-1 Distribution of Pike Killifish ...... 34

1-2 Distribution of Common Snook ...... 35

1-3 Distribution of Pike Killifish within Tampa Bay, FL, with area of highest early- juvenile Common Snook abundance denoted...... 36

2-1 Satellite image of Tampa Bay, FL, with collection locations for lab experiments and ad-hoc sampling of Pike Killifish ...... 62

2-2 Aquarium set-up used for predation trials of Pike Killifish on several prey types ...... 63

2-3 Satellite image of Tampa Bay, FL, with insets of standardized sampling locations for Pike Killifish and early-juvenile Common Snook co-occurring with Pike Killifish: Alafia River and Wildcat Creek, a tributary of the Little Manatee River...... 64

2-4 Satellite image of Wildcat Creek, a tributary of the Little Manatee River in Tampa Bay, FL, one of the standardized sampling locations for Pike Killifish and early-juvenile Common Snook with Pike Killifish co-occurring ...... 65

2-5 Satellite images of the Alafia River in Tampa Bay, FL, one of the standardized sampling locations for Pike Killifish and early-juvenile Common Snook with Pike Killifish co-occurring ...... 65

2-6 Pike Killifish attacks on different prey types based on prey total length (TL) ...... 66

2-7 Pike Killifish consumption of different prey types based on prey total length (TL) ...... 67

3-1 Satellite image of Tampa Bay, FL with insets of standardized sampling locations for early-juvenile Common Snook without Pike Killifish co-occurring: un-named tributary of the Manatee River (2012) and Braden River (2012- 2013) ...... 128

3-2 Satellite image of Tampa Bay, FL with inset of standardized sampling locations for early-juvenile Common Snook without Pike Killifish co-occurring: Frog Creek (also known as Terra Ceia River) (2013)...... 129

3-3 Satellite image of the un-named Manatee River tributary in Tampa Bay, FL, one of the standardized sampling locations for early-juvenile Common Snook without Pike Killifish co-occurring (sampled during 2012) ...... 130

3-4 Satellite image of Frog Creek (Terra Ceia River) in Tampa Bay, FL, one of the standardized sampling locations for early-juvenile Common Snook without Pike Killifish co-occurring (sampled during 2013) ...... 131

3-5 Satellite image of Braden River in Tampa Bay, FL, one of the standardized sampling locations for early-juvenile Common Snook without Pike Killifish co- occurring (sampled during 2012 and 2013) ...... 132

3-6 Cumulative prey curves for early-juvenile Common Snook and Pike Killifish collected from Tampa Bay tidal tributaries...... 133

3-7 Total length (TL) of prey consumed by early-juvenile Common Snook and Pike Killifish as a function of standard length (SL) ...... 134

3-8 Maximum depth (MD) of prey consumed by early-juvenile Common Snook and Pike Killifish as a function of gape width (GW) ...... 135

3-9 Three-dimensional representation of early-juvenile Common Snook diet composition in terms of percent frequency of occurrence (%O), percent numerical abundance (%N), and percent weight (%W) based on functional prey groups...... 136

3-10 Number of planktonic organisms consumed by early-juvenile Common Snook, from Tampa Bay tidal tributaries, based on size ...... 137

3-11 Average relative abundance of fish and shrimp prey functional groups (≤45 mm TL) sampled with the bag seine and minnow seine with and without Pike Killifish present and percent numerical abundance (%N) of these prey groups 138

3-12 Three-dimensional representation of Pike Killifish diet composition in terms of percent frequency of occurrence (%O), percent numerical abundance (%N), and percent weight (%W) based on functional prey groups ...... 139

3-13 Number of planktonic organisms consumed by Pike Killifish, from Tampa Bay tidal tributaries, based on size ...... 140

4-1 Experimental aquarium habitat used to assess the potential interference competition and space resource competition between Pike Killifish and early- juvenile Common Snook ...... 157

4-2 Average proportion (with 95% confidence intervals) of time spent near the bottom by early-juvenile Common Snook without Pike Killifish present, early- juvenile Common Snook with Pike Killifish present, and Pike Killifish with early-juvenile Common Snook present in experimental aquarium habitat replicates ...... 158

4-3 Average proportion (with 95% confidence intervals) of time spent in cover by early-juvenile Common Snook without Pike Killifish present, early-juvenile

Common Snook with Pike Killifish present, and Pike Killifish with early- juvenile Common Snook present in experimental aquarium habitat replicates . 159

4-4 Average proportion (with 95% confidence intervals) of prey consumed by early-juvenile Common Snook without Pike Killifish present, early-juvenile Common Snook with Pike Killifish present, and Pike Killifish with early- juvenile Common Snook present in experimental aquarium habitat replicates . 160

4-5 Average proportion (with 95% confidence intervals) of Eastern Mosquitofish consumed by early-juvenile Common Snook without Pike Killifish present, early-juvenile Common Snook with Pike Killifish present, and Pike Killifish with early-juvenile Common Snook present in experimental aquarium habitat replicates ...... 161

4-6 Average proportion (with 95% confidence intervals) of grass shrimp consumed by early-juvenile Common Snook without Pike Killifish present, early-juvenile Common Snook with Pike Killifish present, and Pike Killifish with early-juvenile Common Snook present in experimental aquarium habitat replicates ...... 162

5-1 Experimental enclosure placed in a small tributary of Mill Bayou in the Little Manatee River, Tampa Bay, FL...... 185

5-2 Experimental enclosure design as viewed from the top ...... 185

5-3 Location of experimental enclosures within Mill Bayou in the Little Manatee River, FL...... 186

5-5 Development of the equation used for daily growth of early-juvenile Common Snook in the population model ...... 188

5-6 Mean daily growth rate (with 95% confidence intervals) of early-juvenile Common Snook under different treatments in experimental enclosures...... 189

5-7 Boxplot of the mean proportion of early-juvenile Common Snook containing stomach contents under different treatments in experimental enclosures ...... 190

5-8 Mean abundance of early-juvenile Common Snook from different sampling locations in Tampa Bay, FL ...... 191

5-9 Mean abundance and adjusted abundance of Pike Killifish from different sampling locations in Tampa Bay, FL ...... 192

5-10 Weight-at-length relationship for early-juvenile Common Snook from locations with (PK) an without (NPK) Pike Killifish co-occurring ...... 193

5-11 Weight-at-length relationship for early-juvenile Common Snook based on sampling location ...... 194

5-12 Contour plot of late stage juvenile Common Snook (exceeding 100 mm SL) production (# of fish) from a single cohort within a tidal tributary under varying reductions in daily growth and simulated Pike Killifish predation...... 195

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

IMPACTS OF A NON-NATIVE PISCIVORE, THE PIKE KILLIFISH, ON JUVENILE COMMON SNOOK

Geoffrey Henry Smith Jr.

May 2019

Chair: Debra Murie Major: Fisheries and Aquatic Sciences

Pike Killifish is an established non-native fish species in Florida, US that was first documented in south Florida in 1957 and secondarily in Tampa Bay tributaries in 1994.

Decreases in small-bodied fish abundances have been linked to the introduction of Pike

Killifish in both of these regions. Increases in the range and abundance of Pike Killifish in the Tampa Bay area and overlap in habitat usage has led to concerns about potential predation on, and competition with, early-juvenile Common Snook (≤100 mm standard length, SL). Several lines of evidence from this dissertation point to minimal or no impacts of Pike Killifish on early-juvenile Common Snook in Tampa Bay tributaries.

Predation trials indicate that Pike Killifish are capable of consuming juvenile Common

Snook up to 48 mm SL, but no Common Snook remains have been found in the diet analysis of Pike Killifish. There is a small degree of diet overlap between these species and declines in the abundance of some prey groups has been detected in locations where Pike Killifish and Common Snook co-occur. However, diet overlap of early- juvenile Common Snook from locations with and without Pike Killifish co-occurring remains high and there is no indication of reduced abundance, condition, or growth of early-juvenile Common Snook in the presence of Pike Killifish. Experimental aquarium

habitats showed that Pike Killifish and early-juvenile Common Snook utilized different microhabitats, which helps to explain the lack of negative impacts observed due to competition. Simulation modelling revealed that if negative impacts were present in the form of reduced abundance of Common Snook from predation by Pike Killifish or reduced growth from competition, large reductions in the production of large juvenile

Common Snook are possible.

CHAPTER 1 GENERAL INTRODUCTION

The first year in the life of a fish is often given a great deal of attention because a number of events during this period are likely to be critical to the survival of an individual

(Able 1999). Small variations in the growth rate and survival of larval and juvenile fish have the potential to greatly influence recruitment to adult populations, and are likely to have a greater impact on recruitment than episodic losses (e.g., aberrant drift, weather related losses, acute toxic events, etc.) (Houde 1989; Able 1999).

Growth rates during the early life of fishes may influence the recruitment of individuals in a number of ways. Larger or faster growing members of a cohort may gain a survival advantage over smaller conspecifics through enhanced resistance to starvation, decreased vulnerability to predation, and better tolerance of environmental extremes (Sogard 1997). Generally, predation in fish is greatest in the smallest individuals and accounts for a large portion of the mortality in juvenile fish (Houde 1987,

1989). Rapid growth of newly settled juveniles may allow individuals to quickly grow out of the size classes that are most vulnerable (i.e., reaching a size refuge from predation)

(Post and Evans 1989; Sogard 1992, 1997; Sogard and Able 1992; Levin et al. 1997). A number of studies have also demonstrated that overwinter survival of juveniles is generally greater in larger individuals, with some species having minimum size thresholds (Henderson et al. 1988; Hurst and Conover 1998; Schultz et al. 1998; Quinn and Peterson 1996). The relative size difference among individuals that may develop in juvenile fish can be maintained to adulthood and may influence maturation and reproductive patterns (Sogard and Able 1992; Sogard 1994).

While growth tends to indirectly influence survival to the adult population, mortality via predation directly removes individuals from the population and decreases recruitment. Mortality of larval and juvenile fish not resulting from episodic events is generally attributed to food availability and predation and, in many cases, food is not likely to be limiting in the nursery areas of fishes, particularly in estuaries (Currin et al.

1984; Kneib 1993).

Organisms will often forage in a manner in which caloric intake is maximized while foraging time is minimized, as predicted by the concept of optimal foraging theory

(Emlen 1966; MacArthur and Pianka 1966). However, this foraging behavior may be modified by factors such as the presence of predators. The degree of predation and growth of juvenile fishes are generally linked to one another, and there are often trade- offs that occur. These trade-offs generally involve sacrificing growth by foraging in less favorable areas where there may be fewer or less energetically profitable prey, more competitors (inter- and intraspecies), or prey that are more difficult to capture (e.g., more refuge for prey or fewer ambush points for predators); with the benefit of encountering fewer predators and decreasing the chances of being consumed (Werner et al. 1983; Werner and Gilliam 1984; Werner and Hall 1988; Sogard 1992, 1994, 1997;

Walters and Juanes 1993; Dahlgren and Eggleston 2000; Halpin 2000). In theory, such trade-offs will tend to optimize the recruitment of juveniles to the adult population

(Anderson 1988; Walters and Juanes 1993). The term foraging arena theory is often applied to this concept and its related models (Walters and Martell 2004).

Predation and growth may be influenced by a number of variables, including abiotic factors, such as depth (Ruiz et al. 1993; Linehan et al. 2001; Manderson et al.

2004; Ryer et al. 2010); temperature and dissolved oxygen (Sogard 1992; Sogard and

Able 1992; Baltz et al. 1998; Phelan et al. 2000; Le Pape et al. 2003; Searcy et al. 2007;

Taylor et al. 2007); the type of structure (Werner et al. 1983; Werner and Hall 1988;

Sogard 1992; Dahlgren and Eggleston 2000; Phelan et al. 2000; Stunz et al. 2002;

Minello et al. 2003) and sediment present (Sogard 1992); as well as density of both intraspecific and interspecific competitors (Mittelback 1988; Searcy et al. 2007) and food availability (Mittelback 1988; Sogard 1992; Levin et al. 1997). Disturbances that may alter these factors, such as habitat alteration, climate change, or the introduction of non- native species, have the potential to shift both predation and growth and their interactions with one another and alter recruitment to the adult population.

In particular, the impacts of non-native species from diverse taxa, including many fish species, are often implicated as a major cause of negative environmental impacts either directly or indirectly (Cucherousset and Olden 2011). The effects of non-native species may be expressed at a number of levels, including: genetic level (transcription, hybridization, and introgression); individual level (behavior, morphology, and vital rates); population level (transmission of pathogens/parasites, demographic effects, and distributional effects); community level (species extinctions, composition change including biotic homogenization, and alteration of food webs); and ecosystem level

(modification of biochemical cycles, modification of energy fluxes between ecosystems, and habitat alteration by engineering species) (Gozlan 2008; Gozlan et al. 2010;

Cucherousset and Olden 2011).

In addition to ecological impacts, species introductions can have societal and economic influence. Although the introduction of non-native species may provide

societal benefits, such as increased food production and economic benefits from the sale of products associated with non-native species (Gozlan 2008), there is also the potential for significant economic losses in the form of lost yields, management, mitigation, eradication, etc. (Cucherousset and Olden 2011). Costs associated with non- native species have been estimated to be nearly $120 billion annually in the United

States, with costs associated with non-native fish in the US conservatively estimated at

$5.4 billion annually (Pimentel et al. 2005).

Despite differences in views about the extent of damage caused by non-native species, most vested parties agree that some species will have negative impacts, while others will be essentially innocuous, and that the potential ecological, societal, and economic benefits need to be weighed against the potential impacts in these areas

(Simberloff 2005; Gozlan 2008). The desire to assess potential impacts and weigh them against potential benefits has led to considerable research in predicting how non-native species will react in new environments and risk assessment models based on such information. There can be no certainty in these assessments (Sagoff 2005; Simberloff

2005; Brown and Sax 2007), although predictions have improved considerably, and some groups of species appear more likely to have negative impacts than others (i.e., certain families, top predators, habitat engineers) (Ruesink 2005; Simberloff 2005,

2007; García-Berthou 2007; Gozlan 2008; Gozlan et al. 2010).

The Pike Killifish Belonesox belizanus is one of a number of established non- native species of fish that has been identified in the freshwaters of Florida where it has been shown to have the potential for significant ecological impacts (Fuller et al. 1999). It has also become established in several estuarine environments (Turner and Snelson

1984; Kerfoot 2009; MacDonald et al. 2010; Schofield et al. 2011; Greenwood 2017).

Relatively few fish introductions become established in estuarine and marine waters

(Baltz 1991; Courtenay et al. 2009); however, those fish species that have become established in these ecosystems often exert a negative impact in their new environments (Baltz 1991; Ruiz et al. 1997; Albins and Hixon 2008; Courtenay et al.

2009).

Pike Killifish are native to Central America, from the Rio Antigua system of

Veracruz, Mexico, south through the Yucatan, Guatemala, Belize, and the Atlantic drainages of Honduras, Nicaragua, and northern Costa Rica where they are found in river drainages and coastal wetlands (Belshe 1961; Rosen and Bailey 1963) (Figure 1-

1A). Two subspecies have been described: B. belizanus maxillosus from the Yucatan and B. belizanus belizanus from the remainder of the Pike Killifish’s range (Hubbs

1936). Belonesox belizanus maxillosus was described as a separate subspecies based on its more robust body and jaws (Hubbs 1936); however, recent genetic analyses suggest that B. belizanus maxillosus may not be a valid taxon (Marchio and Piller 2013).

There does appear to be a genetic divergence between northern and southern populations that occurs along the Rio Grande in Belize (Marchio and Piller 2013).

Belonesox belizanus maxillosus is the form that was believed to have originally been released in south Florida (Belshe 1961) and although genetic evidence points toward this being an invalid taxon it does indicate that the south Florida Pike Killifish population was derived from northern populations, which includes the Yucatan population.

Pike Killifish were first found in Florida waters in November 1957 in several canals in Miami-Dade County in southern Florida (Figure 1-1B) (Belshe 1961). The

introduction was traced back to the release of approximately 50 individuals from the

Medical Research Unit of the University of Miami in April or May of 1957 (Belshe 1961).

From the original introduction, Pike Killifish have spread throughout Miami-Dade,

Collier, Broward, and Monroe counties, including populations in the Everglades National

Park and Big Cypress National Preserve where they are typically found in canals, ditches, and mangrove swamps (Kerfoot et al. 2011; Schofield et al. 2011) (Figure 1-

1B). They have also been found in Key Largo, Florida, but a population never became established. Pike Killifish have been recorded in the Tampa Bay area since 1994

(Greenwood 2012) and are believed to have arisen due to escapes from ornamental fish farms in the area (Schofield et al. 2011) (Figure 1-1B). The only other record of a

Pike Killifish introduction is from near San Antonio, Texas, but this population is believed to have been extirpated (Schofield et al. 2011).

Populations of Pike Killifish in south Florida were large in the areas where they were originally found (up to 20% of the total fish biomass) (Courtenay et al. 1974), but they remained restricted to canals east of the Everglades for over 20 years before experiencing a dramatic expansion in their range in the 1980s and 1990s (Courtenay

1997). Lags in the time it takes for population increase or range expansion to occur are not uncommon in introduced non-native species and may coincide with favorable conditions or overcoming some type of dispersal barrier (Gozlan 2008; Gozlan et al.

2010). The success and continued spread of this species in Florida may be attributable to a number of characteristics related to its size, reproduction, neonate (newly born individuals) traits, and physiological tolerances. The Pike Killifish is the largest member of the family Poeciliidae, reaching 20 cm standard length (SL) (Schofield et al. 2011).

Pike Killifish, like all poeciliids, bear live young. They produce large broods (6-322 young/brood with an average of 99), reproduce year-round, and have a short interbrood interval (mean of 42 days) making them one of the most fecund poeciliids (Turner and

Snelson 1984). Brood size is positively correlated with the size of the female bearing the brood and is often larger in more saline habitats (Turner and Snelson 1984). Females can store sperm for at least 47 days and possibly as long as 83 days (Turner and

Snelson 1984). Such reproductive characteristics may allow for the rapid population increases in this species.

The neonates of Pike Killifish are the largest (13-18 mm total length,TL) of the poeciliids (Belshe 1961; Turner and Snelson 1984). Individuals up to 25 mm SL have been termed to be neonates by some, while individuals between 25 and 55 mm SL are often referred to as juveniles, and individuals >55 mm SL are typically considered to be adults. Their large size at birth likely reduces predation on neonates and also allows

Pike Killifish to be piscivorous from the time they are born. Neonates and smaller juveniles will often float/swim to the surface where they remain motionless when approached by potential predators. This behavior, combined with the dark lateral stripe found in young Pike Killifish, may make them appear to be floating debris to potential predators (Miley 1978; Turner and Snelson 1984; personal observation).

Being a subtropical species, the northern range extent of this species in Florida will likely be limited by its lower lethal temperature (Shafland and Pestrak 1982; Kerfoot

2009), which appears to be around 10° C (Shafland and Pestrak 1982). Based on mean winter freshwater stream temperatures, this corresponds to their potential range extending to just south of Gainesville, FL (Shafland and Pestrak 1982). However,

feeding ceases at slightly higher temperatures (approximately 14-16° C), which could limit their potential northern expansion to near Ocala, FL (Kerfoot 2009). However, individuals have survived and recovered from starvation for up to 4 months (Belshe

1961), which could potentially allow them to survive overnight low temperatures or several days of cold temperatures without feeding as long as the temperatures did not drop below a lethal limit (Kerfoot 2009).There does appear to be some plasticity in the

Pike Killifish’s physiology regarding temperature, as individuals collected from the northern edge of their range in south Florida stopped feeding at lower temperatures compared to individuals from further south (i.e., some individuals may adapt to lower temperatures as they spread northward) (Kerfoot 2009). Juveniles maintain the quickest feeding strikes across all temperatures compared to adults and neonates, and they maintain the lowest temperature tolerance, suggesting that this may be the life stage responsible for dispersal, particularly with regard to its northward expansion (Kerfoot

2009). Pike Killifish are also tolerant of a wide range of salinities with individuals surviving indefinitely at salinities of 0-50 ppt (Belshe 1961), with reproducing populations being found in salinities of 0-35 ppt (Turner and Snelson 1984). Annual changes in Pike Killifish densities in south Florida have been correlated to salinity, temperature, and pH (Kerfoot et al. 2011). In general, higher densities were found at higher pH values, lower temperatures, and lower salinities, but this varied by location and the interaction between some of these variables were often important. In the southernmost sites studied, the interaction between temperature and salinity provided the best predictor of Pike Killifish density, while in the more northern sites variability in pH provided the best model. These correlations suggest that there may be some type of

mediation of physiological, behavioral and/or ecological performance arising from these variables (Kerfoot et al. 2011).

Pike Killifish are essentially an exclusive piscivore from birth, and often will only consume prey other than fish when starved in a lab setting and when prey fish resources have been severely depleted in the wild (Belshe 1961; Miley 1978; Turner and Snelson 1984; Greven and Brenner 2008). They feed diurnally (Miley 1978) and appear to prefer relatively shallow-bodied fishes that live near the surface (Miley 1978;

Greven and Brenner 2008). Several modifications in its jaw morphology allow for a relatively large gape, which enables this species to consume prey that are relatively large in comparison to its own body size from the time they are born (Greven and

Brenner 2008; Ferry-Graham et al. 2010). Pike Killifish generally attack prey in a similar manner to pikes (Esocidae), gars (Lepisosteidae), needlefish (Belonidae), and barracudas (Sphyraenidae), in which minimal body movements are followed by an S- start lunge at prey items (Miley 1978; Greven and Brenner 2008). They also share a number of characteristics with these families that are evolved for this type of attack, such as posteriorly placed dorsal and anal fins, elongate pointed jaws, and many unicuspid teeth) (Greven and Brenner 2008).

In south Florida, Pike Killifish can quickly deplete populations of small prey fish, in particular native cyprinodontiform fishes (Poeciliidae, Fundulidae, and

Cyprinodontidae), and this is especially likely to occur in small, restricted water bodies and/or areas lacking prey refuge (Belshe 1961; Courtenay and Robins 1973; Miley

1978; Loftus and Kushlan 1987; Trexler et al. 2000). This has the potential to reduce the prey items available to native fish species that share similar diets, such as Florida Bass

Micropterus floridanus and Warmouth Lepomis gulosus (Miley 1978). Although both of these species have relatively wide diet breadths (Hill and Cichra 2005) and competition food resource competition with Pike Killifish may be minimal except under certain environmental conditions (e.g., restricted water bodies during drought periods). Recent evidence suggest that Pike Killifish may also be linked to decreased abundance and altered size structure of several species of cyprinodontiform fishes in tidal tributaries of

Tampa Bay (Greenwood 2012).

The Tampa Bay population of Pike Killifish has shown a similar pattern to that of the south Florida populations, in which population levels remained relatively low for a number of years before an increase in both numbers and distribution. Pike Killifish were listed as a known invasive of the greater Tampa Bay ecosystem that was common but not abundant in an assessment of nonindigenous species of Tampa Bay published in

2004 (Baker et al. 2004), and fewer than 200 individuals were collected by the state’s

Fisheries Independent Monitoring (FIM) sampling prior to 2006 (MacDonald et al. 2010).

More recent sampling has shown an increased range in the Tampa Bay area, as well as an increase in abundance, with Pike Killifish being the second most abundant non- native taxon collected (the most abundant taxon were tilapias, which consisted of at least two species) (MacDonald et al. 2010). This increased range and abundance may have come about due to proper conditions (i.e., end of a lag phase), overcoming a dispersal barrier, a proper amount of time passing for spread to occur, or may simply be due to sampling in habitats more likely to contain Pike Killifish. Concern has been expressed about the potential spread of this species to other major estuaries of

Florida’s west coast, such as Charlotte Harbor, either from southward expansion from

Tampa Bay or northward expansion from the estuarine areas of the Everglades

(Idelberger et al. 2011). There has also been concern expressed about potential predation or competition with estuarine species, in particular the Common Snook

Centropomus undecimalis, due to the piscivorous nature of the Pike Killifish and the impacts it has had in freshwater and estuarine habitats in Florida (MacDonald et al.

2010; Greenwood 2011; Schofield et al. 2011).

Common Snook (from here forward referred to simply as Snook) is the most widespread centropomid found in the western Atlantic Ocean (Rivas 1986). Snook are diadromous, and have been referred to as amphidromous or semicatadromous. Both adults and juveniles move between fresh and marine waters, with a general trend of juveniles being located in fresh or inland marine waters and adults moving from these inland waters to inlets and ocean beaches to spawn (Tringali and Bert 1996; Taylor et al. 1998). The primary range of this species extends from Cape Canaveral, Florida, through the Caribbean and the Gulf of Mexico to Rio de Janeiro, Brazil (Gilmore et al.

1983; Rivas 1986) (Figure 1-2A). In Florida, the majority of snook are found south of

Cape Canaveral on the Atlantic Coast and south of Tarpon Springs on the Gulf coast

(Taylor et al. 1998) (Figure 1-2B). Smaller numbers persist to near Cedar Key along the

Gulf coast of Florida and Jacksonville on the Atlantic coast (Taylor et al. 2000; personal observation); however, north of this and extending westward, specimens are rarely taken until Galveston, Texas, where populations also persist (Rivas 1986) (Figure 1-2).

Occasional specimens have been collected in the coastal waters of Georgia (Linton and

Rickards 1965), the Carolinas (Lunz 1953; Martin and Ship 1971; Rivas 1986), and New

York (Schaefer 1972). Lower lethal water temperatures for snook are in the range of 9-

12° C (Shafland and Foote 1983; Howells and Sonski 1990), which results in a northern range restriction. This range restriction is similar to that of the Red Mangrove

Rhizophora mangle, which is one the primary habitats of snook (Marshall 1958; Gilmore et al. 1983).

Historically, snook have supported both commercial and recreational fisheries in

Florida (Marshall 1958; Seaman and Collins 1983; Muller and Taylor 2006). The commercial fishery for snook in Florida began in about 1939 but was never very large and peaked in 1948 (protein short years of WWII) at about 363,000 kg (800,000 lb)

(Marshall 1958). Marshall (1958) speculated that the decline in the catch after 1948 likely indicated a general market decline and not necessarily a decline in abundance, and that if abundance was declining it was likely more attributable to habitat alteration than fishing due to the relatively small harvest of snook. Concerns about the effects of overfishing and habitat degradation resulted in snook being designated as a gamefish in

Florida in 1957 (Table 1-1), and as such they became closed to commercial fishing

(Marshall 1958; Seaman and Collins 1983). Subsequent regulations were placed on the recreational fishery and have continued to become more restrictive with continued concerns of overfishing (Table 1-1). Based on stock assessments, the Gulf coast snook population was deemed to have been overfished from the mid-1980s through at least

2004 (possibly 2006 depending on the assessment) with the possible exception of a few years based on the most recent assessment model (Muller and Taylor 2006, 2013,

2013; Muller et al. 2015). Despite this, snook have remained a highly targeted species contributing substantially to the recreational fishing industry in Florida. Based on the

Marine Recreational Fishing Statistics Survey (MRFSS), snook have ranked in the top 5

most recreationally targeted marine species on the Gulf coast of Florida since at least

2000 (only summary of rankings prior to 2000 occurred in 1987) through 2009 (Muller and Taylor 2013). The rank of snook dropped to 10th place in the 2012 rankings summary; however, this followed an extended closure (January 2010 through August

2013) of snook harvest on the Gulf coast due to a severe cold kill event in January

2010. In 2014, snook were again ranked in the top 5 most targeted species (Muller et al.

2015). Somewhere between 2004 and 2006, assessment models showed that the Gulf snook stock was no longer overfished and remained in this status through 2012 despite the large cold kill in 2010 (Muller and Taylor 2013). The most recent assessment again showed that Gulf coast snook were not overfished in the base model. However, when large natural mortality events were incorporated the stock fell just into an overfished status (Muller et al. 2015).

The importance of Snook to Florida’s economy and concerns about declining populations lead to a number of studies being conducted in an effort to gain knowledge for the better management of this species. These studies revealed several key aspects regarding the population structure, reproduction, and early life history of snook.

Genetics have shown that populations of snook on the Gulf and Atlantic coasts of

Florida are distinct, and that both Florida populations are distinct from populations from

Caribbean islands, which themselves may be genetically different from one another

(Tringali and Bert 1996). Tagging studies also show that migration between the Atlantic and Gulf coasts of Florida is minimal (Volpe 1959, R. Taylor, G. Bruger, and J.

Whittington, Florida Fish and Wildlife Conservation Commission, unpublished data-cited in Tringali and Bert 1996). However, there may be some mixing of the populations in

Florida Bay as individuals in this area appear to be derived in approximately equal numbers from both the Atlantic and Gulf coasts, although it is thought that spawning in in this area of southern Florida is unlikely (Tringali and Bert 1996; Patterson et al. 2005).

These genetic differences, as well as a number of other potential factors such as water temperature regimes and food availability, may account for the differences observed in

Gulf coast and Atlantic coast snook. Snook on the Atlantic coast of Florida are generally larger at age, migrate greater distances along the coast, live longer, and have a lower natural mortality rate than snook on the Gulf coast (Taylor et al. 2000; Muller and Taylor

2006).

Reproductive studies on snook have revealed that they exhibit protandric sex reversal, with all fish beginning life as males and later changing sex (Taylor et al. 2000).

Males may mature as early as age-0 (<200 mm fork length (FL)), and most males mature by age-1. It is not known if these small males are actively spawning, however they showed histological characteristics that indicated they were capable of spawning and were thus considered to be mature (Taylor et al. 2000). On the Atlantic coast of

Florida approximately 50% of males will have transitioned to females at 7.4 years of age and 767 mm FL, and on the Gulf coast the age and size at 50% sex transition was 5.1 years of age and 608 mm FL (Taylor et al. 2000). Histological samples of ovaries indicate that spawning appears to begin in April to May and end in September to

October (Taylor et al. 1998). However, analysis of daily growth rings in juveniles indicates that snook may spawn as late as December (McMichael et al. 1989), and it has been suggested that some spawning may occur year-round (Gilmore et al. 1983). In

Florida spawning occurs in major inlets opening to the Gulf of Mexico and Atlantic

Ocean, secondary passes to larger inland bays, and around nearshore islands (Taylor et al. 1998). Larvae spend approximately 2.5 weeks in nearshore waters prior to arrival at nursery sites, which include both low and high salinity vegetated shorelines of quiet, shallow-water creeks, canals, and lagoons (Peters et al. 1998 a). Transport of larvae to nursery areas appears to be the result of strong flooding tides, two-layer circulation in estuaries due to salinity differences between the surface and bottom, or a combination of these mechanisms (Peters et al. 1998 b). Upon arrival in nursery habitats, juveniles will spend several weeks to several months in a mainly “pelagic” state, which ends when they reach 15-45 mm standard length (SL), with the duration of this stage potentially depending on food availability and predation pressure (Peters et al. 1998 a).

During this stage, juveniles mainly feed on copepods and other microcrustaceans, such as mysids (Harrington and Harrington 1961; Fore and Schmidt 1973; McMichael et al.

1989; Aliaume et al. 1997; Peters et al. 1998 a). With a switch to a more demersal lifestyle there is also a shift in diet, with the main prey items becoming small fishes and grass shrimp Palaemonetes spp. (Fore and Schmidt 1973; McMichael et al. 1989;

Aliaume et al. 1997; Peters et al. 1998 a; Adams et al. 2009; Rock 2009), although nocturnal sampling has shown that mysids may still be important prey items for juveniles <130 mm SL (Rock 2009). Both pelagic juveniles and demersal juveniles up to approximately 100 mm SL (early-juveniles) inhabit similar habitats, which are typically creeks, canals, and lagoons (i.e., tributaries and backwaters) characterized by shallow depths (< 1 m), soft mud bottoms with little or no submerged aquatic vegetation (SAV), slow currents, moderate shoreline slopes (< 0.4 rise/run), and with shoreline vegetation, such as mangrove or emergent marsh grasses, extending into and/or over the water

(Fore and Schmidt 1973; Gilmore et al. 1983; Seaman and Collins 1983; McMichael et al. 1989; Aliaume et al. 1997; Peters et al. 1998 a; Greenwood et al. 2008; MacDonald et al. 2010). At approximately 100 mm SL, juvenile snook may continue to use the same habitats described above but will also move into more open bay and river shorelines and seagrass beds (Gilmore et al. 1983; McMichael et al. 1989; Peters et al. 1998 a).

This ontogenetic shift correlates to a decreased tolerance of low dissolved oxygen (DO) levels, which are characteristic of the former habitats. Early-juvenile snook have been demonstrated to tolerate lower DO levels than larger individuals, as well as showing behavioral adaptations to low DO levels, such as moving towards the water’s surface and decreasing activity (Peterson and Gilmore 1991; Peterson et al. 1991).

The fact that both Pike Killifish and early-juvenile snook (<100 mm SL) have been shown to be most abundant in the same habitats within Tampa Bay (tributaries and backwaters along the eastern shore of the bay, Figure 1-3) has raised concerns about potential impacts of Pike Killifish on early-juvenile snook (MacDonald et al. 2010;

Greenwood 2012), which could ultimately impact recruitment of snook to the adult population. Pike Killifish, as an aggressive piscivore, has the potential to directly prey upon early-juvenile snook. Pike Killifish will prey upon juvenile snook in the lab (E.

Matheson, Florida Fish and Wildlife Conservation Commission and M. Greenwood, ICF

International, personal communication), although whole snook have not been found in the stomachs of Pike Killifish from the wild (Greenwood et al. 2008). However, partial remains of unidentified fish, such as otoliths, have not been examined from Pike Killifish stomachs to look for the presence of snook, and the number of stomachs examined is still relatively low (approximately 250 stomachs with only about half of these containing

stomach contents) (M. Greenwood, ICF International, personal communication). Pike

Killifish may also compete with early-juvenile snook, which could lead to decreased growth and increased predation. The condition, or relative weight at a particular length, which is often assessed as an index of physiological well-being of a fish, may also be influenced by factors such as competition. Interspecific competition may take the form of exploitative competition or interference competition (Connell 1983; Schoener 1983; Mills et al. 2004). Exploitation competition involves individuals using (competing for) the same resources, such as food items, which are limiting in some way (e.g., reduced prey availability) (Connell 1983; Schoener 1983). Interference competition involves the production of toxins in sessile organisms, fighting, and other aggressive behaviors

(Connell 1983; Schoener 1983). Some cases of competition, such as for space, may involve components of both types of competition (i.e., space is a resource and thus exploitation competition is involved, but competition for space often involves aggression and thus interference competition is involved) (Schoener 1983). Exploitation competition may exist between Pike Killifish and juvenile snook, since Pike Killifish are piscivores feeding mainly on cyprinodontiform fish, which are also common prey items of juvenile snook, and Pike Killifish have been shown to severely deplete such prey items in freshwater systems in Florida. There is also evidence that some prey items, Eastern

Mosquitofish Gambusia holbrooki and Sailfin Molly Poecilia latipinna in particular, can have reduced abundances and tend to be larger in Tampa Bay tributaries that have been invaded by Pike Killifish (Greenwood 2012).

This study aims to determine the potential extent of both predation on and competition with early-juvenile snook by Pike Killifish in the Tampa Bay area. The

specific objectives of this study include: 1) examining the extent of Pike Killifish predation on juvenile snook through diet analysis and predation trials; 2) assessing the potential for exploitative competition for food resources between these two species through diet analysis and prey availability; 3) assessing the potential for interference competition (aggressive behaviors) or exploitative competition for space between Pike

Killifish and snook using experimental mesocosms; 4) assessing potential competition among these two species by determining if growth and condition of snook is reduced in the presence of Pike Killifish; and 5) integrating data from the previous objectives into simulated population models to examine how the presence of Pike Killifish may influence the number of early-juvenile snook that would survive to become late-stage juveniles (>100 mm SL).

Table 1-1. History of Common Snook regulations in Florida Year Regulations 1947 Snook haul seines made illegal in Lee County 1951 Snook haul seines made illegal in Collier County 1953 18 in (457 mm) fork length (FL) minimum size limit 1957 Designated a gamefish; commercial harvest prohibited; hook and line capture only; 4/person daily bag limit; possession limit of 8 1981 2/person daily bag limit; 26 in (660 mm) FL maximum size limit in June and July (1982- 1986) 1982 Designated species of special concern in Florida; June and July closed to harvest in 1982 1983 January and February closed to harvest (1983-1986); June and July closed to harvest (1983-1986) 1985 January, February, June and July permanently closed to harvest; 24 in (610 mm) total length (TL) minimum size limit; 1 fish may be >34 in (864 mm) TL; August close to harvest in 1985-1986 1987 All Centropomus covered by regulations; August permanently closed to harvest; fish must be landed whole; no treble hooks may be used with natural baits 1989 Snook stamp required for possession for those that are required to have a Florida fishing license 1994 Winter closure changed to December 15 through January 31 1999 26-34 in (660-864 mm) TL slot limit 2000 Clarified gear regulations (clear definitions of hook and line gear and spearing) 2001 Removed species of special concern designation 2002 Gulf coast only: 1/person daily bag limit; May closed to harvest 2006 27 in (686 mm) TL minimum size limit; TL defined as tip of snout to end of pinched tail 2007 Atlantic coast: 28-32 in (711-813 mm) TL slot limit, 1/person daily bag limit Gulf coast, Monroe County, and Everglades National Park: 28-33 in (711-838 mm) TL slot limit, 1/person daily bag limit; winter closed season change December 1 through February 28 2010 Atlantic coast: extended closure due to fish kills in winter of 2009/2010; opened briefly from September 1-17 Gulf coast: extended closure due to fish kills in winter of 2009/2010 2011 Atlantic coast: September: season reopens after 2010 closure Gulf coast: cold kill closure still in effect 2013 Gulf coast: September: first opening since cold kill closure enacted in 2010 2018 Gulf coast: August: temporary harvest closure enacted in areas of Collier, Lee, Charlotte, Sarasota, and Manatee counties due to red tide fish kill

United States

Florida Gulf of Mexico

Mexico Tampa Bay Belize Guatemala Honduras

El Salvador Nicaragua

Panama Costa Rica

Figure 1-1. Distribution of Pike Killifish: A) Native coastal distribution of Pike Killifish in Central America. B) Non-native distribution of Pike Killifish in Florida by county, with date of first detection for each county.

Atlantic Ocean

Figure 1-2. Distribution of Common Snook: A) Native coastal distribution of Common Snook. B) Distribution of Common Snook in Florida, red denotes primary range and orange denotes areas where smaller but relatively persistent populations are found.

Figure 1-3. Distribution of Pike Killifish within Tampa Bay, FL, with area of highest early- juvenile Common Snook abundance denoted.

CHAPTER 2 PREDATION OF JUVENILE COMMON SNOOK BY PIKE KILLIFISH

Introduction

The natural mortality of juvenile fish is typically quite high, often on the order of

10-20 times greater than that of adults (Lorenzen 1996). In general, most mortality not attributed to episodic losses (e.g., aberrant drift, abnormal weather condition, acute toxic events, etc.) are related to food availability and predation (Houde 1987, 1989).

Generally, predation in fish is greatest in the smallest individuals and accounts for a large portion of the mortality in juvenile fish (Houde 1987, 1989).

Common Snook (hereafter Snook) are important both economically and ecologically in the estuarine waters of Florida. The Gulf coast population of Snook has experienced long periods of being designated as overfished (Taylor and Muller 2012,

2013; Muller et al. 2015). It has been speculated that loss of habitat, particularly that of juveniles, may play a more significant role in the reduced population size of Snook than actual overfishing. This may be particularly true for early-juvenile Snook (≤100 mm SL), which are the life stage with the most restricted habitat, and further stressors to this stage have the potential to further reduce populations and delay recovery rates. Early- juvenile Snook utilize protected shallow estuarine backwaters and tributaries primarily lined by mangroves and emergent marsh vegetation. These areas provide a low energy environment with ample food while also reducing the risk of predation (Peters et al.

1998 a; Taylor and Muller 2012). The shoreline vegetation provides cover both for ambushing prey and avoiding predation while the shallowness and often restricted entrances to these areas reduce the presence of larger fish predators, particularly

during low tides when the juvenile Snook may have to leave the protection of their shoreline cover. Early-juvenile Snook are also tolerant of low dissolved oxygen levels

(Peterson and Gilmore 1991; Peterson et al. 1991), which may occur periodically in these areas and be unfavorable to potential predators. In addition, to living in locations that may be unfavorable to predators, it has been suggested that the enlarged anal spine of juvenile Snook may serve as a deterrent to predation (Brennan 2008).

The Pike Killifish is a non-native fish species that has been established in south

Florida since 1957 (Belshe 1961). Like juvenile Snook, Pike Killifish can tolerate a wide range of salinities (Belshe 1961; Turner and Snelson 1984) and are noted to be tolerant of low dissolved oxygen levels (Hensley and Courtenay 1980; Page and Burr 1991).

This species is primarily piscivorous and has been linked to decreases in abundance and altered size structure in native small-bodied fishes in both fresh and estuarine waters in Florida (Miley 1978; Greenwood 2012). A second established population that was first detected in 1994 now exists in the estuarine waters of Tampa Bay (Greenwood

2017). Recent work has revealed that the distribution and abundance of Pike Killifish in

Tampa Bay waters has either increased in recent years or is greater than previously thought due to inadequate sampling in their preferred habitats within the Tampa Bay area (MacDonald et al. 2010; Greenwood 2017). It was found that Pike Killifish were relatively common along the southwestern shore of Tampa Bay and were most often found in quiet, shallow estuarine backwaters and tributaries (Greenwood et al. 2008;

MacDonald et al. 2010; Greenwood 2017), which not only overlaps with the general area of highest juvenile Snook recruitment in Tampa Bay but also the preferred habitat of early-juvenile Snook (Figure 1-3) (McMichael et al. 1989; Peters et al. 1998 a;

Stevens et al. 2007; MacDonald et al. 2010). The piscivorous diet of Pike Killifish, previous implications in the decrease of small-bodied fish abundance, and the overlap in distribution and habitat use of Pike Killifish and early-juvenile Snook has raised concerns about the potential impact of Pike Killifish on Snook through competition for food and predation (MacDonald et al. 2010; Greenwood 2012, 2017).

This study aimed to evaluate the potential extent of predation on juvenile Snook by Pike Killifish through predation trials and diet analysis. Predation trials were utilized to determine the size of Snook that Pike Killifish were capable of consuming in comparison to consumption of conspecifics and a commonly consumed prey species

(Eastern Mosquitofish Gambusia holbrooki, hereafter Mosquitofish), and to explore whether the enlarged anal spine of Snook aids in deterring predation by Pike Killifish.

Diet analysis of Pike Killifish captured from areas with early-juvenile Snook co-occurring was utilized to estimate how often Snook are consumed by Pike Killifish where they co- occur in the wild.

Methods

Predation Trials

This portion of the study was conducted at the University of Florida’s Fisheries and Aquatic Sciences (FAS) facility in Gainesville, FL. Fish were maintained in tanks with a temperature of 28-30 °C, salinity of 5-10 ppt, and a 12L:12D light cycle (based on data available from the Tampa Bay Water Atlas

(http://www.tampabay.wateratlas.usf.edu) and field observations during the standardized sampling period of this study). Predators consisted of large adult Pike

Killifish (≥ 90 mm SL) and prey consisted of juvenile Snook, juvenile Snook with their anal spine clipped, Mosquitofish, and Pike Killifish. Mosquitofish were selected as potential prey to compare Pike Killifish consumption rates of a known and commonly consumed prey item (Belshe 1961; Miley 1978; Greenwood 2012) to Pike Killifish consumption of Snook. Pike Killifish were selected as potential prey items due to the fact that they are: a) similar in body shape to Mosquitofish, b) reach sizes equivalent to the largest Snook presented as prey (Mosquitofish are noted to occasionally reach these sizes (Nico and Fuller 2015) but none were collected during the course of this study), and c) are noted to be cannibalistic (Belshe 1961; Miley 1978; Anderson 1980;

Greenwood et al. 2008). Prey from four size classes (21-40 mm TL, 41-60 mm TL, 61-

80 mm TL, and >80 mm TL) were presented to predators. Total length was used for the analysis of predation trial data to allow for comparison among the different prey species as the SL to TL ratios vary by species, but SL were also recorded allowing for comparison with most published literature on juvenile Snook in which SL is reported.

Pike Killifish and juvenile Snook used in predation trials were collected from locations outside of the standardized sampling areas for diet analysis (Figure 2-1); all

Pike Killifish that served as predators were collected from locations where juvenile

Snook co-occurred and Mosquitofish were collected on site from FAS ponds. Both predators and potential prey were collected with cast nets (1.5 m radius with 4.8 mm mesh and 2.1 m radius with 6.4 mm mesh) and various sized dip nets.

Predation trials were conducted in 37.8 L (10 gal) aquaria in a recirculating system (Figure 2-2). A black plastic divider was placed between each aquarium to prevent the predators from viewing the Pike Killifish in the tank next to them, and the

aquaria were wrapped in a sheet of black plastic to prevent the predators from being disturbed by investigator movements in the lab. Predators were acclimated to lab conditions. Acclimation to lab temperature and salinity was relatively short (2-6 hours), as the lab and field conditions did not typically differ greatly. Predators were then measured to the nearest mm and placed in aquaria and allowed to acclimate until they started feeding on small poeciliid fish in captivity, after which the predation trials were initiated. Prey were also acclimated to the lab conditions and placed in 570-680 L recirculating holding tanks that were approximately ¼ - ½ filled.

To initiate a predation trial, predators were fasted for approximately 12 hours (an entire light cycle after an initial feeding within the first ½ hour of the light cycle). A potential prey item (species and size class) was randomly selected and anesthetized with a 70 mg/L solution of MS-222 buffered with sodium bicarbonate. All anesthetized prey had their anal fin extended, and for those Snook selected to have their anal spine clipped, the anal fin was extended, and the anal spine was clipped approximately 1 mm from the body with a pair of dissecting scissors sterilized in 95% ethanol. After anal fin extension, potential prey fish were placed in an aerated bucket to recover for a minimum of 9 hours. Recovered prey were measured to the nearest mm SL and TL prior to being placed in a predation trial aquarium with a predator. Prey were placed in the predation trial aquaria 0-2.5 hours after the end of the light cycle and left overnight bringing the potential fasting time to approximately 24 hours, as Pike Killifish are noted to not feed nocturnally (Miley 1978). Prey were left in the aquaria for 48 hours (2 dark and 2 light cycles). At the end of the first light cycle, it was noted if a prey had been consumed or attacked but not consumed (visible bite marks, lost scales, torn fins, etc.)

and whether it was alive or deceased (consumed or mortality due to an attack). This was noted again at the conclusion of the predation trial. Any prey items that had not been consumed during the predation trial were then removed. If prey were not consumed during the predation trial, a Mosquitofish (25-40 mm TL) was presented to the predator and another predation trial was not initiated until that prey item had been consumed to avoid a predation trial being conducted with a predator that had been fasted for more than 24 hours. At the conclusion of the predation trials, all predators and remaining prey items were euthanized with a 300 mg/L solution of tricaine methanesolfonate (MS-222) buffered with sodium bicarbonate (American Veterinary

Medical Association 2013; UF IACUC Protocol 201207451).

Attack data and consumption data, based on TL, after 48 hours were fit to a negative logistic function using the glm procedure in R with 95% confidence intervals to determine the size at which 50% of a prey species (Snook, Snook without anal spine,

Mosquitofish, or Pike Killifish) had been attacked or consumed respectively. The confidence intervals of the logistic curves for each prey species were visually compared to determine if there were any significant differences. In addition, a glm logistic model for each pair of prey species was created. This model fit attack or consumption data based on TL, prey type, and an interaction factor. An analysis of deviance was run to determine if the slopes and intercepts of each curve differed (Ogle 2016). A significant interaction factor indicates that the slope of the separate logit-transformed models differ, while a significant prey type factor indicates that the intercept of the separate logit- transformed models differ (Ogle 2016). Pairwise Fisher exact tests were also performed on this data in R (pairwise.Fisher.test procedure, α = 0.05) to compare the

proportion of both attacks and consumption between prey types (species and size classes). A Benjamini and Hochberg correction was used to adjust p-values as it corrects for the multiple Fisher tests without being overly conservative (Kallio et al.

2011).

Fish Collection for Diet Analysis

Pike Killifish stomachs analyzed for the presence of juvenile Snook remains were obtained from standardized sampling events as well as ad-hoc sampling. Standardized sampling was conducted in Wildcat Creek, a tributary of the Little Manatee River and backwaters of the Alafia River (Figure 2-3), which are both areas known to be utilized by both Pike Killifish and early-juvenile Snook (Greenwood et al. 2008; MacDonald et al.

2010; Greenwood 2012). In each of these locations, twelve fixed sampling sites were selected. Fixed sampling sites consisted of shoreline stretches of approximately 100 m in length. Wildcat Creek is located approximately 8.5 km from the mouth of the Little

Manatee River. Its entire length consists of suitable habitat for both early-juvenile Snook and Pike Killifish, thus the fixed sampling sites consisted of continuous sections of shoreline along both the east and west forks starting approximately 500-600 m from the mouth upstream until access became overly restricted by the narrow width of the creek and overgrowth of vegetation (Figure 2-4). Backwater areas of the Alafia River are shorter in length and separated by stretches of the mainstem of the river. Fixed sites began approximately 4.75 km from the mouth of the river and were placed along continuous sections of shoreline when possible (Figure 2-5).

A 21.3 m long, 1.8 m high center bag seine with 3.2 mm (#35) knotless nylon mesh and a 1.8 m3 bag was used for the standardized sampling. Each of the 12 fixed

sites in each river system was sampled once between August and October, the period of peak juvenile Snook recruitment in Tampa Bay (McMichael et al. 1989; Peters 1998 a), in 2012 and 2013. Sampling occurred during the second half of a falling tide through the first half of a rising tide, as the sampling efficiency of this type of gear in these backwater habitats is often decreased during flood tides. Four of the twelve fixed sites were randomly selected without replacement to be sampled each month from August to

October. In some cases, tidal cycles in August and September did not allow all four sites to be sampled within a given month, and in these cases the un-sampled sites were sampled in one of the following months when the low tide cycles had a greater duration.

In addition to the sampling with the bag seine, a small minnow seine (3.5 m long x 1.2 m tall with 3.2 mm mesh) was also utilized. Window screening (1.3 mm) was attached to the mesh of this seine to retain smaller prey items. It was hauled adjacent to the bag seine sampling location to collect potential prey items for a co-occurring study. The minnow seine was pulled for 6.1 m parallel to the shoreline adjacent to bag seine hauls in which either Pike Killifish or early-juvenile Snook or both had been captured and where bottom and shoreline conditions permitted deployment of the minnow seine. Both

Pike Killifish and early-juvenile Snook were occasionally captured with this gear. Pike

Killifish captured in the standardized sampling were given a unique identification number and placed in individual bags, which were placed in an ice bath for euthanasia

(UF IFAS Non-regulatory Animal Research Protocol 006-12FAS). Upon returning from the field, these samples were frozen for later stomach content analysis.

To supplement samples obtained from the standardized sampling, Pike Killifish were randomly collected from several locations between Coachroach Bay Aquatic

Preserve and the Alafia River (Figure 2-1), where both Pike Killifish and early-juvenile

Snook co-occurred. Additional samples were also collected from some of the standardized sampling locations after the completion of the standardized sampling.

These samples were collected with dip nets (roughly oval in shape with a flat bottom:

0.36 m along bottom frame, height 0.48 m, and 4.8 mm mesh) and cast nets (1.5 m radius with 4.8 mm mesh and 2.1 m radius with 6.4 mm mesh).

Stomach Content Analysis

Pike Killifish were measured with digital calipers (to the nearest 0.1 mm) and weighed to the nearest (0.0001 g) prior to their stomach being removed. Once removed, the stomachs were opened, and prey items were examined for the presence of juvenile

Snook under a dissecting microscope (8x-80x magnification). The otoliths of partially digested fish were removed when present to determine if those particular remains were that of a juvenile Snook. Partially digested fish remains without otoliths present were closely examined for key characteristics of early-juvenile Snook (enlarged anal spine, jaw/head morphology, etc.).

Results

Predation Trials

A total of 275 predation trials were conducted (70 Snook, 69 Snook with anal spine clipped, 68 Mosquitofish, and 68 Pike Killifish). A total of 35 Pike Killifish ranging in size from 90-135 mm SL were used as predators. Due to availability of certain sizes of prey items, not all Pike Killifish predators were exposed to the entire size range of

potential prey of each species, but they were all exposed to each species of prey (with similar sizes of prey between each prey species). Size ranges of each prey species were as follows: Snook 21-80 mm TL, Snook with anal spine clipped 22-83 mm TL,

Mosquitofish 21-57 mm TL, and Pike Killifish 21-89 mm TL).

Pike Killifish Attacks on Prey

During the course of the predation trials there were only two instances of a prey item mortality resulting from a Pike Killifish attack without consumption, and no statistical analyses were conducted on this type of outcome due to the low number of instances. Both instances involved relatively large Snook prey, one with and one without its anal spine (64 mm TL and 57 mm TL respectively). There were also few instances

(21 of 386 predation trials) of prey either being attacked or consumed after 48 hours that had not already been attacked or consumed after 24 hours.

Pike Killifish attacked Snook with an intact anal spine ranging in size from 21 to

64 mm TL, and the size at which 50% of Snook were attacked was 53.9 mm TL (~43 mm SL) (Figure 2-6). Attacked Snook without an anal spine ranged in size from 22 to 66 mm TL with a size at 50% attack of 52.8 mm TL (~42 mm SL) (Figure 2-6). The 95% confidence intervals for the negative logistic curves for each of these prey types overlap over the entire range of prey size and the sizes at 50% attack are nearly identical

(Figure 2-6). The interaction and prey type factors for a model containing both Snook with and without their anal spine were both non-significant based on analysis of deviance (Table 2-1). This would indicate that the slope and intercept of the logit- transformed model were not significantly different between these two prey types. Based on these lines of evidence, there does not appear to be a significant difference in the

attack rate of Pike Killifish on Snook of a particular size regardless of the presence of the anal spine.

All but one Mosquitofish (a 57 mm TL individual) was attacked during the course of the predation trials (Figure 2-6), indicating that Mosquitofish of all sizes are susceptible to attack by Pike Killifish. Because of this a logistic function could not be fit to the data and subsequent analyses comparing logistic regressions could not be completed. A plot of the attack data for both Mosquitofish and Snook, both with and without their anal spine, shows that Snook within and even slightly larger than the largest Mosquitofish tested were attacked, but there were also a number of Snook in this size range that were not attacked by Pike Killifish (Figure 2-6).

Pike Killifish attacked smaller conspecifics ranging in size from 21 to 87 mm TL.

The size at 50% attack for Pike Killifish was 84.7 mm TL (~71 mm SL) (Figure 2-6). The

95% confidence intervals for Pike Killifish overlapped those of Snook with an anal spine to just over 30 mm TL and those of Snook without their anal spine up to just over 40 mm

TL (Figure 2-6). The model containing both Pike Killifish and Snook with their anal spine had a non-significant interaction factor but a significant prey type factor (Table 2-1). This would indicate that the separate logit-transformed models had similar slopes but different intercepts. This was also true for the model containing both Pike Killifish and

Snook without an anal spine (Table 2-1). When Pike Killifish and Mosquitofish attack data are plotted together it is clear that Pike Killifish in the same size range as

Mosquitofish were attacked (Figure 2-6).

There were no significant differences in the number of attacks on Snook with and without an anal spine within the same size class based on Pairwise Fisher exact tests

(Table 2-2), which corroborates the results of the logistic regressions. The largest size class of Snook (61-80 mm TL), both with and without an anal spine, were attacked significantly less than two smaller size classes of conspecifics tested (Table 2-2). The comparison of the smallest and middle size class of Snook showed a significant difference (smaller size attacked more than middle size) for Snook without an anal spine but not for Snook with an anal spine. In general, Mosquitofish were attacked by Pike

Killifish significantly more than Snook regardless of size and Pike Killifish of an equivalent size were attacked significantly more than Snook (Table 2-2). There was not a significant difference in the number of attacks with any combination of Mosquitofish and the two smallest size classes of Pike Killifish, but the largest two size classes of

Pike Killifish (61-80 and >80 mm TL) were attacked significantly less than Mosquitofish of either size class (21-40 or 41-60 mm TL) (Table 2-2).

Pike Killifish Consumption of Prey

Pike Killifish consumed Snook with an intact anal spine ranging in size from 21 to

60 mm TL, and the size at which 50% of Snook were consumed was 42.4 mm TL (~33 mm SL) (Figure 2-7). Consumed Snook without an anal spine ranged in size from 22 to

56 mm TL with a size at 50% consumption of 45.4 mm TL (~37 mm SL) (Figure 2-7).

The 95% confidence intervals for the negative logistic curves for each of these prey types overlap over the entire range of prey size and the sizes at 50% consumption are similar (Figure 2-7). As with the attack data, a model containing both Snook with and without their anal spine did not have significant interaction or prey type factors, which would suggest the separate logit-transformed models have similar slopes and intercepts

(Table 2-1). Overall, there does not appear to be a significant difference in the

consumption of Snook, of similar sizes, by Pike Killifish based on the presence of an anal spine.

The one Mosquitofish that was not attacked by a Pike Killifish during the predation trials was also the only Mosquitofish that was not consumed (Figure 2-7).

Again, a logistic function could not be fit to the data and subsequent statistical comparison could not be done. The consumption data for both Mosquitofish and Snook were plotted and mirror the results of the attack data with many Snook in the size range of the Mosquitofish being consumed, but a number of the Snook in this size range also not being consumed (Figure 2-7).

Pike Killifish cannibalized smaller conspecifics ranging in size from 21 to 84 mm

TL. The size at 50% consumption for Pike Killifish was 69.7 mm TL (~57 mm SL)

(Figure 2-7). The 95% confidence intervals for Pike Killifish and Snook with their anal spine intact do not overlap indicating significant difference in consumption between the two species across the entire size range tested (Figure 2-7), while Pike Killifish and

Snook that had their anal spine clipped have confidence intervals that just barely overlap only up to about 25 mm TL (Figure 2-7). As with the attack data, there was a significant prey type factor but non-significant interaction factor for a model with both

Pike Killifish and Snook with an anal spine data (Table 2-1). This was also true for the model with Pike Killifish and Snook without an anal spine (Table 2-1). This suggests that the intercept, but not the slope, of the logit-transformed models differed between Pike

Killifish and Snook, both with and without an anal spine. When plotted together the Pike

Killifish and Mosquitofish consumption data mirrors the attack data, with Pike Killifish in the same size range as Mosquitofish being consumed (Figure 2-7).

As with the attack data, there were no significant differences in the number of

Snook consumed by Pike Killifish within a specific size class regardless of anal spine presence based on Fisher exact tests (Table 2-3). The two smaller size classes of

Snook, both with and without an anal spine, were consumed significantly more than the largest size class of Snook (Table 2-3). The smallest size class of Snook was consumed significantly more than the middle size class for fish without anal spines but not for Snook with intact anal spines (Table 2-3). Overall, Mosquitofish were consumed significantly more than Snook regardless of size and Pike Killifish of an equivalent size were consumed significantly more than Snook (Table 2-3). No significant differences were detected in the number of fish consumed with any combination of Mosquitofish and the two smallest size classes of Pike Killifish, however, the largest two size classes of Pike Killifish (61-80 and >80 mm TL) were consumed significantly less than

Mosquitofish of either size class (21-40 or 41-60 mm TL) (Table 2-3).

Fisher exact tests also revealed that 41-60 mm TL Snook, both with and without anal spines, were, in general, less likely to be consumed after an attack than both

Mosquitofish and Pike Killifish of an equivalent or smaller size (Table 2-4). Large Pike

Killifish, 61-80 mm TL, were significantly less likely to be consumed after an attack than nearly all other prey types (species and size), although they could not be compared to

Snook of an equivalent size as few equivalent sized Snook (5 with anal spine intact and

2 with anal spine clipped) were attacked (Table 2-4).

Stomach Content Analysis

A total of 422 Pike Killifish stomachs were analyzed, with 160 (37.9%) containing prey items that were <90% digested. No identifiable Snook remains were found in any

of the Pike Killifish stomachs examined. There were also relatively few, N = 8, fish prey items that could not be identified to at least an order, and 1 unidentified perciform fish that did not appear to be a Snook based on its otolith shape.

Discussion

This study demonstrated that large adult Pike Killifish (>90 mm SL) would attack juvenile Common Snook up to 66 mm TL and were capable of consuming Snook up to

60 mm TL under fasted conditions. Attacks where prey were not consumed rarely lead to mortality, and the two instances where this occurred involved larger Snook that had clearly been attacked repeatedly but were likely just above the gape limitation of the predator in those particular trials. It is likely that in the wild these Snook would have found refuge after escaping the initial attack, rather than being repeatedly attacked as was the case in the predation trials due to the confined nature of the experimental tanks. The damage caused to other prey that were attacked but not consumed was minimal and consisted of small patches of missing scales and small tears in the fins.

Based on these observations, it is likely that Pike Killifish attacks on prey that do not lead to consumption in the wild would not lead to delayed mortality from wounds or secondary infections in most cases.

The fact that both Mosquitofish and conspecifics were generally both attacked and consumed significantly more by Pike Killifish than Snook of similar sizes suggests that Pike Killifish either have a preference for the other two prey species tested, Snook avoided attacks, or Snook possess some deterrent to attack by predators (Figures 2-6 and 2-7, Tables 2-2 and 2-3). It has been suggested that the enlarged anal spine of juvenile Snook may act as a deterrent to predation (Brennan 2008). This spine could

deter potential predation in the decision of whether a predator choses to attack a prey

(i.e., a visual deterrent) and it could also deter predation by preventing actual consumption once an attack has been made (i.e., physical deterrent). Preliminary work investigating Pike Killifish prey preferences suggested that they may preferentially consume Snook without an anal spine to those with an anal spine when both prey were presented, but this was only based on 3 trials (E. Matheson, Florida Fish and Wildlife

Conservation Commission, and M. Greenwood, ICF International, personal communication). However, the current study indicated that there were no significant differences in the attack or consumption rate between Snook with and without an intact anal spine of similar sizes (Figures 2-6 and 2-7 and Tables 2-2 and 2-3). This result is somewhat of a surprise as Pike Killifish typically consume their prey tail first, a position in which an anal spine would typically remain erect and potentially obstruct consumption in comparison to swallowing a prey head first in which the anal spine could be folded down more easily. It is possible that the anal spine of Snook in the size range that were consumed (≤60 mm TL, ≤ 48 mm SL) was not rigid enough to deter Pike Killifish consumption. It is also quite possible that the anal spine may not completely prevent predation (i.e., Snook can be consumed), but may increase handling time and allow for escape from a predation event. In the confines of the predation trials, Pike Killifish could repeatedly attack and eventually consume a Snook as there was no place for the Snook to escape as would be the case in the wild. Future studies investigating the predation deterrence of Snook anal spines may want to not only look at consumption, but handling time and number of attacks before successful consumption as well as choice trials

between Snook prey with and without an anal spine (i.e., are Snook without an anal spine consumed over those with a spine when both are present).

Other possibilities that could explain the lower Pike Killifish attack and consumption rates of Snook compared to Mosquitofish and conspecifics relate to prey behavior and a preference for a particular prey type. Pike Killifish are generally surface dwelling fishes that have been noted to feed more heavily on other surface dwelling fishes than benthic or demersal fishes (Belshe 1961; Miley 1978; Greenwood 2012), and this was also observed in the diet analysis of Pike Killifish collected in this study

(see Chapter 3). Early-juvenile Snook often form loose pelagic schools in the water column and steadily become more and more demersal as they grow, by the time they are approximately 40 mm SL they have become fully demersal (Peters et al. 1998a).

This behavior was also observed in the predation trials, with Snook, especially larger individuals, often staying near the bottom of the tanks while the Mosquitofish and Pike

Killifish tended to spend more time on the surface. An unpublished preliminary study investigating prey preferences by Pike Killifish showed that Pike Killifish had a strong preference for Mosquitofish and only consumed Snook after all other potential prey had been consumed and not feeding for a prolonged period after the other prey had been consumed (E. Matheson, Florida Fish and Wildlife Conservation Commission and M.

Greenwood, ICF International, personal communication). This lends some support to the notion that Snook may be a less preferred prey item for Pike Killifish. Larger juvenile

Snook, both with and without their anal spine clipped, were also attacked by Pike

Killifish without being consumed more often than similar sized Mosquitofish (Table 2-4), which may indicate that even if the anal spine of Snook does not deter consumption,

some other feature may reduce consumption. The dorsal and pelvic spines are nearly vertically aligned in Snook and occur at the deepest part of the body, which could potentially provide more protection than the anal spine even though the anal spine is typically larger and more rigid. It is also possible that Snook are in general stronger than either Mosquitofish or Pike Killifish and hence more difficult for Pike Killifish predators to hold onto and manipulate in their mouths during consumption.

Although Pike Killifish consumed a number of Snook in the predation trials, no

Snook remains were identified in the stomach contents of Pike Killifish collected in the field during the course of this study. Of the 268 prey items, including 100 fish prey,

<90% digested that were found in Pike Killifish stomachs collected from the field, only 8 were fish prey that could not be identified to at least an order. Of these unidentified fish prey items, most appeared to be some type of cyprinodontiform, but there were no otoliths remaining or the individuals were too small (post-larvae) to confirm this. There was also one fish prey item that was classified as an unidentified perciform based on its otolith shape, but the otoliths did not resemble those of a juvenile Snook and most resembled the otoliths of some type of juvenile cichlid (several of which were found in

Pike Killifish stomachs). Previous data on Pike Killifish diet from the Tampa Bay area also did not reveal Snook remains, however a large number of the prey items were classified as unidentified fish with no attempt to identify them to species via otolith morphology (Greenwood et al. 2008; M. Greenwood, ICF International, personal communication).

The majority of the Pike Killifish used for diet analysis were collected between

7:30 and 18:30, so it is possible that if a particular prey item was commonly consumed

between 18:30 and approximately 7:30 it would not have been detected in this study.

There is some disparity in the literature as to whether Pike Killifish feed nocturnally.

Miley (1978) noted that prey left in aquaria overnight with Pike Killifish were not consumed until the room’s lights came on the following morning. However, Anderson

(1980) noted that Pike Killifish were observed pursuing prey both during the day and night but appeared to be more active at night. There was no further indication of how these observations were made (i.e., if there was artificial or moonlight present at night) or if diet analysis confirmed this feeding periodicity. During the predation trials in this experiment, some prey items were consumed almost immediately upon being placed in aquaria despite the room lights being out. However, there was some dim lighting from

UV filters on the recirculating systems and the red headlamp light of the investigator placing prey in the tanks, and these Pike Killifish had also been fasted for approximately

12 hours. Twelve Pike Killifish were collected for diet analysis after sunset, and of those only 2 contained identifiable prey that appeared to have likely been consumed at or before sunset based on their level of digestion. On several occasions, Pike Killifish were collected nocturnally via headlamps and dip nets for the lab studies as they were generally less active and, in many cases, required minimal effort to capture compared to daytime collections. During these collections, it did not appear that the Pike Killifish were actively searching for prey. In general, juveniles would be located within vegetation remaining relatively still while larger individuals would be located just above the bottom and also remaining relatively still. No “eye-shine” was observed in Pike

Killifish collected at night, which indicates that Pike Killifish likely do not have a tapetum lucidum, which is generally a feature of visual predators that feed nocturnally. Based on

these observations, it would appear that in most cases, Pike Killifish do not feed nocturnally unless some type of lighting is present (e.g., moonlight around full moons).

It cannot be ruled out that juvenile Snook may occasionally be consumed by Pike

Killifish, especially if other preferred prey are not available. However, juvenile Snook do not appear to make up any significant portion of the Pike Killifish’s diet in the Tampa

Bay area. Pike Killifish are noted to be cannibalistic with adults preying on juveniles and juveniles consuming other smaller conspecifics (Belshe 1961; Miley 1978; Anderson

1980; Greenwood et al. 2008). This study demonstrated that large adult Pike Killifish can not only consume juvenile conspecifics but also smaller adults, some of which were

2 nearly /3 the size of the predator Pike Killifish. It was also shown that Pike Killifish consumed significantly more conspecifics than Snook of similar sizes (Table 2-2). This was also reflected in the stomach content analysis of Pike Killifish, in which no Snook were found but 5 Pike Killifish (3.13% of their diet by occurrence, 1.87% of their diet by numerical abundance, and 12.63% of their diet by weight) were consumed. Both juveniles and smaller adult Pike Killifish were cannibalized. This could potentially lead to a somewhat self-regulating situation in which Pike Killifish would keep their numbers in check through cannibalism, after other preferred prey items had been depleted, prior to reaching abundances in which they would begin consuming Snook on a regular basis. It has also been noted that larger juvenile Snook (>140 mm SL) and adult Snook may consume adult Pike Killifish (personal observation; M. Greenwood, ICF International, personal communication). Smaller juvenile Snook (as small as 60 mm SL) have been observed preying on juvenile Pike Killifish on occasion (personal observation). Early- juvenile Snook and Pike Killifish share similar habitats and some common prey species

within Tampa Bay (McMichael et al. 1989; Peters et al. 1998a; Greenwood et al. 2008;

Greenwood 2012). Because of this, it cannot be ruled out that although there does not appear to be a direct impact of Pike Killifish on Snook through predation there may still be indirect impacts on early-juvenile Snook growth and survival resulting from competition for space and food resources.

Table 2-1. Comparison of logistic regression curves for Pike Killifish attacks on and consumption of different prey types based on size. Significance (α = 0.05) was determined by analysis of deviance and is indicated by bold: SNK = Common Snook, SNK-NS = Common Snook with anal spine removed, PK = Pike Killifish. Attacks Interaction Factor Prey Type Factor Comparison (Slope) (Intercept) SNK and SNK-NS 0.086 0.91 SNK and PK 0.36 <0.001 SNK-NS and PK 0.69 <0.001

Consumption Interaction Factor Prey Type Factor Comparison (Slope) (Intercept) SNK and SNK-NS 0.234 0.601 SNK and PK 0.096 <0.001 SNK-NS and PK 0.0575 <0.001

Table 2-2. Comparison of Pike Killifish 48 h attacks on different prey species in 20 mm TL size bins. Significance (α = 0.05) was determined using Fisher exact tests: SNK = Common Snook, SNK-NS = Common Snook with anal spine removed, MF = Eastern Mosquitofish, PK = Pike Killifish. Sample sizes are given in parenthesis. Italics indicate a significantly lower attack rate and bold indicates a significantly higher attack rate when reading across a row. SNK- SNK- SNK- SNK SNK NS NS NS MF MF PK PK PK PK 41-60 61-80 21-40 41-60 61-80 21-40 41-60 21-40 41-60 61-80 >80 (31) (15) (20) (33) (15) (32) (36) (24) (15) (23) (6) SNK 21-40 (24) 0.736 0.004 0.162 0.375 0.001 0.011 0.029 0.043 0.104 0.936 0.819 SNK 41-60 (31) . 0.023 0.035 0.797 0.005 <0.001 0.003 0.004 0.020 0.948 1.000 SNK 61-80 (15) . . <0.001 0.052 1.000 <0.001 <0.001 <0.001 <0.001 0.016 0.179 SNK-NS 21-40 . . (20) . 0.011 <0.001 0.540 1.000 0.612 1.000 0.083 0.181 SNK-NS 41-60 . . . (33) . 0.013 <0.001 <0.001 <0.001 0.006 0.566 1.000 SNK-NS 61-80 . . . . (15) . <0.001 <0.001 <0.001 <0.001 0.004 0.055 MF 21-40 (32) ...... 1.000 1.000 1.000 0.004 0.043 MF 41-60 (36) ...... 1.000 1.000 0.011 0.083 PK 21-40 (24) ...... 1.000 0.011 0.060 PK 41-60 (15) ...... 0.053 0.112 PK 61-80 (23) ...... 1.000

Table 2-3. Comparison of Pike Killifish 48 h consumption of different prey species in 20 mm TL size bins. Significance (α = 0.05) was determined using Fisher exact tests: SNK = Common Snook, SNK-NS = Common Snook with anal spine removed, MF = Eastern Mosquitofish, PK = Pike Killifish. Sample sizes are given in parenthesis. Italics indicate a significantly lower attack rate and bold indicates a significantly higher attack rate when reading across a row. SNK- SNK- SNK- SNK SNK NS NS NS MF MF PK PK PK PK 41-60 61-80 21-40 41-60 61-80 21-40 41-60 21-40 41-60 61-80 >80 (31) (15) (20) (33) (15) (32) (36) (24) (15) (23) (6) SNK 21-40 (24) 0.141 <0.001 0.577 0.144 <0.001 0.001 0.004 0.007 0.024 0.031 0.085 SNK 41-60 (31) . 0.007 0.017 1.000 0.007 <0.001 <0.001 <0.001 <0.001 0.485 0.459 SNK 61-80 (15) . . <0.001 0.004 1.000 <0.001 <0.001 <0.001 <0.001 0.044 0.363 SNK-NS 21-40 (20) . . . 0.017 <0.001 0.029 0.071 0.052 0.157 0.004 0.017 SNK-NS 41-60 (33) . . . . 0.004 <0.001 <0.001 <0.001 <0.001 0.485 0.460 SNK-NS 61-80 (15) . . . . . <0.001 <0.001 <0.001 <0.001 0.044 0.363 MF 21-40 (32) ...... 1.000 1.000 1.000 <0.001 <0.001 MF 41-60 (36) ...... 1.000 1.000 <0.001 <0.001 PK 21-40 (24) ...... 1.000 <0.001 <0.001 PK 41-60 (15) ...... <0.001 <0.001 PK 61-80 (23) ...... 0.737

Table 2-4. Comparison of Pike Killifish 48 h ratio consumption to attack rates of different prey species in 20 mm TL size bins. Significance (α = 0.05) was determined using Fisher exact tests: SNK = Common Snook, SNK-NS = Common Snook with anal spine removed, MF = Eastern Mosquitofish, PK = Pike Killifish. Sample sizes are given in parenthesis. Italics indicate a significantly lower attack rate and bold indicates a significantly higher attack rate when reading across a row. SNK SNK-NS SNK-NS MF MF PK PK PK 41-60 21-40 41-60 21-40 41-60 21-40 41-60 61-80 (20) (19) (19) (32) (35) (24) (15) (16) SNK 21-40 (18) 0.224 1.000 0.561 0.224 0.210 0.291 0.652 0.033 SNK 41-60 (20) . 0.410 0.908 0.004 0.003 0.010 0.038 0.450 SNK-NS 21-40 . (19) . 0.891 0.112 0.101 0.166 0.373 0.083 SNK-NS 41-60 . . (19) . 0.020 0.017 0.038 0.118 0.188 MF 21-40 (32) . . . . . 1.000 1.000 <0.001 MF 41-60 (35) . . . . . 1.000 1.000 <0.001 PK 21-40 (24) ...... 1.000 <0.001 PK 41-60 (15) ...... 0.005 PK 61-80 (16) ......

Figure 2-1. Satellite image of Tampa Bay, FL, with collection locations for lab experiments and ad-hoc sampling of Pike Killifish. Yellow indicates an area where fish were collected for lab experiments, green indicates an area where ad-hoc sampling for diet analysis occurred, and red indicates an area where both of the previous occurred. Black circles denote the collection of early- juvenile Common Snook, black squares denote the collection of Pike Killifish, and black stars indicate the collection of both species.

Figure 2-2. Aquarium set-up used for predation trials of Pike Killifish on several prey types. Photo taken by author.

Figure 2-3. Satellite image of Tampa Bay, FL, with insets of standardized sampling locations for Pike Killifish and early-juvenile Common Snook co-occurring with Pike Killifish: Alafia River and Wildcat Creek, a tributary of the Little Manatee River.

Figure 2-4. Satellite image of Wildcat Creek, a tributary of the Little Manatee River in Tampa Bay, FL, one of the standardized sampling locations for Pike Killifish and early-juvenile Common Snook with Pike Killifish co-occurring: The red lines represent the boundaries between the 12 fixed sampling sites (~100 m shoreline length). The yellow line is a 100 m scale bar.

Figure 2-5. Satellite images of the Alafia River in Tampa Bay, FL, one of the standardized sampling locations for Pike Killifish and early-juvenile Common Snook with Pike Killifish co-occurring: The red boxes in A represent the locations pictured in B and C. The red lines in B and C represent the boundaries between the 12 fixed sampling sites (~100 m shoreline length). The yellow lines are 100 m scale bars.

Snook, N = 70 Snook w/o Anal Spine, N = 69 Eastern Mosquitofish, N = 68 Pike Killifish, N = 68 50% Attack

Figure 2-6. Pike Killifish attacks on different prey types based on prey total length (TL). Dots denote the attack data (jitter added to show overlapping data points), solid lines represent negative logistic regressions fit to the attack data, and dashed lines form the 95% confidence intervals for their respective logistic regression. The red dots denotes the size at 50% attack for each prey species that could have a negative logistic model fit to it: Snook with anal spine (53.9 mm TL), Snook without anal spine (52.8 mm TL), and Pike Killifish (84.7 mm TL).

Snook, N = 70 Snook w/o Anal Spine, N = 69 Eastern Mosquitofish, N = 68 Pike Killifish, N =68 50% Consumption

Figure 2-7. Pike Killifish consumption of different prey types based on prey total length (TL). Dots denote the consumption data (jitter added to show overlapping data points), solid lines represent negative logistic regressions fit to the consumption data, and dashed lines form the 95% confidence intervals for their respective logistic regression. The red dots denotes the size at 50% consumption for each prey species that could have a negative logistic model fit to it: Snook with anal spine (42.4 mm TL), Snook without anal spine (45.4 mm TL), and Pike Killifish (69.7 mm TL).

CHAPTER 3 DIET ANALYSIS AND POTENTIAL FOOD RESOURCE COMPETITION BETWEEN EARLY-JUVENILE COMMON SNOOK AND PIKE KILLIFISH

Introduction

Juveniles of most fish species experience high levels of natural mortality, which may reach 10-20 times that which is experienced by adults (Lorenzen 1996). This mortality is most often related to episodic losses (aberrant weather, pollution, etc.), predation, and food availability (Houde 1987, 1989).

Natural mortality resulting from starvation is typically of greater importance in larval stages than post-settlement stages and, in many cases, food is not likely to be limiting in nursery areas, particularly estuaries (Currin et al. 1984; Kneib 1993).

Reduced intake of food resulting from competition or reduced foraging due to predation risk, however, can lead to slower growth (Werner et al. 1983; Werner and Gilliam 1984;

Werner and Hall 1988; Sogard 1992, 1994, 1997; Walters and Juanes 1993; Dahlgren and Eggleston 2000; Halpin 2000). Such reduced growth can lead to increased mortality via predation, as more rapid growth of newly settled juveniles may allow individuals to grow out of the size classes that are most vulnerable (i.e., reaching a size refuge from predation) (Post and Evans 1989; Sogard 1992, 1997; Sogard and Able 1992; Levin et al. 1997). Additional stressors, such as competition with non-native species, that could potentially lead to reduced food intake and growth may ultimately lead to reduced populations of a species of particular importance or concern.

Snook are important both economically and ecologically in the estuarine waters of Florida. The Gulf coast stock of Snook has experienced long periods of being classified as overfished (Taylor and Muller 2012, 2013; Muller et al. 2015). It is believed that habitat loss, especially juvenile habitat, has likely played a more important role in

the reduced population size of Gulf coast Snook than actual overfishing. This may be particularly true for early-juvenile Snook (≤100 mm SL), as this is the life stage with the most restricted habitat usage. Additional stressors to the early-juvenile stage of Snook could lead to further population reductions and delay recovery rates.

Initially, early-juvenile Snook feed primarily on planktonic organisms, such as copepods, and small benthic invertebrates, such as mysids (Harrington and Harrington

1961; Fore and Schmidt 1973; Gilmore et al. 1983; McMichael et al. 1989; Aliaume et al. 1997; Peters et al. 1998 a). However, small fish are still regularly consumed by post- larval Snook, especially when they are locally abundant (Harrington and Harrington

1961; Peters et al. 1998 a). As Snook grow, there is a gradual shift towards larger prey including fish, such as cyprinodontiforms, and shrimp, mainly grass shrimp

Palaemonetes with few planktonic organisms being consumed once they are greater than 40 mm SL (Harrington and Harrington 1961; Fore and Schmidt 1973; Gilmore et al.

1983; McMichael et al. 1989; Aliaume et al. 1997; Peters et al. 1998 a).

Pike Killifish, which are not native to Florida, have been established in freshwaters of south Florida since 1957 (Belshe 1961). They have also been found in estuarine waters in south Florida (Kerfoot 2011) and more recently an estuarine population has been documented in tidal tributaries of Tampa Bay (Greenwood 2017).

Pike Killifish are a large live-bearer that is piscivorous from birth (Belshe 1961; Miley

1978; Turner and Snelson 1984; Greven and Brenner 2008). They have been linked to decreases in abundance and altered size structure in native small-bodied fishes in both fresh and estuarine waters in Florida (Miley 1978; Greenwood 2012). The Tampa Bay population of Pike Killifish was first documented in 1994; however, recent work has

revealed that the distribution and abundance of Pike Killifish in Tampa Bay waters has either increased in recent years or is greater than previously thought due to inadequate sampling in their preferred habitats within the Tampa Bay area (MacDonald et al. 2010;

Greenwood 2017). Pike Killifish are relatively common along the southwestern shore of

Tampa Bay and were most often found in quiet, shallow estuarine backwaters and tributaries (Greenwood et al. 2008; MacDonald et al. 2010; Greenwood 2017). This overlaps both with the general area of highest juvenile Snook recruitment in Tampa Bay and also the preferred habitat of early-juvenile Snook (Figure 1-3) (McMichael et al.

1989; Peters et al. 1998 a; Stevens et al. 2007; MacDonald et al. 2010). The piscivorous diet of Pike Killifish, previous implications in the decrease of small-bodied fish abundance, and the overlap in distribution and habitat use of Pike Killifish and early- juvenile Snook has raised concerns about the potential impact of Pike Killifish on Snook through competition for food and predation (MacDonald et al. 2010; Greenwood 2012,

2017).

The goals of this study were to 1) examine the diet of Pike Killifish and early- juvenile Snook in Tampa Bay tidal tributaries; 2) assess the overlap in diet between these two species; 3) determine if early-juvenile Snook diet differs in locations with and without Pike Killifish co-occurring; and 4) determine if the fish and shrimp prey species abundance and composition differs between locations with and without Pike Killifish co- occurring with early-juvenile Snook.

Methods

Fish Collection for Diet Analysis

Pike Killifish and early-juvenile Common Snook utilized for diet analysis were obtained from standardized sampling events as detailed in Chapter 2. Pike Killifish and co-occurring early-juvenile Snook were collected from two locations in Tampa Bay.

Early-juvenile Snook were also collected from two locations without Pike Killifish co- occurring during each year of the study. One location from each treatment (Pike Killifish co-occurring and Pike Killifish not co-occurring) consisted of a smaller tributary with minimal shoreline urbanization, which provided suitable early-juvenile Snook and Pike

Killifish habitat along its entire length. The other location from each treatment consisted of backwater areas of a larger tributary where there was moderate shoreline urbanization and suitable habitat was fragmented by the mainstem of the river. The fixed sites within these sampling locations had similar salinity, temperature, dissolved oxygen, and water clarity regimes. They also had similar shoreline habitats, particularly among the sites within each of the smaller creeks and among the sites within the backwaters of each of the larger rivers. Shoreline vegetation was typically dominated by

Black Needle Rush Juncus roemerianus or a mixture of mangrove species. The mangroves present were primarily Black Mangrove Avicennia germinans and White

Mangrove Laguncularia racemosa, but Red Mangrove Rhizophora mangle were also abundant in a few locations. Other shoreline habitats that were occasionally dominant consisted of Brazilian Pepper Schinus terebinthifolius and mixed emergent marsh vegetation (Leather Fern Acrostichum danaeifolium, Cattails Typha spp., and other mixed marsh grasses). Hardened shorelines, seawalls and rock, were present in very

few locations and when present they were accompanied by one or more of the plant species previous mentioned.

The locations where Pike Killifish and co-occurring early-juvenile Snook were collected consisted of Wildcat Creek, a tributary of the Little Manatee River, and backwaters of the Alafia River (Figures 2-3, 2-4, and 2-5) (see Chapter 2 for full site description). In 2012, Snook without Pike Killifish were collected from an un-named tributary of the Manatee River and the Braden River, the largest tributary to the Manatee

River (Figure 3-1). This un-named tributary of the Manatee River produced relatively low catches of early-juvenile Snook, possibly related to being located too far upstream, relatively strong currents in several locations, greater abundance of larger juvenile

Snook, poor recruitment to this particular area in that particular year, or some other unknown reason. Due to these low catches, this site was replaced with Frog Creek (also known as Terra Ceia River), a moderate sized tributary of Tampa Bay that drains into a secondary embayment, Terra Ceia Bay, in 2013 (Figure 3-2). This creek is known to consistently have a high abundance of early-juvenile Snook (Greenwood et al. 2008;

MacDonald et al. 2010; Brame 2012; Brame et al. 2014). Similar to Wildcat Creek, the un-named Manatee River tributary and Frog Creek contain suitable early-juvenile Snook habitat along their entire length. Sampling in the un-named tributary began approximately 100 m from the mouth (which is located approximately 20 km upstream from the mouth of the Manatee River) and consisted of continuous stretches of shoreline upstream until access was restricted by the narrow width of the creek (Figure

3-3). Sampling of Frog Creek began approximately 2.5 km upstream from its mouth

(lower portions of the creek contain suitable early-juvenile Snook habitat, but it differed

from other locations in the study with a greater abundance of oyster bars and Red

Mangroves along the shoreline) and extended upstream along continuous stretches of shoreline until the dominant shoreline habitat shifted to oaks Quercus spp. (Figure 3-4).

Backwater areas of the Braden River, like the Alafia River, are more fragmented and separated by stretches of the mainstem river and boating channels. Fixed-sampling sites within the Braden River began approximately 1.5 km from its mouth (which is about 13 km from the mouth of the Manatee River) and extended upstream to about 2 km before the dam forming Ward Lake (Figure 3-5).

Pike Killifish and Common Snook captured in the standardized sampling were placed in aerated buckets upon capture. The first 20 Pike Killifish and Snook were given a unique identification number and placed in individual bags, which were placed in an ice bath for euthanasia (UF IFAS Non-regulatory Animal Research Protocol 006-

12FAS). One exception to this occurred with a particularly large catch of Snook in which approximately one third of the catch was randomly selected to be retained. Additionally, any early-juvenile Snook that died during the collection process were also retained for diet analysis. Fish that were not retained, were given a unique identification number and measured to the nearest mm (SL) in the field with a measuring board prior to release at their capture location. As described in Chapter 2, additional targeted sampling of Pike

Killifish was conducted at several locations both within and outside of the standardized sampling locations to increase the number of stomachs analyzed for Pike Killifish.

Collection of Prey for Abundance Estimates

Potential prey items (fish, decapods, aquatic insects, etc.) up to approximately 45 mm TL (a few larger individuals were also retained for creating prey regression curves)

were retained from each standard bag seine haul in which early-juvenile Snook or Pike

Killifish were captured. To capture smaller prey items that may have escaped the bag seine sampling, a fine mesh minnow seine (see Chapter 2 for description) was hauled for 6.1 m parallel to the shoreline adjacent to any standard bag seine haul in which Pike

Killifish or early-juvenile Snook were captured where shoreline and bottom conditions permitted. Retained prey items from each seine haul were placed in a bag in an ice bath and upon returning to the lab were frozen for later identification, enumeration, and measurement.

Diet Analysis

Snook and Pike Killifish that were retained for diet analysis had their SL measured with digital calipers (to the nearest 0.01 mm) and were weighed (to the nearest 0.0001 g). Stomachs were then removed, and the contents were extracted, a coarse identification and enumeration of prey items was recorded, and the contents were placed either in 1.5 mL microcentrifuge tubes or 20 mL scintillation vials with water and frozen for detailed analysis at a later date.

Detailed analysis of the stomach content samples involved thawing the samples and placing them in a transparent weigh boat for viewing under a stereomicroscope (8-

50x magnification) with an attached imaging system (QImaging MicroPublisher 5.0

RTV). Prey were enumerated and identified to the lowest taxonomic level. Otoliths were removed from partially digested fish to aid in their identification. Each prey item was placed in a 10% digestion bin (i.e., ≤10%, 11-20%, …, >90%). Prey that were >90% digested were not included in further diet analysis (this typically consisted of fish scales and bones or small bits of exoskeleton from crustaceans and insects). Prey items that

were ≤10% digested and large enough were weighed on a balance to the nearest

0.0001 g. Maximum length and depth measurements (nearest 0.01 mm) of prey were taken when possible to examine relative prey size to predator size and gape.

Several measurements (Table 3-1: total length (TL), standard length (SL), maximum depth (MD), eye diameter (ED), caudal peduncle height (CPH), otolith length

(OL), otolith depth (OD), gape width (GW), post-orbital length (POL), and weight (Wt)) were also taken when possible from partially digested fish and shrimp to reconstruct their lengths and weights from regression equations developed from the prey item samples. Measurements of smaller organisms (mainly planktonic organisms and larval dipterans) that could not be accurately weighed on the balance were also taken to reconstruct weights based on their calculated biovolume (Table 3-2) (Edler 1979;

Hillebrand et al. 1999; Sun and Liu 2003).

When weights of individual prey items could not be reconstructed, the average weight of all prey of the same taxa within a particular year (or over the whole study when numbers were low) was assigned to these individuals. The average weight of all identified fish was applied to unidentified fish, this was also done for shrimp, mysids, and terrestrial insects. In some cases, published weight estimates or estimates based on closely related taxa were utilized to estimate the weights of rare, partially digested prey items, such as adult damselflies.

Prey total lengths (either from whole prey or prey regressions) were plotted against the SL of each species to compare maximum prey size to predator size.

Additionally, the gape width (nearest 0.01 mm with digital calipers), was measured as the distance between the maxillary bones when the mouth is closed and served as a

proxy for the pharyngeal gape (Luczkovich et al. 1995, Hill et al. 2004), was plotted against the maximum depth of prey (either from whole prey or prey regressions). These plots were used to determine if Pike Killifish and Snook consume prey near or exceeding their gape limitations.

Traditional diet measures including, frequency of occurrence, numerical abundance of prey, and prey weight (Hynes 1950, Hyslop 1980) were calculated for

Pike Killifish and Common Snook. For Snook, these measures were also calculated for individuals from locations both with and without Pike Killifish co-occurring. These measures were calculated on a percentage basis for easier and more accurate comparisons between the species: percent numerical abundance [%N = (total number of a specific prey item / total number of prey items) * 100], percent frequency of occurrence [%O = (number of stomachs containing a specific prey item / total number of non-empty stomachs) * 100], and percent weight [%W = (total weight of a specific prey item/total weight of all prey items) * 100]. An index of relative importance [IRI= (%N +

%W) * %O] (Pinkas et al. 1971) was also calculated as a percentage [%IRI = (IRI for a specific prey item / IRI for all prey items) * 100] as %IRI provides more robust results and allows for easier comparisons among species than IRI (Cortés 1997). IRI is a compound index that incorporates all three traditional measures of diet composition in an attempt to assess the importance of a particular prey type, whether they be small regularly consumed prey or larger, less frequently consumed prey. An IRI measure, was calculated for comparison with other diet studies, as a number of more recent studies often only report some form of this metric. These measures (%O, %N, %W, and %IRI)

were calculated for each individual prey type and also with prey grouped into several functional prey groups based on habitat use, morphology, and taxonomy (Table 3-3).

To ensure that the diet of each species was well characterized, cumulative prey curves were constructed for each species. For Snook, cumulative prey curves were created both for Snook collected from locations with and without Pike Killifish co- occurring. An adequate number of stomach samples is considered to have been achieved when the curve reaches an asymptote (Ferry and Cailliet 1996), which in this case was considered to be a <5% increase in new prey species over the last 10 stomachs (Baremore et al. 2010). The order in which stomachs were sampled was randomized 10 times and the number of new prey items in each stomach per randomization was calculated (Baremore et al. 2010). The mean of these randomizations was used to create the cumulative prey curves.

To compare the potential overlap in diet between Pike Killifish and early-juvenile

Snook, and in diet between Snook from sites with and without Pike Killifish co-occurring,

Morisita’s index of similarity (C), which ranges from 0 (no similarity) to 1 (compete similarity), was calculated (Morisita 1959). Morisita’s index of similarity is expressed as:

푛 2 ∑ (푝푖푗 × 푝푖푘) 퐶 = 푖 푛 푛푖푗 − 1 푛 푛푖푘 − 1 ∑푖 [푝푖푗 × ( )] + ∑푖 [푝푖푘 × ( )] 푁푗 − 1 푁푘 − 1

Where pij is the proportion resource i is of the total resources used by species j, pik is the proportion resource i is of the total resources used by species k, nij is the number of individuals of species j that use resource i, nik is the number of individuals of species k that use resource i, Nj is the total number of species j, and Nk is the total number of species k. This index was selected as it gives little bias regardless of sample size,

number of resources, or number of prey categories (Cailliet and Barry 1979; Smith and

Zaret 1982; Krebs 2014). Morisita’s index of similarity can only be calculated based on numerical counts of prey items, which were calculated in this study. When diet cannot be calculated based on numerical counts, it is suggested that Horn’s index be used to minimize bias (Smith and Zaret 1982; Krebs 2014). A value of >0.6 is generally considered to indicate a high degree of dietary overlap when using Morisita’s index

(Zaret and Rand 1971). Morisita’s index was calculated for all individual prey taxa and also based on the functional prey groups outlined in Table 3-3. Functional groups were assessed in addition to individual taxa, as there is a possibility that a specific taxa may not be present from a particular sampling location but a closely related and similar taxa be present instead.

Levin’s index is one of the most commonly used metrics to assess dietary breadth in fishes (Saikia 2012) and was calculated for comparison with other diet studies. The diet breadth of Pike Killifish, Snook, and each Snook treatment (with and without Pike Killifish co-occurring) was assessed using Levin’s standardized index (BA)

2 (Hurlbert 1978): BA = (B -1) / (n – 1), where B is equal to 1 / Σpij and pij is the proportion of the diet of predator i that contains prey j, and n is the number of prey categories. This index is expressed on a scale from 0 (minimum diet breadth) to 1

(maximum niche breadth) allowing for comparison among species (Krebs 2014). As with diet overlap, diet breadth was calculated using the numerical abundance of each prey group both for all individual prey taxa and prey functional groups. Levin’s index does not take into account prey abundance and may give the false impression of a specialized diet if a few highly abundant prey groups are commonly consumed

compared to other less abundant prey types. To incorporate prey abundance into the estimation of diet breadth for Snook and Pike Killifish, both a visual comparison and numerical index were calculated.

The relative abundance of each functional fish prey group and the shrimp functional prey group (≤45 mm TL) were plotted with the %N of these same groups

(calculated only for those groups plotted) for Pike Killifish and Snook with and without

Pike Killifish co-occurring as a means of visually assessing diet breadth and prey selectivity. Only fish and shrimp prey groups were analyzed in these plots as the other prey taxa were not well represented in the prey sampling and these are the prey groups that would most likely be adversely impacted by Pike Killifish based on their diet.

The Ivlev electivity index (Ei) (Ivlev 1961), was also calculated for these prey groups for both Pike Killifish and Snook as a numerical measure of prey selectivity. This index is calculated as: Ei = (ri – Pi) / (ri + Pi), where ri is the relative abundance (or proportion) of a prey item in a predator’s diet and Pi is the prey’s relative abundance (or proportion) within an ecosystem. An Ei value of -1 is indicative of complete avoidance of a particular prey item, a value of 1 is indicative of complete selection for a particular prey item, and a value of zero is indicative of non-selective feeding on a particular prey item. For this study, Ei values greater than 0.5 and less than -0.5 were used to indicate potential selection or avoidance of a particular prey type. Prey characteristics such as transience (i.e., may have been present recently but left area prior to sampling) and location in relation to potential predators should be considered in the assessment of selection and avoidance.

Prey Abundance

Prey samples collected from both the bag seine and minnow seine were examined to determine the identification, number, and size of several prey groups available to Pike Killifish and early-juvenile Snook. For each sample, potential prey items were identified. At least the first 50 randomly selected individuals of a species from a bag seine sample were measured for total length (TL) and maximum depth/height (MD) with digital calipers (0.01 mm). For the minnow seine prey samples, at least the first 25 randomly selected individuals of each species were measured for TL and MD. The remaining individuals of each species from these samples were enumerated. The ratio of measured individuals in each 15 mm TL size bin and 5 mm

MD size bin were applied to this number to estimate the size distribution of the unmeasured individuals of each species in each sample. Prey species were grouped into the functional groups outlined in Table 3-3 and placed in the size bins previously described. The abundance of each size class of each functional group was calculated based on the area sampled in each seine haul. Prey abundance between locations with and without Pike Killifish was only compared for prey groups that were adequately sampled by the seine nets and that were represented in the diet of early-juvenile Snook and/or Pike Killifish (e.g., mysids and amphipods were not regularly collected in the seine nets and Hogchoker Trinectes maculatus were adequately sampled by the seine nets but never found in the stomach contents of Snook or Pike Killifish). These were also the prey groups that were most likely to be impacted by Pike Killifish (i.e., a concerted effort to sample planktonic and small benthic invertebrate prey was not made because they make up a small portion of Pike Killifish diet and would likely not be impacted by their presence). Prey abundances were compared by analyzing the

community structure of these prey groups with generalized linear models using the mvabund package in R (Statistical Methods for Analyzing Multivariate Abundance

Data). Post-hoc univariate tests were conducted for each prey group (species and size class) with p-values being adjusted for multiple tests using a step-down resampling procedure (as in Westfall and Young 1993, Algorithm 2.8).

Additional measurements (Table 3-1: total length (TL), standard length (SL), maximum depth (MD), eye diameter (ED), caudal peduncle height (CPH), otolith length

(OL), otolith depth (OD), gape width (GW), post-orbital length (POL), and weight (Wt)) were collected from 10 (occasionally more for rarely encountered prey species or from all individuals if fewer than 10 were collected) individuals of each species across a range of the sizes represented in a particular bag seine sample to develop prey regression curves to reconstruct the weights of partially digested prey items. Weights and additional measurements were also taken on 15 individuals (occasionally more for rare prey items or from all individuals if fewer than 10 were collected) of each prey species collected in a particular minnow seine sample for inclusion in the prey regression curves.

Results

Prey Regression Curves

Prey regression curves used to reconstruct the size and approximate weight of partially digested prey items were calculated for all commonly encountered potential fish and decapod prey items collected during the course of this study. In most cases, reconstructed lengths and weights could be determined from several different partial

prey measurements, with most having a coefficient of determination (R2) greater than

0.9 (Table 3-1).

Early-juvenile Common Snook Diet Analysis

A total of 1132 Snook (502 from locations without Pike Killifish co-occurring and

630 from locations with Pike Killifish co-occurring) ranging from 5 to 119 mm SL were retained for diet analysis. Of these fish, 962 (85%; 86% for Snook without Pike Killifish co-occurring and 84% for Snook with Pike Killifish co-occurring) contained prey that were less than 90% digested. Overall, the diet of early-juvenile Snook was well characterized in this study based on the cumulative prey curve, with only a 0.14% increase in unique prey items over the last 10 stomachs analyzed (Figure 3-6). This held true both for Snook with and without Pike Killifish co-occurring, with a 0.52% and

0.33% increase in unique prey items over the last 10 stomachs analyzed, respectively.

Early-juvenile Snook consumed a large range of prey sizes with some being planktonic and others being nearly the same size as the Snook consuming the prey (Figure 3-7A).

There were even a few instances of prey having a maximum depth that exceeded the measured gape width of an individual Snook (about 6% of the prey items that had a MD measurement was acquired directly or from regressions) (Figure 3-8A).

A total of 74 unique prey types were found in the diet of early-juvenile Snook

(Table 3-4). In general, early-juvenile Snook diet is dominated by planktonic crustaceans (copepods and several cladoceran species), small benthic crustaceans

(mainly mysids), aquatic insects (mainly chironomid larvae), and clupeiform fish (Bay

Anchovy Anchoa mitchilli) (Tables 3-4 and 3-5 and Figure 3-9A). Although non-goby perciform fish (mainly mojarra), cyprinodontiform fish, shrimp (mainly grass shrimp), and

gobies (mainly Clown Goby Microgobius gulosus) also contributed largely to the diet in terms of weight and IRI (Tables 3-4 and 3-5 and Figure 3-9A).

An ontogenetic shift in diet occurred around 35 mm SL for early-juvenile Snook.

Fish smaller than this fed heavily on planktonic crustaceans (Figures 3-9B and 3-10).

Snook larger than 35 mm SL consumed few planktonic organisms and more fish and shrimp (Figure 3-9C). Small benthic crustaceans were a dominant prey source for both of these size classes (Table 3-5 and Figure 3-9). This shift in diet is apparent when comparing the dietary overlap of these two size classes with Morisita’s index, both for all prey types (0.33) and the functional prey groups (0.45). These values show a moderate degree of overlap in the diet, but also indicate that there are some large differences in the diet as previously summarized.

One main difference that was detected in terms of diet between Snook from locations with and without Pike Killifish co-occurring is that cyprinodontiform fish and aquatic insects are consumed less in locations with Pike Killifish co-occurring while clupeiform fish appear to be consumed more in these locations (Tables 3-6, 3-7, and 3-

8). This was most apparent in larger Snook but also occurred to a lesser degree in small

Snook (Tables 3-7 and 3-8).

Levin’s standardized index calculated for all prey types for all early-juvenile

Snook that contained prey items <90% digested was 0.05 and differed little between locations with and without Pike Killifish co-occurring, 0.07 and 0.03 respectively (Table

3-9). When only larger Snook (>35 mm SL) were analyzed, this index increased to 0.11

(Table 3-9). The value of Levin’s standardized index calculated for the prey functional groups increased slightly for each Snook group (Table 3-9). These relatively low values

for Levin’s index point towards a narrow diet breadth in early-juvenile snook, regardless of Pike Killifish presence. Larger Snook generally had larger Levin’s index values than smaller Snook (Table 3-9).

When plotted against the average relative abundance of prey items captured in the bag seine and minnow seine samples, the %N of early-juvenile Snook diet

(calculated based only on those groups plotted) generally follows the same trends as the prey abundance (Figure 3-11A and B). The two main differences are that considerably more non-perciform fish were captured in the bag seines in comparison to how many were consumed by Snook and a large number of clupeiform fish were consumed by Snook in locations with Pike Killifish co-occurring despite low catches of clupeiforms in the seine nets (Figure 3-11A and B).

Ivlev electivity index values for Snook from locations without Pike Killifish present generally point to non-selective feeding on most of the prey groups sampled with the bag seine (Table 3-10). The exception is atheriniform and non-goby perciform fish, which appear to be avoided by Snook. Snook from locations with Pike Killifish present appeared to select clupeiform fish and avoid atheriniform and cyprinodontiform fish based on bag seine collections of prey items (Table 3-10). Ivlev electivity values from the minnow seine prey collections also point towards relatively non-selective feeding by

Snook form locations without Pike Killifish present (Table 3-10). There is some indication of selective feeding on gobies by Snook from locations without Pike Killifish.

Electivity values based on minnow seine collections from locations with Pike Killifish present point toward selection of clupeiform and gobiid fish and avoidance of atheriniform and cyprinodontiform fish (Table 3-10).

Pike Killifish Diet Analysis

A total of 422 Pike Killifish ranging from 14 to 127 mm SL were retained for diet analysis, of which 160 (38%) contained prey that were <90% digested. Based on the cumulative prey curve, Pike Killifish diet was well characterized in this study, with a

1.69% increase in unique prey items over the last 10 stomachs analyzed (Figure 3-6B).

As with early-juvenile Snook, Pike Killifish consume a large range of prey sizes with some being planktonic and others being nearly the same size as the Pike Killifish consuming the prey (Figure 3-7B). Approximately 20% of the prey items that had a maximum depth measurement taken or estimated from the prey regression curves had maximum depths that exceeded the gape width of the Pike Killifish that had consumed the prey (Figure 3-8B).

A total of 36 unique prey types were found in the diet of Pike Killifish (Table 3-4).

In general, Pike Killifish diet was numerically dominated by shrimp (grass shrimp), cyprinodontiform fish (mainly Sailfin Molly Poecilia latipinna and Gulf Killifish Fundulus grandis), and planktonic crustaceans (the cladoceran, Scapholeberis sp.) (Tables 3-4 and 3-11 and Figure 3-12A). The cladoceran, Scapholeberis sp. only contributed significantly to the %N, while shrimp and cyprinodontiform fish made up the majority of

%O, %W, and %IRI (Table 3-11 and Figure 3-12A). An ontogenetic shift in diet occurred around 30 mm SL for Pike Killifish (Figure 3-13), with fish smaller than this feeding heavily on the surface dwelling cladoceran Scapholeberis sp. as well as consuming some other small surface-dwelling insect larvae in addition to shrimp and cyprinodontiform fish. Larger Pike Killifsh consumed almost exclusively grass shrimp and fish, mainly various cyprindontiform fish and to a lesser extent several perciform

fish (Table 3-11). Morisita’s index shows a very low degree of dietary overlap between these two size classes of Pike Killifish, 0.03 for all prey types and 0.09 for the functional prey groups.

Levin’s standardized index based on all prey types for Pike Killifish that contained prey items <90% digested was 0.09 (Table 3-9). The Levin’s index was even smaller for small (≤30 mm SL) and large (>30 mm SL) Pike Killifish, although the sample size for small Pike Killifish was very low (Table 3-9). These values increased by about 4 times when Levin’s index was calculated based on the functional prey groups (Table 3-9).

These relatively low Levin’s index values point towards a low diet breadth.

Unlike early-juvenile Snook, the %N of Pike Killifish diet did not generally track well with the relative prey abundance captured in the bag seine and minnow seine samples, except for shrimp (Figure 3-11C). Cyprinodontiform fish were consumed in much higher numbers in comparison to the number collected in the seines (Figure 3-

11C). Ivlev electivity index values based on prey collected from bag seines indicate that

Pike Killifish generally selected shrimp and cyprinodontiform fish while avoiding clupeiforms, atheriniforms, and gobies. Prey data from the minnow seine resulted in electivity values that showed Pike Killifish selection of cyprinodotiforms, avoidance of clupeiforms, and non-selective feeding on the other prey types sampled.

Dietary Overlap

Dietary overlap between early-juvenile Snook and Pike Killifish was relatively low when Morisita’s index was calculated for all prey groups (0.13) but increased substantially (0.40) when calculated for the functional prey groups (Table 3-12). This pattern held true for the small size class of each species (i.e., Snook ≤35 mm SL and

Pike Killifish ≤30 mm SL), but the overlap of larger individuals of each species remained low regardless of which prey designation was used in calculating Morisita’s index (Table

3-2).

Morisita’s index calculated for Snook with and without Pike Killifish co-occurring was quite high when calculated for all prey types (0.80) and increased to nearly full overlap when calculated for the functional prey groups (0.99) This was also the case for both the smaller (≤35 mm SL) and larger (>35 mm SL) size classes of early-juvenile

Snook (Table 3-12).

Prey Abundance

The prey community structure measured through prey group abundances calculated based on TL size groups from bag seine samples showed a significant difference (p = 0.02) between sites with and without Pike Killifish co-occurring (Table 3-

10). Univariate tests showed that the abundance of cyprinodontiforms (≤30 mm TL) and perciforms (>30 mm TL and ≤45 mm TL), both of which were higher on average in locations without Pike Killifish present, was significantly affected by Pike Killifish presence (Table 3-13). There was a significant difference (p = 0.05) in the prey community structure based on TL size groups from the minnow seine samples, but none of the univariate tests were significant (Table 3-13). Prey species abundance calculated based on MD size groups from bag seine samples also showed a significant difference (p = 0.01) between locations with and without Pike Killifish co-occurring.

Similar to the results from abundances based on TL size groups, the differences in abundance based on MD size groups were driven by differences in moderate sized cyprinodontiforms and perciforms (>5 mm MD and ≤10 mm MD) (Table 3-14). No

significant differences were detected in the prey community structure based on abundance of MD size groups from the minnow seine samples between locations with and without Pike Killifish co-occurring (Table 3-14).

Discussion

Early-juvenile Snook collected in this study had diets similar to those described in previous studies, with smaller individuals preying heavily on planktonic organisms and small benthic invertebrates with a shift away from planktonic organisms toward larger fish and shrimp prey with increasing size (Fore and Schmidt 1973; Gilmore 1983;

McMichael et al. 1989; Peters et al. 1998). The greatest contribution to the planktonic organisms and benthic invertebrates in these previous studies were copepods and mysids respectively. This held true for this study as well; however, a greater contribution of cladocerans to the planktonic organisms and amphipods and chironomid larvae to the benthic invertebrates was noted. Differences in the spatial and temporal scope of these studies may in part explain these differences in diet. The previously cited studies generally covered a greater spatial scale, as well as collections being made throughout the year, while this study focused on a few smaller areas during peak Snook recruitment in late summer through early fall. This temporal scale alone, likely accounts for some of these differences in the diet. Most of the identified cladocerans and chironomid larvae tend to be more associated with freshwaters. Higher rainfall, which typically occurs in the summer and fall in the Tampa Bay area, results in lower salinities in tidal tributaries.

A greater diversity of fish (between 1.5 and 9 times the number of species) was also noted in the fish species that contributed to a large portion of early-juvenile Snook diet in this study compared to previous studies (Fore and Schmidt 1973; Gilmore 1983;

McMichael et al. 1989; Peters et al. 1998 a). This most likely occurred because of the better taxonomic resolution acquired by using otoliths to identify partially digested fish prey. Unidentified fish made up approximately 10% or less of the diet depending on the metric used in this study, compared to other studies where unidentified fish generally made up 40-50% of the diet (Fore and Schmidt 1973; Gilmore 1983; McMichael et al.

1989).

Levin’s standardized index for early-juvenile Snook was generally low, regardless of the presence of Pike Killifish or the size of the early-juvenile Snook (≤35 mm or >35 mm SL). Although larger Snook did have higher values likely resulting from the heavy feeding on planktonic organisms by Snook ≤35 mm SL. These low Levin’s index values would suggest early-juvenile Snook have a relatively specialized diet, however, several lines of evidence actually point to early-juvenile Snook having a relatively diverse diet. A total of 74 unique prey items were identified in this study, and the most commonly consumed prey items (e.g., mysids, planktonic crustaceans, chironomid larvae, and bay anchovies) are dissimilar prey types (i.e., early-juvenile Snook are not feeding on a single prey type or in a single microhabitat). The disjunct between Levin’s index and the diversity in common prey items likely occurs due to Snook feeding on highly abundant but diverse prey types and Levin’s index does not incorporate prey abundance into it calculation, it is based solely on the diet composition of the species being studied.

When comparing the relative abundance of prey types to the %N of these same prey types in early-juvenile Snook diet, the %N generally corresponded well with the abundance of prey regardless of whether or not Pike Killifish co-occur. If pelagic crustaceans and small benthic invertebrates had been targeted for prey sampling,

similar trends likely would have occurred. The biggest discrepancies occurred with clupeiforms and non-goby perciforms. The difference between %N and clupeiform (Bay

Anchovy) abundance was most likely a result of the schooling and transient nature of this species. Bay Anchovy form large schools that move in and out of areas with the tides and currents, thus it is quite possible that Snook could have fed heavily on Bay

Anchovy prior to a seine haul and that the school of anchovies could have moved out of the sampling area by the time the seine was hauled. Most of the other prey groups are less transient overall. The discrepancies with non-goby perciforms may have occurred because a number of the perciforms are deeper bodied and may have exceeded the gape limitations of the early-juvenile Snook. Ivlev electivity index values show that among the prey items sampled with the bag seine and minnow seine, Snook are generally non-selective in feeding on most prey types (i.e., if a prey item is abundant they will consume it). There was some evidence of selection of clupeiform fish, but as described above, this may be a result of the transient nature of these schooling fishes

(i.e., Snook consumed them but many had left the area prior to a seine haul). There was also some evidence of avoidance of cyprinodontiform fish by Snook from locations with

Pike Killifish. Cyprinodontiform fish abundance was generally low in these areas, as was their consumption by Snook, and those that were present were generally larger and may have exceeded the gape limitations of many of the Snook collected. This apparent avoidance of atheriniform fish may be attributed to them typically being located in open surface waters further away from the early-juvenile Snook are foraging. While the apparent avoidance of perciforms in some locations may be tied to gape limitations (i.e.,

many of the non-goby perciforms were too large to be consumed by many of the early juvenile snook that were sampled).

An interesting side note in the diet analysis of early-juvenile Common Snook was the identification of four conspecifics that had been consumed (i.e., intra-cohort cannibalism). Cannibalism in Common Snook is considered by some to be a factor that can strongly influence young of the year (YOY) abundance (Brennan 2008; Brennan et al. 2008) , despite few published instances of Common Snook cannibalism (Adams and

Wolfe 2006; Brennan 2008). Of these published instances of Snook cannibalism, six

(3.44 %O and 0 %O) involved inter-cohort cannibalism with older/larger Snook feeding on YOY, and only one (0 %O and 0.24 %O) involved intra-cohort cannibalism with YOY feeding on smaller YOY recruits. The present study would suggest that intra-cohort cannibalism (0.42 %O) may occur slightly more frequently than would be thought based on previous studies, but as mentioned previously, the use of otoliths to identify partially digested prey greatly increased the diet resolution. It is quite possible that more cannibalism was present in previous diet studies but was not detected. Nevertheless, it still appears that intra-cohort cannibalism in early-juvenile Snook is relatively rare in the wild.

Pike Killifish are often referred to as exclusive piscivores from birth, which in general, only feed on other prey resources when starved in lab conditions or when fish prey resources in the wild are depleted (Belshe 1961; Miley 1978; Turner and Snelson

1984; Greven and Brenner 2008). More recent evidence has shown that naïve Pike

Killifish may not switch prey in the lab, however, individuals that are starved and “learn” to consume an alternate prey resource (in this case grass shrimp as opposed to

mosquitofish) will readily consume that alternate prey resource when available (Harms

2011; Harms and Turingan 2012). These authors also showed that a preference for fish prey over shrimp prey, at least under certain lab conditions, appeared to diminish after

Pike Killifish began to consume shrimp prey (Harms 2011; Harms and Turingan 2012).

Pike Killifish were also found to be highly piscivorous in the current study. However, invertebrate prey were also an important component of the diet in both small juveniles

(≤30 mm SL), which consumed a large number cladocerans, and larger individuals that commonly consumed grass shrimp. There was very little overlap in diets of small juvenile and larger Pike Killifish, however, the sample size for the small juveniles containing stomach contents was quite low. This combined with the large number of cladocerans consumed by several of these individuals may have biased the results, as personal observations of small specimens in captivity and previous studies have shown that small juvenile Pike Killifish regularly consume the same types of prey as larger individuals (Belshe 1961; Miley 1978; Turner and Snelson 1984; Greven and Brenner

2008). Other prey that were less commonly consumed were juvenile Bluegill, tilapias, terrestrial insects, and mosquito larvae and pupae. One common thread among almost every prey type consumed by Pike Killifish was an association with the water’s surface.

Cyprinodontiform fish are noted for spending much of their time near the surface of the water, particularly poeciliids and fundulids. Both juvenile Bluegill and tilapia will form loose schools near the surface. The cladocerans consumed by small Pike Killifish were all Scapholeberis, a surface-dwelling species. Mosquito larvae and pupae rest at the water’s surface unless disturbed and terrestrial insects consumed by Pike Killifish are likely those that landed or fell on the water’s surface. The one discrepancy in this trend

is grass shrimp, which are generally thought of as being a benthic species. However, in the tidal tributaries sampled during this study, grass shrimp could be very abundant in localized areas and many individuals were observed and captured near the water’s surface on shoreline vegetation (Juncus stems, mangrove roots and branches, floating grasses, etc.) and flotsam (mainly Juncus debris). A large percentage of Pike Killifish in this study had empty stomachs, although this does not appear to be a function of sampling outside of their peak feeding period. It has been noted that piscivores, especially as adults, may regularly have empty stomachs (Arrington et al. 2002). This may be particularly true after having recently consumed and digested a relatively large fish meal. In a review of empty stomachs in diet studies, Pike Killifish had similar levels of empty stomach (~60%) to this study both in their native and introduced ranges

(Arrington et al. 2002). In many cases the prey that were present within the Pike Killifish stomachs in this study had been recently consumed (i.e., stomachs were often either empty or had freshly consumed prey).

As with Snook, Pike Killifish appeared to have a narrow diet breadth as indicated by their low Levin’s index values. Unlike early-juvenile Snook, these low index values do actually appear to represent diet specialization as the number of unique prey items consumed by Pike Killifish was less than half of that consumed by early-juvenile Snook

(36 versus 74), despite all of these prey groups being available to the Pike Killifish. The

%N of fish and shrimp prey in Pike Killifish diet as compared to the abundance of the fish and shrimp prey groups also showed what appears to be selection of certain prey items over others despite their high abundance. Cyprinodontiforms were clearly selected by Pike Killifish despite being less abundant than other fish prey such as

clupeiforms and perciforms. It is unclear whether grass shrimp are being selected as they are often consumed but they are also quite abundant compared to other prey types.

Both early-juvenile Common Snook and Pike Killifish consumed prey across a wide range of sizes, with prey exceeding the maximum size that would be estimated based on gape width. These larger prey were consumed somewhat regularly, particularly in the case of Pike Killifish. This was unexpected because both of these species would be considered to be gape-limited in their feeding (i.e., they can only consume whole prey that will fit in their mouth/through and do not bite prey into smaller pieces for consumption). The maximum depth of a particular prey item may not be the best measurement for estimating the size of prey that can be consumed based on a predator’s gape, as the maximum depth, particularly for gravid individuals, can be compacted in many cases. Applying slight pressure when measuring the maximum depth (i.e., maximum rigid depth) would likely give a more accurate estimate as to whether a particular prey could be consumed or not based on a predator’s gape. It was also noted several times, both in the lab and the field, particularly for Pike Killifish, that a predator could “digest down” particularly large prey items. Predators would partially swallow prey with the remaining portion of the prey in their oral cavity or even sticking out of their mouth, while part of the prey was digested prior to swallowing it the rest of the way. The greater occurrence (over double) of prey that exceeded the estimated maximum prey size and “digesting down” in Pike Killifish compared to early-juvenile

Snook may be attributed to their unique jaw morphology and kinematics (rotating premaxilla, lower jaw rotation, etc.) that has been noted in several previous studies

(Greven and Brenner 2008; Ferry-Graham et al. 2010; Harms 2011; Harms and

Turingan 2012).

Three requirements for competition to occur have been outlined in previous competition research. These requirements include: 1) shared resource use (i.e., the species in question need to share some resource); 2) the shared resource must be limited/depleted (i.e., the resource(s) being shared by the species in question need to be depleted over time); and 3) the depletion of this shared resource needs to lead to reduced growth, condition, or survival of one or both of the species sharing the resource

(Wiens 1989; Prins 2000). Overall, there is little evidence of food resource competition between Pike Killifish and early-juvenile Common Snook. First, there are few shared prey resources. When all prey groups are considered, the dietary overlap between these two species is quite low regardless of the size group being compared. The dietary overlap of small individuals increases to nearly full overlap when prey functional groups are used to calculate Morisita’s index of similarity, however, as discussed before the sample size for small juvenile Pike Killifish was quite low. This overlap was driven by the high numbers of planktonic organisms consumed by both species when they are smaller. It is unlikely, however, that either of these species would limit the abundance of planktonic organisms in a tidal tributary system as there are other planktivores that are much more abundant, such as Bay Anchovy. In addition, tidal cycles and rainfall likely move new planktonic organisms into the tidal tributaries from the mainstem rivers and/or bays and from upstream freshwater sources (e.g., ponds and ditches). Morisita’s index increased slightly for all individuals combined and larger individuals when based on prey functional groups rather than all unique prey groups, but it was still indicative of low

dietary overlap. Along with a generally low dietary overlap between Pike Killifish and early-juvenile Snook was a high degree of dietary overlap between Snook from locations with and without Pike Killifish co-occurring regardless of the size group of

Snook analyzed. Morisita’s index values ranged from 0.73 to 0.80, which were considerably higher than the value of 0.6, which is often considered to be indicative of high dietary overlap (Zaret and Rand 1971). These values all increased to over 0.9, near complete overlap, when prey functional groups were used instead of each unique prey species. Some differences in diet between Snook with and without Pike Killifish co- occurring were noted. The higher number of cyprinodontiformes consumed by Snook in locations without Pike Killifish present compared to locations with Pike Killifish does appear to be attributed to the presence of Pike Killifish based on both the diet data and prey availability data. However, the greater number of aquatic insects, mainly larval chironomids, consumed by Snook in locations without Pike Killifish is likely not a result of Pike Killifish presence, as Pike Killifish rarely fed on aquatic insects. It is more likely that some other factor likely contributed to this difference (insect spraying, extent of flood plain, abundance of predators on adult insects, etc.).

The main indication of potential food resource competition between Pike Killifish and early-juvenile Common Snook comes from the analysis of fish and shrimp prey community structure between locations with and without Pike Killifish co-occurring with

Snook, which shows some indication of a shared prey resource being depleted. Prey community structure from the bag seine samples was shown to be significantly different from locations where Snook were collected with and without Pike Killifish present, with the abundance of larger perciforms and small to moderate sized cyprinodontiforms

being significantly altered by the presence of Pike Killifish. The difference in the larger perciforms is likely not actually attributed to Pike Killifish, as they rarely consumed this prey group. This difference could occur because juvenile Snook, one of the most abundant perciforms, was not included in the prey abundance estimations as their abundance was being analyzed separately in a concurrent study (see Chapter 5) and they were never found in Pike Killifish stomachs and rarely consumed by conspecifics. It is also possible that early-juvenile Snook were competing with some of these other perciforms. However, one of the most plausible explanations is that it may be an artefact of how the prey samples were retained in the field, as not every potential prey was measured in the field. Some larger individuals were measured and others were either retained or returned to the sampling area based on their general size in relation to those measured individuals (i.e., the cut-off for whether a larger potential prey item was retained was determined by eyesight approximation). This likely resulted in some perciforms >30 mm TL and ≤45 mm TL being discarded. On the other hand, the difference in cyprindontiform abundance does actually appear to be related to Pike

Killifish abundance, as Pike Killifish fed heavily upon cyprinodontiforms and appeared to select them over other more abundant fish prey groups. Several previous studies have noted a decrease in cyprinodontiform fish, most notably mosquitofish, in several freshwater areas of south Florida where Pike Killifish are present (Belshe 1961;

Courtenay and Robins 1973; Miley 1978; Loftus and Kushlan 1987; Trexler et al. 2000).

Greenwood (2012) also found similar results in several tidal tributaries of Tampa Bay. In the current study, mosquitofish were nearly non-existent in Wildcat Creek, one of the sampling locations with Snook and Pike Killifish co-occurring (4 individuals collected

over 2 years), and very few mosquitofish were collected in the backwaters of the Alafia

River, the other sampling location with both Snook and Pike Killifish present (24 individuals over 2 years). While observations along the mainstem of the Alafia and Little

Manatee River, where few if any Pike Killifish are caught/observed, revealed a high abundance of mosquitofish. Such observations were made along similar shoreline habitats, with similar depths and salinities, and were often within a few kilometers of the standardized sampling sites and some of them occurring just outside the mouths of tributaries with Pike Killifish present. On the other hand, Mosquitofish were regularly observed along the entire stretch of Frog Creek and the un-named tributary of the

Manatee River, and were observed both in backwaters and along the mainstem of the

Braden River, all of which do not contain Pike Killifish.

The lower abundance of cyprinodontiforms from locations with Pike Killifish was also reflected in the diet of early-juvenile Snook, which consumed 11-18 times more cyprinodontiformes (depending on if %N, %O, or %W is compared; 183 times more based on %IRI) in locations without Pike Killifish co-occurring (Table 3-3). This is likely one source, albeit a relatively small one, in the separation of the diet overlap of early- juvenile Snook from locations with and without Pike Killifish co-occurring. It is possible that this small shift in diet could influence the condition or growth of early-juvenile Snook in locations with Pike Killifish co-occurring, as cyprinodontiforms represent a large, potentially energy dense (e.g., gravid females) meal. However, it is also possible that early-juvenile Snook could switch to consuming other abundant fish prey such as gobies, non-goby perciforms (such as Bluegill and mojarra), and Bay Anchovy which also represent large meals that are potentially energy dense. This is particularly true for

Bay Anchovy, as Clupeiformes including Bay Anchovy, are noted for being a particularly energy dense prey item due to their high lipid content (Wang and Houde 1994; Hartman and Brandt 1995; Lawson et al. 1998; Spitz et al. 2010). Bay Anchovy mature quickly

(<90 days, 30-45 mm fork length), thus even at the small size consumed by early- juvenile Snook, Bay Anchovy are likely quite an energy rich meal. Data collected for a concurrent study has been analyzed to address whether decreased condition or growth was apparent in early-juvenile Snook when Pike Killifish co-occurred with them (i.e., whether the third requirement of competition is being met). Based solely on the diet analysis and prey availability, it would appear that Pike Killifish may be competing with

Snook for some food resources. However, this competition is minimal and has little to no effect on early-juvenile Snook diet composition overall.

Table 3-1. Regression equations for various fish and invertebrate prey species used to back-calculate the weight of partially digested prey items based on a variety of measurements. Measurements included total length (TL), standard length (SL), maximum depth (MD), eye diameter (ED), caudal peduncle height (CPH), otolith length (OL), otolith depth (OD), gape width (GW), post-orbital length (POL), and weight (Wt). Prey Species and Coefficient Intercept R2 N MinTL MaxTL Model (SL) (SL) Bay Anchovy Anchoa mitchilli MD~TL 0.2092 -1.4963 0.79 2321 16 58 TL~ED 10.3021 9.2250 0.88 199 16 47 TL~CPH 8.8845 7.5473 0.91 201 16 47 TL~SL 1.2203 -0.7510 0.99 201 16 47 TL~OL 23.5242 10.6244 0.96 197 17 47 TL~OD 33.8760 8.4352 0.93 197 17 47 TL~MD 3.7759 11.9664 0.79 2321 16 58 logWt~logTL 3.6139 -14.3319 0.96 201 16 47 Bluegill Lepomis macrochirus MD~TL 0.3312 -2.1218 0.96 150 12 46 TL~ED 9.0585 3.9623 0.91 88 11 46 TL~CPH 8.4258 4.5041 0.96 88 11 46 TL~SL 1.2620 -0.3584 0.99 88 11 46 TL~OL 19.3697 3.1313 0.97 88 11 46 TL~OD 29.7841 1.5059 0.96 88 11 46 TL~MD 2.8820 7.4550 0.96 150 12 46 logWt~logTL 3.3956 -12.7163 0.97 88 11 46 Blue Crab Callinectes sapidus MD~TL 0.4808 0.1679 0.97 63 8 26 TL~MD 2.0101 0.1409 0.97 63 8 26 logWt~logTL 3.0733 -9.9415 0.98 62 8 26 Blue Tilapia Oreochromis aureus MD~TL 0.3221 -0.8322 0.88 10 27 43 TL~ED 9.0740 3.9710 0.89 10 27 43 TL~CPH 7.9550 3.8810 0.83 10 27 43 TL~SL 1.2947 -0.7677 0.97 10 27 43 TL~OL 15.1320 12.2010 0.94 10 27 43 TL~OD 20.3780 12.1980 0.89 10 27 43 TL~MD 2.7419 6.6513 0.88 10 27 43 logWt~logTL 2.7947 -10.3350 0.96 10 27 43

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Table 3-1. Continued. Prey Species and Coefficient Intercept R2 N MinTL MaxTL Model (SL) (SL) Brook Silverside Labidesthes sicculus TL~ED 15.4200 -2.4160 0.94 16 17 53 TL~CPH 14.7880 2.3070 0.93 16 17 53 TL~SL 1.2092 -0.8336 1 16 17 53 TL~OL 40.2720 1.4270 0.96 16 17 53 TL~OD 64.6680 -2.7830 0.97 16 17 53 TL~MD 7.2434 6.4831 0.94 16 17 53 logWt~logTL 3.3860 -13.6755 0.99 16 17 53 Clown Goby Microgobius gulosus MD~TL 0.1350 0.4979 0.6 528 11 49 TL~ED 9.1888 11.0735 0.49 210 11 49 TL~CPH 10.7904 7.7783 0.71 210 11 49 TL~SL 1.2759 -0.4000 0.97 210 11 49 TL~OL 28.5763 0.6576 0.78 210 11 49 TL~OD 30.2140 -1.8477 0.86 210 11 49 TL~MD 4.4030 10.0969 0.6 528 11 49 logWt~logTL 3.0307 -12.0001 0.94 210 11 49 Common Snook Centropomus undecimalis MD~SL 0.2424 0.3251 0.96 54 8 47 SL~ED 10.3836 0.5123 0.95 28 8 47 SL~CPH 9.5360 -0.6255 0.96 28 8 47 SL~OL 16.4632 -0.6594 0.98 70 8 47 SL~OD 30.3913 -4.7306 0.97 70 8 47 SL~MD 3.9620 -0.2569 0.96 54 8 47 logWt~logSL 2.9183 -10.8617 0.99 1129 8 119 TL~SL 1.2319 0.8680 0.98 143 8 75 SL~GW 11.2500 0.7214 0.97 1129 8 119 Diamond Killifish Fundulus xenicus MD~TL 0.4069 -3.9434 0.99 13 18 45 TL~ED 12.0417 0.0508 0.91 13 18 45 TL~CPH 4.5937 9.6879 0.98 13 18 45 TL~SL 1.1748 0.6127 0.99 13 18 45 TL~OL 40.6880 -3.5570 0.96 13 18 45 TL~OD 38.4020 -5.6370 0.94 13 18 45 TL~MD 2.4370 9.8187 0.99 13 18 45 logWt~logTL 3.4716 -12.7355 0.98 13 18 45 Eastern Mosquitofish Gambusia holbrooki MD~TL 0.2118 -0.7743 0.82 681 8 47 TL~ED 15.6785 -4.0526 0.91 123 8 45 TL~CPH 8.4944 2.0485 0.96 124 8 45

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Table 3-1. Continued. Prey Species and Coefficient Intercept R2 N MinTL MaxTL Model (SL) (SL) Eastern Mosquitofish Gambusia holbrooki TL~SL 1.1575 0.8579 0.99 172 8 45 TL~OL 38.4996 -1.4461 0.97 124 8 45 TL~OD 31.0469 -3.7866 0.98 124 8 45 TL~MD 3.8648 7.8758 0.82 681 8 47 logWt~logTL 3.1680 -12.0781 0.99 124 8 45 Gobiosoma spp. MD~TL 0.1713 -0.2716 0.79 153 10 37 TL~ED 11.9820 6.2850 0.57 103 10 35 TL~CPH 7.4131 7.0545 0.83 103 10 35 TL~SL 1.2015 0.1297 0.96 103 10 35 TL~OL 33.7070 -4.4940 0.85 103 10 35 TL~OD 43.8930 -11.6280 0.79 103 10 35 TL~MD 4.5893 6.7498 0.79 153 10 37 logWt~logTL 3.1989 -12.1404 0.93 102 10 35 Grass Shrimp Palaemonetes spp. MD~TL 0.1631 -0.2866 0.85 1262 4 38 TL~POL 3.6126 4.2566 0.83 347 7 34 TL~MD 5.1827 4.6764 0.85 1262 4 38 logWt~logTL 3.1253 -12.2462 0.91 348 4 34 Gulf/Marsh Killifish Fundulus grandis/F. confluentus MD~TL 0.2383 -2.1434 0.98 83 17 71 TL~ED 12.8594 0.2246 0.93 80 17 71 TL~CPH 6.6288 10.0340 0.97 80 17 71 TL~SL 1.1290 1.6263 1 80 17 71 TL~OL 35.0550 2.6400 0.94 80 17 71 TL~OD 37.5700 1.3410 0.94 80 17 71 TL~MD 4.0936 9.7605 0.98 83 17 71 logWt~logTL 3.3999 -12.8648 0.99 80 17 71 Hogchoker Trinectes maculatus MD~TL 0.4357 -0.7721 0.93 893 9 38 TL~CPH 8.2109 3.5342 0.85 216 9 33 TL~SL 1.2431 0.1965 0.97 220 9 33 TL~MD 2.1458 2.9847 0.93 893 9 38 logWt~logTL 3.3079 -12.1935 0.94 220 9 33 Inland/Tidewater Silverside Menidia beryllina/M. peninsulae MD~TL 0.1283 0.0973 0.79 1016 6 75 TL~ED 12.2307 3.3561 0.84 197 10 64 TL~CPH 12.8017 4.4543 0.9 198 10 64 TL~SL 1.0613 5.3632 0.9 199 10 64

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Table 3-1. Continued. Prey Species and Coefficient Intercept R2 N MinTL MaxTL Model (SL) (SL) Inland/Tidewater Silverside Menidia beryllina/M. peninsulae TL~OL 34.9305 1.4524 0.9 198 10 64 TL~OD 52.3035 -0.5587 0.91 198 10 64 TL~MD 6.1796 8.5921 0.79 1016 6 75 logWt~logTL 3.3774 -13.4754 0.94 199 10 64 Least Killifish Heterandria formosa MD~TL 0.2439 -0.5276 0.97 17 7 28 TL~ED 10.2570 2.2810 0.74 16 7 28 TL~CPH 8.6059 0.3608 0.94 17 7 28 TL~SL 1.1708 0.3380 0.99 17 7 28 TL~OL 33.6160 2.5750 0.86 17 7 28 TL~OD 33.8862 -0.0090 0.79 17 7 28 TL~MD 3.9741 2.6051 0.97 17 7 28 logWt~logTL 3.6045 -12.8276 0.87 17 7 28 Mojarra Eucinostomus spp. MD~TL 0.2504 -0.4990 0.91 2449 9 62 TL~ED 10.1515 0.0529 0.94 279 12 54 TL~CPH 11.0601 2.4646 0.96 279 12 54 TL~SL 1.3086 -1.1755 0.99 293 12 54 TL~OL 25.2294 -3.7630 0.95 283 12 54 TL~OD 33.7826 -1.5856 0.94 283 12 54 TL~MD 3.6292 4.9429 0.91 2449 9 62 logWt~logTL 3.0955 -11.7738 0.98 282 12 54 Mud Crab Superfamily Xanthoidea MD~TL 0.7587 0.3241 0.97 160 3 15 TL~MD 1.2808 -0.1975 0.97 160 3 15 logWt~logTL 2.8936 -7.7329 0.93 117 3 15 Pike Killifish Belonesox belizanus MD~SL 0.1889 -0.9963 0.88 85 14 77 SL~ED 13.8100 -2.9480 0.9 19 15 35 SL~CPH 7.2187 6.4328 0.96 103 14 77 SL~OL 46.4138 -2.8865 0.99 103 14 77 SL~OD 49.6034 -9.1978 0.99 103 14 77 SL~GW 11.4863 8.9827 0.92 433 14 127 MD~TL 0.1656 -1.2138 0.89 83 15 89 TL~ED 15.3590 -1.8290 0.91 19 19 40 TL~CPH 8.4355 7.4601 0.97 83 15 89 TL~OL 53.5382 -2.6002 0.99 83 15 89 TL~OD 57.0987 -9.7509 0.99 83 15 89 TL~GW 15.8087 2.5416 0.97 83 15 89

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Table 3-1. Continued. Prey Species and Coefficient Intercept R2 N MinTL MaxTL Model (SL) (SL) Pike Killifish Belonesox belizanus TL~SL 1.1499 0.7375 1 83 15 89 SL~MD 4.6576 10.4101 0.88 85 14 77 TL~MD 5.3818 12.5906 0.89 83 15 89 logWt~logTL 3.2317 -12.9327 0.99 83 15 89 logWt~logSL 3.1632 -12.1775 0.99 440 14 127 Pink Shrimp Farfantepenaeus duorarum MD~TL 0.1372 -0.0968 0.93 203 10 64 TL~POL 3.7428 1.5099 0.97 101 11 64 TL~MD 6.8051 2.4172 0.93 203 10 64 logWt~logTL 3.2326 -12.8590 0.98 101 11 64 Rainwater Killifish Lucania parva MD~TL 0.2290 -0.6553 0.91 261 13 42 TL~ED 10.7388 3.8832 0.8 110 13 42 TL~CPH 7.2002 5.9809 0.93 111 13 42 TL~SL 1.1496 1.4393 0.96 111 13 42 TL~OL 36.6442 -1.8816 0.9 111 13 42 TL~OD 41.9158 -3.3350 0.92 111 13 42 TL~MD 3.9843 4.9962 0.91 261 13 42 logWt~logTL 3.2442 -12.3007 0.98 111 13 42 Red Drum Sciaenops occelatus MD~TL 0.1946 0.5045 0.93 101 16 37 TL~ED 10.8510 2.6736 0.82 81 16 37 TL~CPH 10.5885 2.5483 0.91 81 16 37 TL~SL 1.2334 0.1137 0.98 81 16 37 TL~OL 17.6251 -2.0892 0.97 81 16 37 TL~OD 27.2683 -1.8750 0.96 81 16 37 TL~MD 4.7958 -0.8112 0.93 101 16 37 logWt~logTL 2.9718 -11.4692 0.93 81 16 37 Sailfin Molly Poecilia latipinna MD~TL 0.2850 -1.2241 0.94 316 11 56 TL~ED 14.9670 -4.5611 0.88 128 11 56 TL~CPH 5.8266 5.2030 0.97 128 11 56 TL~SL 1.1915 0.1728 0.98 135 11 56 TL~OL 29.3200 2.3364 0.95 128 11 56 TL~OD 33.8450 -2.1220 0.95 128 11 56 TL~MD 3.2842 6.1191 0.94 316 11 56 logWt~logTL 3.1306 -11.6277 0.99 128 11 56

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Table 3-1. Continued. Prey Species and Coefficient Intercept R2 N MinTL MaxTL Model (SL) (SL) Sand Seatrout Cynoscion arenarius MD~TL 0.1913 0.1586 0.92 116 19 56 TL~ED 11.2040 5.1230 0.87 84 21 56 TL~CPH 12.2271 2.0457 0.91 84 21 56 TL~SL 1.2264 1.2473 0.98 84 21 56 TL~OL 17.8902 0.6010 0.95 83 21 56 TL~OD 30.8750 -2.1370 0.95 83 21 56 TL~MD 4.8191 1.8834 0.92 116 19 56 logWt~logTL 3.3353 -13.0101 0.95 84 21 56 Seminole Killifish Fundulus seminolis MD~TL 0.1624 -0.6976 0.97 151 14 71 TL~ED 15.8271 -2.9944 0.94 78 16 71 TL~CPH 9.2440 6.1565 0.98 78 16 71 TL~SL 1.1411 0.8786 1 78 16 71 TL~OL 42.2724 2.5244 0.97 78 16 71 TL~OD 45.0298 -0.0582 0.98 78 16 71 TL~MD 5.9714 5.4112 0.97 151 14 71 logWt~logTL 3.3444 -13.0540 0.99 78 16 71 Striped Mojarra Eugerres plumieri MD~TL 0.2714 -0.3706 0.93 180 16 52 TL~ED 10.4715 0.1042 0.88 54 18 47 TL~CPH 11.7097 -0.9063 0.92 54 18 47 TL~SL 1.3453 -0.7051 0.96 55 18 47 TL~OL 29.0477 -6.2982 0.94 54 18 47 TL~OD 37.6250 -5.4260 0.91 54 18 47 TL~MD 3.4230 3.7706 0.93 180 16 52 logWt~logTL 3.0327 -11.7310 0.95 54 18 47

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Table 3-2. Geometric shapes and equations used to estimate the biomass of small organisms including planktonic crustaceans, some small benthic crustacean, and some aquatic insects. Abbreviations used in equations: BMD = body/head maximum depth (maximum depth of animal when no tail present or tail measurements not made separately); BLD = body/head least depth (least depth of animal when no tail present or tail measurements not made separately); BTL = body/head total length (total animal length when tail no tail present or tail measurements not made separately); TTL = tail total length; TMD = tail maximum depth; TLD = tail least depth; BW = body width (length along third axis). Biovolume was converted to biomass by using the estimation that planktonic organisms have approximately the same density as water (i.e. 0.001 g/mm3). A conversion factor of 1.11 for small crustaceans (crab megalops, small isopods, etc.) based on the actual weights and estimated biomass (biovolume*0.001g/mm3). A conversion factor of 1.07 for small aquatic insect larvae/pupae was used based on data in Smit et al. 1993. Organism Shape Equation Copepods Truncated cone for body and 0.001*((π*1/3*BTL*(((BMD/2)^2)+((BLD/2)^2)+((BMD/2)*(BLD/2))))+(π cylinder for tail *TTL*(TMD/2)^2))) Cladocerans Ellipsoid 0.001*(4/3*π*((BTL/2)*(BMD/2)*(2/3*BMD/2))) Caridean shrimp zoea/mysis Cylinder for head and 1.11*0.001*((π*BTL*(BMD/2)^2)+(π*1/3*TTL*(((TMD/2)^2)+((TLD/2)^ truncated cone for tail 2)+((TMD/2)*(TLD/2))))) Caridean shrimp zoea/mysis Ellipsoid 1.11*0.001*(4/3*π*((TL/2)*(MD/2)*(2/3*MD/2))) Crab zoea and megalops Cylinder for head/body and 1.11*0.001*((π*BTL*(BMD/2)^2)+(π*TTL*(TD/2)^2)) tail Crab megalops Cylinder 1.11*0.001*(π*BTL*(BMD/2)^2) Ostracods Ellipsoid 1.11*0.001*(4/3*π*((BTL/2)*(BMD/2)*(BW/2))) Small unidentified isopods Elliptical cylinder 1.11*0.001*(π*BTL*(BW/2)*(BMD/2)) Aquatic Mites Ellipsoid 0.001*(4/3*π*((TL/2)*(BW/2)*(BD/2))) Culicidae (mosquito) larvae Truncated cone 1.07*0.001*(π/3*BTL*((BMD/2)^2)+((BLD/2)^2)+((BMD/2)*(BLD/2))) Culicidae (mosquito) larvae and pupae Cylinder for head and 1.07*0.001*((π*BTL*(BMD/2)^2)+(π*1/3*TTL*(((TMD/2)^2)+((TLD/2)^ truncated cone for tail 2)+((TMD/2)*(TLD/2))))) Chironomidae (midge) larvae Truncated cone 1.07*0.001*(π/3*BTL*((BMD/2)^2)+((BLD/2)^2)+((BMD/2)*(BLD/2))) Chaoboridae (phantom midge) larvae Truncated cone 1.07*0.001*(π/3*BTL*((BMD/2)^2)+((BLD/2)^2)+((BMD/2)*(BLD/2)))

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Table 3-2. Continued. Organism Shape Equation Small Chironomidae/Chaoboridae pupae Cylinder for head and 1.07*0.001*((π*BTL*(BMD/2)^2)+(π*1/3*TTL*(((TMD/2)^2)+((TLD/2)^ truncated cone for tail 2)+((TMD/2)*(TLD/2))))) Small Chironomidae/Chaoboridae pupae Truncated cone 1.07*0.001*(π/3*BTL*((BMD/2)^2)+((BLD/2)^2)+((BMD/2)*(BLD/2)))

Gyrinidae (whirligig beetle) larvae Truncated cone 1.07*0.001*(π/3*BTL*((BMD/2)^2)+((BLD/2)^2)+((BMD/2)*(BLD/2)))

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Table 3-3. Prey functional groups used in diet and prey availability analysis. Prey Functional Group Planktonic Crustaceans (copepods, cladocerans, decapod larvae) Small Benthic Crustaceans (mysids, amphipods, isopods, tanaids, etc.) Annelids (polychaetes and leeches) Aquatic Insects and Arachnids Terrestrial/Unidentified Insects and Arachnids Shrimp Crabs Clupeiformes (pelagic schooling fish without spines) Atheriniformes (pelagic surface schooling fish with small spines) Cyprinodontiformes (surface/benthopelagic littoral fish without spines) Order Perciformes excluding Gobiidae (benthopelagic fish with spines) Family Gobiidae (benthic fish with weak spines) Unidentified Fish

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Table 3-4. Diet composition of early-juvenile Common Snook and Pike Killifish collected from Tampa Bay tidal tributaries. Percent frequency of occurrence (%O), percent numerical abundance (%N), percent weight (%W), and percent index of relative importance (%IRI) are reported for both Common Snook and Pike Killifish for all prey items identified in their diet analysis. The %IRI is reported for different taxonomic levels, designated by the IRI level. Early-juvenile Common Snook (N=962) Pike Killifish (N=160) IRI Prey Item %O %N %W %IRI %O %N %W %IRI Level Phylum Annelida 1 0.94 0.25 1.08 0.01 0.00 0.00 0.00 0.00 Class Clitellata 2 0.10 0.02 0.05 <0.01 0.00 0.00 0.00 0.00 Subclass Hirudinea 3 0.10 0.02 0.05 <0.01 0.00 0.00 0.00 0.00 Hirudinea 4 0.10 0.02 0.05 <0.01 0.00 0.00 0.00 0.00 Hirudinea 5 0.10 0.02 0.05 <0.01 0.00 0.00 0.00 0.00 Class Polychaeta 2 0.83 0.23 1.03 0.02 0.00 0.00 0.00 0.00 Subclass Errantia 3 0.83 0.23 1.03 0.03 0.00 0.00 0.00 0.00 Errantia 4 0.83 0.23 1.03 0.04 0.00 0.00 0.00 0.00 Errantia 5 0.83 0.23 1.03 0.04 0.00 0.00 0.00 0.00 Subphylum Chelicerata 1 0.21 0.01 <0.01 <0.01 0.63 0.37 0.03 <0.01 Class Arachnida 2 0.21 0.01 <0.01 <0.01 0.63 0.37 0.03 <0.01 Order Araneae 3 0.00 0.00 0.00 0.00 0.63 0.37 0.03 <0.01 Araneae 4 0.00 0.00 0.00 0.00 0.63 0.37 0.03 <0.01 Araneae 5 0.00 0.00 0.00 0.00 0.63 0.37 0.03 0.01 Order Trombidiformes 3 0.21 0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Suborder Prostigmata 4 0.21 0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Prostigmata 5 0.21 0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Subphylum Hexapoda 1 25.88 10.68 3.11 3.24 17.50 15.67 0.63 3.22 Class Insecta 2 25.88 10.68 3.11 5.33 17.50 15.67 0.63 3.51 Order Coleoptera 3 0.21 0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Family Gyrinidae 4 0.21 0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Gyrinidae larvae 5 0.21 0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Order Diptera 3 23.49 10.26 1.91 8.25 7.50 6.34 0.09 0.95 Family Chaoboridae 4 4.78 1.32 0.73 0.34 0.00 0.00 0.00 0.00 Chaoboridae larvae 5 4.78 1.32 0.73 0.38 0.00 0.00 0.00 0.00 Family Chironomidae 4 17.26 7.40 0.77 4.90 1.88 1.12 0.01 0.07 Chironomidae larvae 5 17.26 7.40 0.77 5.51 0.00 0.00 0.00 0.00 Chironomidae adult 5 0.00 0.00 0.00 0.00 1.88 1.12 0.01 0.10 Family Chironomidae/ 4 8.63 1.50 0.40 0.57 1.25 1.12 0.03 0.05 Chaoboridae Chironomidae/ 5 8.63 1.50 0.40 0.64 1.25 1.12 0.03 0.07 Chaoboridae pupae Family Culicidae 4 0.62 0.04 <0.01 <0.01 4.38 4.10 0.05 0.61 Culicidae larvae 5 0.42 0.03 <0.01 <0.01 3.75 2.99 0.04 0.56 Culicidae pupae 5 0.21 0.01 <0.01 <0.01 1.25 1.12 <0.01 0.07 Order Ephemeroptera 3 0.31 0.02 0.01 <0.01 1.25 0.75 0.02 0.02 Ephemeroptera 4 0.31 0.02 0.01 <0.01 1.25 0.75 0.02 0.03 Ephemeroptera naiad 5 0.31 0.02 0.01 <0.01 1.25 0.75 0.02 0.05 Order Hemiptera 3 0.83 0.06 0.40 0.01 5.00 2.99 0.03 0.30 Family Corixidae 4 0.62 0.04 <0.01 <0.01 1.25 0.75 0.01 0.03 Corixidae 5 0.62 0.04 <0.01 <0.01 1.25 0.75 0.01 0.05

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Table 3-4. Continued. Early-juvenile Common Snook (N=962) Pike Killifish (N=160) IRI Prey Item %O %N %W %IRI %O %N %W %IRI Level Family Gerridae 4 0.00 0.00 0.00 0.00 3.13 1.87 0.02 0.20 Gerridae 5 0.00 0.00 0.00 0.00 3.13 1.87 0.02 0.29 Family Naucoridae 4 0.10 <0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Naucoridae 5 0.10 <0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Family Notonectidae 4 0.10 <0.01 <0.01 <0.01 0.63 0.37 0.01 <0.01 Notonectidae 5 0.10 <0.01 <0.01 <0.01 0.63 0.37 0.01 0.01 Order Hymenoptera 3 0.00 0.00 0.00 0.00 0.63 0.37 <0.01 <0.01 Family Formicidae 4 0.00 0.00 0.00 0.00 0.63 0.37 <0.01 <0.01 Formicidae 5 0.00 0.00 0.00 0.00 0.63 0.37 <0.01 0.01 Order Lepidoptera 3 0.00 0.00 0.00 0.00 1.88 1.12 0.25 0.05 Lepidoptera 4 0.00 0.00 0.00 0.00 1.88 1.12 0.25 0.09 Lepidoptera adult (moth) 5 0.00 0.00 0.00 0.00 1.88 1.12 0.25 0.13 Order Odonata 3 0.73 0.06 0.43 0.01 1.88 1.12 0.10 0.05 Suborder Anisoptera 4 0.42 0.04 0.32 <0.01 0.00 0.00 0.00 0.00 Anisoptera naiad 5 0.42 0.04 0.32 <0.01 0.00 0.00 0.00 0.00 Suborder Zygoptera 4 0.31 0.02 0.12 <0.01 1.88 1.12 0.10 0.08 Zygoptera naiad 5 0.31 0.02 0.12 <0.01 0.00 0.00 0.00 0.00 Zygoptera adult 5 0.00 0.00 0.00 0.00 1.88 1.12 0.10 0.11 Order Trichoptera 3 1.04 0.14 0.17 <0.01 0.00 0.00 0.00 0.00 Family Hydropsychidae 4 0.52 0.08 0.17 <0.01 0.00 0.00 0.00 0.00 Hydropsychidae larvae 5 0.52 0.08 0.17 <0.01 0.00 0.00 0.00 0.00 Unidentified Trichoptera 4 0.62 0.06 <0.01 <0.01 0.00 0.00 0.00 0.00 Trichoptera larvae 5 0.62 0.06 <0.01 <0.01 0.00 0.00 0.00 0.00 Unidentified Insecta 3 1.87 0.13 0.19 0.02 4.38 2.99 0.14 0.27 Insecta 4 1.87 0.13 0.19 0.02 4.38 2.99 0.14 0.46 Insecta 5 1.87 0.13 0.19 0.02 4.38 2.99 0.14 0.67 Subphylum Crustacea 1 74.32 85.18 17.55 69.38 36.25 46.64 8.71 22.68 Unidentified Crustacea Nauplii 2 0.73 0.30 <0.01 <0.01 0.00 0.00 0.00 0.00 Nauplii 3 0.73 0.30 <0.01 <0.01 0.00 0.00 0.00 0.00 Nauplii 4 0.73 0.30 <0.01 <0.01 0.00 0.00 0.00 0.00 Nauplii 5 0.73 0.30 <0.01 <0.01 0.00 0.00 0.00 0.00 Class Branchiopoda 2 13.41 29.26 0.19 5.90 3.13 17.91 <0.01 0.69 Suborder Cladocera 3 13.41 29.26 0.19 11.40 3.13 17.91 <0.01 1.10 Family Bosminidae 4 1.04 0.08 <0.01 <0.01 0.00 0.00 0.00 0.00 Bosminidae 5 1.04 0.08 <0.01 <0.01 0.00 0.00 0.00 0.00 Family Chydoridae 4 0.83 0.14 <0.01 <0.01 0.00 0.00 0.00 0.00 Chydoridae 5 0.83 0.14 <0.01 <0.01 0.00 0.00 0.00 0.00 Family Daphniidae 4 3.64 9.80 0.05 1.25 3.13 17.91 <0.01 1.89 Scapholeberis 5 3.43 9.79 0.05 1.32 3.13 17.91 <0.01 2.76 Simocephalus 5 0.21 0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Family Moinidae 4 0.10 <0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Moina 5 0.10 <0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Family Sididae 4 7.69 18.93 0.13 5.09 0.00 0.00 0.00 0.00 Diaphanosoma 5 7.69 18.93 0.13 5.73 0.00 0.00 0.00 0.00 Unidentified Cladocera 4 1.66 0.30 <0.01 0.02 0.00 0.00 0.00 0.00 Cladocera 5 1.66 0.30 <0.01 0.02 0.00 0.00 0.00 0.00 Class Maxillopoda 2 24.32 35.63 0.25 13.03 0.00 0.00 0.00 0.00

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Table 3-4. Continued. Early-juvenile Common Snook (N=962) Pike Killifish (N=160) IRI Prey Item %O %N %W %IRI %O %N %W %IRI Level Subclass Copepoda 3 24.32 35.62 0.24 25.19 0.00 0.00 0.00 0.00 Copepoda 4 24.32 35.62 0.24 30.29 0.00 0.00 0.00 0.00 Copepoda 5 24.32 35.62 0.24 34.12 0.00 0.00 0.00 0.00 Subclass Branchiura 3 0.10 <0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Family Argulidae 4 0.10 <0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Argulus 5 0.10 <0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Class Ostracoda 2 7.48 1.03 0.02 0.12 0.00 0.00 0.00 0.00 Ostracoda 3 7.48 1.03 0.02 0.23 0.00 0.00 0.00 0.00 Ostracoda 4 7.48 1.03 0.02 0.27 0.00 0.00 0.00 0.00 Ostracoda 5 7.48 1.03 0.02 0.31 0.00 0.00 0.00 0.00 Class Malacostraca 2 56.96 18.97 17.09 30.67 33.13 28.73 8.71 15.24 Order Mysida 3 36.69 11.18 5.25 17.41 0.00 0.00 0.00 0.00 Family Mysidae 4 36.69 11.18 5.25 20.94 0.00 0.00 0.00 0.00 Americamysis almyra 5 21.73 7.52 2.61 8.61 0.00 0.00 0.00 0.00 Taphromysis spp. 5 11.33 1.44 1.33 1.23 0.00 0.00 0.00 0.00 Unidentified Mysidae 5 18.40 2.22 1.31 2.54 0.00 0.00 0.00 0.00 Order Isopoda 3 6.34 1.51 0.32 0.34 0.00 0.00 0.00 0.00 Family Anthuridae 4 0.21 0.01 0.07 <0.01 0.00 0.00 0.00 0.00 Cyathura polita 5 0.21 0.01 0.07 <0.01 0.00 0.00 0.00 0.00 Family Sphaeromatidae 4 3.12 0.39 0.21 0.07 0.00 0.00 0.00 0.00 Sphaeromatidae 5 3.12 0.39 0.21 0.07 0.00 0.00 0.00 0.00 Unidentified Isopoda* 4 4.16 1.11 0.04 0.17 0.00 0.00 0.00 0.00 Isopoda 5 4.16 1.11 0.04 0.19 0.00 0.00 0.00 0.00 Order Tanaidacea 3 2.70 0.84 0.05 0.07 0.00 0.00 0.00 0.00 Family Leptocheliidae 4 2.08 0.63 0.04 0.05 0.00 0.00 0.00 0.00 Hargeria rapax 5 2.08 0.63 0.04 0.05 0.00 0.00 0.00 0.00 Family Parapseudidae 4 0.62 0.18 <0.01 <0.01 0.00 0.00 0.00 0.00 Halmyrapseudes 5 0.62 0.18 <0.01 <0.01 0.00 0.00 0.00 0.00 bahamensis Family Tanaididae 4 0.42 0.03 <0.01 <0.01 0.00 0.00 0.00 0.00 Sinelobus stanfordi 5 0.42 0.03 <0.01 <0.01 0.00 0.00 0.00 0.00 Order Amphipoda 3 16.32 4.19 3.53 3.64 1.25 0.75 0.02 0.02 Suborder Corophiida 4 8.94 1.19 0.56 0.54 0.63 0.37 <0.01 <0.01 Apocorophium 5 3.53 0.46 0.10 0.08 0.63 0.37 <0.01 0.01 louisianum Grandidierella 5 4.57 0.48 0.36 0.15 0.00 0.00 0.00 0.00 bonnieroides Unidentified 5 2.39 0.25 0.11 0.03 0.00 0.00 0.00 0.00 Corophiida Suborder Gammaridea/ 4 10.19 3.00 2.98 2.11 0.63 0.37 0.01 <0.01 Senticaudata Gammaridea/ 5 10.19 3.00 2.98 2.38 0.63 0.37 0.01 0.01 Senticaudata Order Decapoda (larvae) 3 2.39 0.34 0.02 0.02 0.00 0.00 0.00 0.00 Decapoda (larvae) 4 2.39 0.34 0.02 0.03 0.00 0.00 0.00 0.00 Brachyura zoea 5 1.14 0.23 <0.01 0.01 0.00 0.00 0.00 0.00 Brachyura megalops 5 0.31 0.02 <0.01 <0.01 0.00 0.00 0.00 0.00

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Table 3-4. Continued. Early-juvenile Common Snook (N=962) Pike Killifish (N=160) IRI Prey Item %O %N %W %IRI %O %N %W %IRI Level Caridea zoea/mysis 5 1.04 0.07 <0.01 <0.01 0.00 0.00 0.00 0.00 Upogebia larvae 5 0.10 0.02 <0.01 <0.01 0.00 0.00 0.00 0.00 Order Decapoda (adults) 3 10.81 0.92 7.91 2.76 32.50 27.99 8.69 23.53 Family Palaemonidae 4 8.32 0.71 5.84 1.89 31.88 26.87 8.55 38.01 Palaemonetes 5 8.32 0.71 5.84 2.13 31.88 26.87 8.55 55.59 Family Penaeidae 4 0.73 0.06 0.45 0.01 0.00 0.00 0.00 0.00 Farfantepenaeus 5 0.73 0.06 0.45 0.01 0.00 0.00 0.00 0.00 duorarum Unidentified Shrimp 4 1.46 0.10 0.74 0.04 1.88 1.12 0.15 0.08 Shrimp 5 1.46 0.10 0.74 0.05 1.88 1.12 0.15 0.12 Family Ocypodidae 4 0.31 0.03 0.65 <0.01 0.00 0.00 0.00 0.00 Uca 5 0.31 0.03 0.65 <0.01 0.00 0.00 0.00 0.00 Family Portunidae 4 0.10 <0.01 0.07 <0.01 0.00 0.00 0.00 0.00 Callinectes sapidus 5 0.10 <0.01 0.07 <0.01 0.00 0.00 0.00 0.00 Superfamily Xanthoidea 4 0.10 <0.01 0.06 <0.01 0.00 0.00 0.00 0.00 Xanthoidea 5 0.10 <0.01 0.06 <0.01 0.00 0.00 0.00 0.00 Unidentified Brachyura 4 0.10 <0.01 0.11 <0.01 0.00 0.00 0.00 0.00 Brachyura 5 0.10 <0.01 0.11 <0.01 0.00 0.00 0.00 0.00 Class Actinopterygii 1 36.49 3.87 78.65 27.36 51.25 37.31 90.60 74.09 Infraclass Teleostei 2 36.49 3.87 78.65 44.95 51.25 37.31 90.60 80.56 Order Atheriniformes 3 0.21 0.02 0.90 <0.01 1.25 0.75 3.74 0.11 Family Atherinopsidae 4 0.21 0.02 0.90 <0.01 1.25 0.75 3.74 0.19 Menidia peninsulae/ 5 0.21 0.02 0.90 <0.01 1.25 0.75 3.74 0.28 Menidia beryllina Order Clupeiformes 3 21.10 2.20 33.89 21.98 2.50 1.49 0.98 0.12 Family Engraulidae 4 21.10 2.20 33.89 26.44 2.50 1.49 0.98 0.21 Anchoa mitchilli 5 21.10 2.20 33.89 29.78 2.50 1.49 0.98 0.30 Order Cyprinodontiformes 3 5.20 0.52 11.73 1.84 35.63 24.63 74.86 69.95 Family Fundulidae 4 1.56 0.12 5.36 0.30 18.75 12.31 41.65 34.07 Fundulus grandis/ 5 0.83 0.06 3.23 0.11 10.00 6.34 29.47 17.64 Funduls confluentus Fundulus seminolis 5 0.31 0.03 0.73 <0.01 3.13 2.61 8.19 1.66 Fundulus xenicus 5 0.21 0.01 1.20 <0.01 0.00 0.00 0.00 0.00 Lucania parva 5 0.21 0.01 0.21 <0.01 5.63 3.36 3.99 2.04 Family Poeciliidae 4 3.74 0.35 6.27 0.86 15.00 10.07 32.31 21.41 Belonesox belizanus 5 0.00 0.00 0.00 0.00 3.13 1.87 12.63 2.23 Gambusia holbrooki 5 2.49 0.22 1.63 0.18 1.88 1.12 2.28 0.31 Heterandria formosa 5 0.31 0.02 0.14 <0.01 0.00 0.00 0.00 0.00 Poecilia latipinna 5 1.25 0.10 4.51 0.23 10.00 7.09 17.41 12.06 Unidentified 4 0.52 0.06 0.10 <0.01 3.13 2.24 0.90 0.33 Cyprinodontiformes Cyprinodontiformes 5 0.52 0.06 0.10 <0.01 3.13 2.24 0.90 0.48 Order Perciformes 3 7.48 0.65 21.55 4.80 10.00 7.46 8.77 3.20 Family Centrachidae 4 1.46 0.11 0.68 0.04 3.13 1.87 4.04 0.62 Lepomis macrochirus 5 1.46 0.11 0.68 0.04 3.13 1.87 4.04 0.91 Family Centropomidae 4 0.42 0.03 0.23 <0.01 0.00 0.00 0.00 0.00 Centropomus 5 0.42 0.03 0.23 <0.01 0.00 0.00 0.00 0.00 undecimalis

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Table 3-4. Continued. Early-juvenile Common Snook (N=962) Pike Killifish (N=160) IRI Prey Item %O %N %W %IRI %O %N %W %IRI Level Family Cichlidae 4 0.62 0.08 1.79 0.04 3.75 3.73 2.79 0.82 Hemichromis 5 0.00 0.00 0.00 0.00 0.63 0.37 0.95 0.04 letourneuxi Oreochromis aureus 5 0.10 <0.01 1.57 <0.01 0.00 0.00 0.00 0.00 Sarotherodon 5 0.00 0.00 0.00 0.00 0.63 0.37 1.52 0.06 melanotheron O. aureus/ S. melanotheron 5 0.52 0.07 0.22 <0.01 2.50 2.99 0.32 0.41 juveniles Family Gerreidae 4 1.35 0.11 11.69 0.55 1.25 0.75 0.38 0.05 Eucinostomus 5 1.35 0.11 11.69 0.62 1.25 0.75 0.38 0.07 Family Gobiidae 4 3.01 0.27 6.05 0.66 1.25 0.75 1.52 0.10 Gobiosoma 5 0.42 0.03 0.61 0.01 0.63 0.37 0.14 0.02 Microgobius gulosus 5 2.60 0.24 5.45 0.58 0.63 0.37 1.38 0.05 Family Scianidae 4 0.52 0.03 1.04 0.02 0.00 0.00 0.00 0.00 Cynoscion arenarius 5 0.21 0.01 0.61 <0.01 0.00 0.00 0.00 0.00 Sciaenops ocellatus 5 0.31 0.02 0.44 <0.01 0.00 0.00 0.00 0.00 Unidentified 4 0.31 0.02 0.07 <0.01 0.63 0.37 0.03 <0.01 Perciformes Perciformes 5 0.31 0.02 0.07 <0.01 0.63 0.37 0.03 0.01 Unidentified_Teleostei 3 6.24 0.49 10.58 1.99 3.13 2.99 2.25 0.32 Teleostei 4 6.24 0.49 10.58 2.40 3.13 2.99 2.25 0.55 Teleostei 5 6.24 0.49 10.58 2.70 3.13 2.99 2.25 0.81 * The majority of the unidentified isopods were subsequenty identified as Munnidae; however, these had not been separated from other unidentified isopods upon initial analysis.

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Table 3-5. Diet composition of early-juvenile Common Snook of different size classes based on prey functional groups. Percent frequency of occurrence (%O), percent numerical abundance (%N), percent weight (%W), and percent index of relative importance (%IRI) are reported for each size group. All (N = 962) ≤35 mm SL (N = 495) >35 mm SL (N = 467)

Prey Functional Group %O %N %W %IRI %O %N %W %IRI %O %N %W %IRI

Planktonic Crustaceans 31.70 65.52 0.46 41.86 50.91 79.13 3.39 57.16 11.35 18.40 0.04 3.97 Small Benthic 53.64 18.75 9.17 29.97 57.17 10.31 25.33 27.73 Crustaceans 49.89 47.97 6.86 51.95 Annelids 0.94 0.25 1.08 0.02 0.61 0.03 0.40 <0.01 1.28 1.02 1.18 0.05 Aquatic Insects and 25.16 10.56 2.92 6.79 30.91 9.28 15.70 10.51 Arachnids 19.06 15.01 1.09 5.83 Terrestrial/Unidentified 1.87 0.13 0.19 0.01 2.22 0.11 1.02 0.03 Insects and Arachnids 1.50 0.22 0.07 0.01 Shrimp 10.40 0.86 7.03 1.64 4.65 0.24 5.95 0.39 16.49 3.02 7.19 3.20

Crabs 0.52 0.06 0.88 0.01 0.00 0.00 0.00 0.00 1.07 0.25 1.01 0.03

Clupeiformes 21.10 2.20 33.89 15.24 9.70 0.45 21.14 2.85 33.19 8.26 35.72 27.72

Atheriniformes 0.21 0.02 0.90 0.00 0.20 0.01 0.09 0.00 0.21 0.06 1.01 <0.01

Cyprinodontiformes 5.20 0.52 11.73 1.27 2.22 0.13 1.91 0.06 8.35 1.85 13.14 2.38 Order Perciformes 4.47 0.38 15.50 1.42 2.63 0.13 3.51 0.13 excluding Gobiidae 6.42 1.23 17.22 2.25 Family Gobiidae 3.01 0.27 6.05 0.38 0.61 0.03 1.80 0.02 5.57 1.11 6.66 0.82

Unidentified Fish 6.24 0.49 10.58 1.38 3.64 0.17 22.44 1.12 8.99 1.60 8.88 1.79

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Table 3-6. Diet composition of early-juvenile Common Snook with and without Pike Killifish co-occurring. Percent frequency of occurrence (%O), percent numerical abundance (%N), percent weight (%W), and percent index of relative importance (%IRI) are reported for both Common Snook and Pike Killifish for all prey items identified in their diet analysis. The %IRI is reported for different taxonomic levels, designated by the IRI level. Snook w/o Pike Killifish (N=431) Snook w/Pike Killifish (N=531) IRI Prey Item %O %N %W %IRI %O %N %W %IRI Level Phylum Annelida 1 0.93 0.08 0.24 <0.01 0.94 0.41 1.70 0.02 Class Clitellata 2 0.23 0.04 0.12 <0.01 0.00 0.00 0.00 0.00 Subclass Hirudinea 3 0.23 0.04 0.12 <0.01 0.00 0.00 0.00 0.00 Hirudinea 4 0.23 0.04 0.12 <0.01 0.00 0.00 0.00 0.00 Hirudinea 5 0.23 0.04 0.12 <0.01 0.00 0.00 0.00 0.00 Class Polychaeta 2 0.70 0.04 0.12 <0.01 0.94 0.41 1.70 0.03 Subclass Errantia 3 0.70 0.04 0.12 <0.01 0.94 0.41 1.70 0.05 Errantia 4 0.70 0.04 0.12 <0.01 0.94 0.41 1.70 0.06 Errantia 5 0.70 0.04 0.12 <0.01 0.94 0.41 1.70 0.07 Subphylum Chelicerata 1 0.23 0.01 <0.01 <0.01 0.19 0.01 <0.01 <0.01 Class Arachnida 2 0.23 0.01 <0.01 <0.01 0.19 0.01 <0.01 <0.01 Order Araneae 3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Araneae 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Araneae 5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Order Trombidiformes 3 0.23 0.01 <0.01 <0.01 0.19 0.01 <0.01 <0.01 Suborder Prostigmata 4 0.23 0.01 <0.01 <0.01 0.19 0.01 <0.01 <0.01 Prostigmata 5 0.23 0.01 <0.01 <0.01 0.19 0.01 <0.01 <0.01 Subphylum Hexapoda 1 35.27 14.95 5.14 6.30 18.27 6.49 1.62 1.35 Class Insecta 2 35.27 14.95 5.14 10.62 18.27 6.49 1.62 2.12 Order Coleoptera 3 0.00 0.00 0.00 0.00 0.38 0.03 <0.01 <0.01 Family Gyrinidae 4 0.00 0.00 0.00 0.00 0.38 0.03 <0.01 <0.01 Gyrinidae larvae 5 0.00 0.00 0.00 0.00 0.38 0.03 <0.01 <0.01 Order Diptera 3 34.11 14.52 3.39 16.02 14.88 6.08 0.80 2.61 Family Chaoboridae 4 9.98 2.62 1.70 1.36 0.56 0.04 0.02 <0.01 Chaoboridae larvae 5 9.98 2.62 1.70 1.60 0.56 0.04 0.02 <0.01 Family Chironomidae 4 24.13 9.74 1.01 8.17 11.68 5.10 0.59 2.02 Chironomidae larvae 5 24.13 9.74 1.01 9.63 5.10 5.10 0.59 2.18 Chironomidae adult 5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Family Chironomidae/ 4 13.46 2.11 0.68 1.18 0.90 0.90 0.19 0.16 Chaoboridae Chironomidae/ 5 13.46 2.11 0.68 1.39 0.90 0.90 0.19 0.17 Chaoboridae pupae Family Culicidae 4 0.70 0.04 <0.01 <0.01 0.04 0.04 <0.01 <0.01 Culicidae larvae 5 0.23 0.01 <0.01 <0.01 0.04 0.04 <0.01 <0.01 Culicidae pupae 5 0.46 0.03 <0.01 <0.01 0.00 0.00 0.00 0.00 Order Ephemeroptera 3 0.70 0.04 0.03 <0.01 0.00 0.00 0.00 0.00 Ephemeroptera 4 0.70 0.04 0.03 <0.01 0.00 0.00 0.00 0.00 Ephemeroptera naiad 5 0.70 0.04 0.03 <0.01 0.00 0.00 0.00 0.00 Order Hemiptera 3 0.93 0.06 0.68 0.02 0.05 0.05 0.19 <0.01 Family Corixidae 4 0.70 0.04 0.01 <0.01 0.04 0.04 <0.01 <0.01 Corixidae 5 0.70 0.04 0.01 <0.01 0.04 0.04 <0.01 <0.01

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Table 3-6. Continued. Snook w/o Pike Killifish (N=431) Snook w/Pike Killifish (N=531) IRI Prey Item %O %N %W %IRI %O %N %W %IRI Level Family Gerridae 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Gerridae 5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Family Naucoridae 4 0.23 0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Naucoridae 5 0.23 0.01 <0.01 <0.01 0.00 0.00 0.00 0.00 Family Notonectidae 4 0.00 0.00 0.00 0.00 0.19 0.01 0.02 <0.01 Notonectidae 5 0.00 0.00 0.00 0.00 0.19 0.01 0.02 <0.01 Order Hymenoptera 3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Family Formicidae 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Formicidae 5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Order Lepidoptera 3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Lepidoptera 4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Lepidoptera adult (moth) 5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Order Odonata 3 1.16 0.07 0.48 0.02 0.38 0.05 0.40 <0.01 Suborder Anisoptera 4 0.46 0.03 0.20 <0.01 0.38 0.05 0.40 <0.01 Anisoptera naiad 5 0.46 0.03 0.20 <0.01 0.38 0.05 0.40 <0.01 Suborder Zygoptera 4 0.70 0.04 0.27 <0.01 0.00 0.00 0.00 0.00 Zygoptera naiad 5 0.70 0.04 0.27 <0.01 0.00 0.00 0.00 0.00 Zygoptera adult 5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Order Trichoptera 3 1.39 0.18 0.35 0.02 0.75 0.10 0.04 <0.01 Family Hydropsychidae 4 0.46 0.10 0.34 <0.01 0.56 0.07 0.04 <0.01 Hydropsychidae larvae 5 0.46 0.10 0.34 <0.01 0.56 0.07 0.04 <0.01 Unidentified Trichoptera 4 1.16 0.08 <0.01 <0.01 0.19 0.03 <0.01 <0.01 Trichoptera larvae 5 1.16 0.08 <0.01 <0.01 0.19 0.03 <0.01 <0.01 Unidentified Insecta 3 1.39 0.08 0.21 0.01 2.26 0.18 0.18 0.02 Insecta 4 1.39 0.08 0.21 0.01 2.26 0.18 0.18 0.02 Insecta 5 1.39 0.08 0.21 0.02 2.26 0.18 0.18 0.03 Subphylum Crustacea 1 80.74 82.14 20.61 73.73 69.11 88.16 15.27 65.10 Unidentified Crustacea Nauplii 2 1.39 0.59 <0.01 0.01 0.19 0.01 <0.01 <0.01 Nauplii 3 1.39 0.59 <0.01 0.02 0.19 0.01 <0.01 <0.01 Nauplii 4 1.39 0.59 <0.01 0.03 0.19 0.01 <0.01 <0.01 Nauplii 5 1.39 0.59 <0.01 0.03 0.19 0.01 <0.01 <0.01 Class Branchiopoda 2 10.90 18.58 0.15 3.06 15.44 39.71 0.22 8.83 Suborder Cladocera 3 10.90 18.58 0.15 5.35 15.44 39.71 0.22 15.73 Family Bosminidae 4 1.39 0.11 <0.01 <0.01 0.75 0.05 <0.01 <0.01 Bosminidae 5 1.39 0.11 <0.01 <0.01 0.75 0.05 <0.01 <0.01 Family Chydoridae 4 1.39 0.22 <0.01 0.01 0.38 0.05 <0.01 <0.01 Chydoridae 5 1.39 0.22 <0.01 0.01 0.38 0.05 <0.01 <0.01 Family Daphniidae 4 0.46 0.03 <0.01 <0.01 6.21 19.37 0.09 3.68 Scapholeberis 5 0.00 0.00 0.00 0.00 6.21 19.37 0.09 3.97 Simocephalus 5 0.46 0.03 <0.01 <0.01 0.00 0.00 0.00 0.00 Family Moinidae 4 0.00 0.00 0.00 0.00 0.19 0.01 <0.01 <0.01 Moina 5 0.00 0.00 0.00 0.00 0.19 0.01 <0.01 <0.01 Family Sididae 4 7.66 17.87 0.14 4.34 7.72 19.96 0.13 4.72 Diaphanosoma 5 7.66 17.87 0.14 5.12 7.72 19.96 0.13 5.09 Unidentified Cladocera 4 1.86 0.35 <0.01 0.02 1.51 0.26 <0.01 0.01 Cladocera 5 1.86 0.35 <0.01 0.02 1.51 0.26 <0.01 0.01 Class Maxillopoda 2 28.77 45.75 0.31 19.86 20.72 25.71 0.20 7.68

116

Table 3-6. Continued. Snook w/o Pike Killifish (N=431) Snook w/Pike Killifish (N=531) IRI Prey Item %O %N %W %IRI %O %N %W %IRI Level Subclass Copepoda 3 28.77 45.75 0.31 34.74 20.72 25.70 0.19 13.68 Copepoda 4 28.77 45.75 0.31 41.70 20.72 25.70 0.19 16.33 Copepoda 5 28.77 45.75 0.31 49.17 20.72 25.70 0.19 17.62 Subclass Branchiura 3 0.00 0.00 0.00 0.00 0.19 0.01 <0.01 <0.01 Family Argulidae 4 0.00 0.00 0.00 0.00 0.19 0.01 <0.01 <0.01 Argulus 5 0.00 0.00 0.00 0.00 0.19 0.01 <0.01 <0.01 Class Ostracoda 2 9.28 1.30 0.03 0.18 6.03 0.77 <0.01 0.07 Ostracoda 3 9.28 1.30 0.03 0.32 6.03 0.77 <0.01 0.12 Ostracoda 4 9.28 1.30 0.03 0.39 6.03 0.77 <0.01 0.14 Ostracoda 5 9.28 1.30 0.03 0.46 6.03 0.77 <0.01 0.15 Class Malacostraca 2 60.32 15.93 20.12 32.59 54.24 21.95 14.84 28.57 Order Mysida 3 45.24 11.53 6.70 21.62 29.76 10.84 4.17 11.39 Family Mysidae 4 45.24 11.53 6.70 25.95 29.76 10.84 4.17 13.60 Americamysis almyra 5 28.31 7.69 3.14 11.37 16.38 7.35 2.22 5.15 Taphromysis spp. 5 14.39 1.55 1.88 1.83 8.85 1.34 0.92 0.66 Unidentified Mysidae 5 21.58 2.29 1.68 3.18 15.82 2.15 1.04 1.65 Order Isopoda 3 3.25 0.31 0.19 0.04 8.85 2.69 0.42 0.70 Family Anthuridae 4 0.00 0.00 0.00 0.00 0.38 0.03 0.11 <0.01 Cyathura polita 5 0.00 0.00 0.00 0.00 0.38 0.03 0.11 <0.01 Family Sphaeromatidae 4 2.09 0.24 0.18 0.03 3.95 0.53 0.24 0.09 Sphaeromatidae 5 2.09 0.24 0.18 0.03 3.95 0.53 0.24 0.10 Unidentified Isopoda* 4 1.16 0.07 <0.01 <0.01 6.59 2.13 0.07 0.44 Isopoda 5 1.16 0.07 <0.01 <0.01 6.59 2.13 0.07 0.48 Order Tanaidacea 3 0.70 0.04 <0.01 <0.01 4.33 1.61 0.09 0.19 Family Leptocheliidae 4 0.23 0.01 <0.01 <0.01 3.58 1.23 0.08 0.14 Hargeria rapax 5 0.23 0.01 <0.01 <0.01 3.58 1.23 0.08 0.15 Family Parapseudidae 4 0.00 0.00 0.00 0.00 1.13 0.36 0.01 0.01 Halmyrapseudes 5 0.00 0.00 0.00 0.00 1.13 0.36 0.01 0.01 bahamensis Family Tanaididae 4 0.46 0.03 <0.01 <0.01 0.38 0.03 <0.01 <0.01 Sinelobus stanfordi 5 0.46 0.03 <0.01 <0.01 0.38 0.03 <0.01 <0.01 Order Amphipoda 3 12.53 2.85 3.19 1.98 19.40 5.50 3.79 4.59 Suborder Corophiida 4 6.26 0.60 0.34 0.19 11.11 1.76 0.72 0.84 Apocorophium 5 2.78 0.31 0.08 0.04 4.14 0.62 0.10 0.10 louisianum Grandidierella 5 3.02 0.20 0.19 0.04 5.84 0.75 0.48 0.24 bonnieroides Unidentified 5 1.62 0.10 0.07 0.01 3.01 0.40 0.13 0.05 Corophiida Suborder Gammaridea/ 4 7.66 2.25 2.84 1.23 12.24 3.73 3.07 2.54 Senticaudata Gammaridea/ 5 7.66 2.25 2.84 1.45 12.24 3.73 3.07 2.74 Senticaudata Order Decapoda (larvae) 3 2.78 0.35 0.02 0.03 2.07 0.33 0.02 0.02 Decapoda (larvae) 4 2.78 0.35 0.02 0.03 2.07 0.33 0.02 0.02 Brachyura zoea 5 1.39 0.27 <0.01 0.01 0.94 0.19 <0.01 <0.01 Brachyura megalops 5 0.23 0.01 <0.01 <0.01 0.38 0.03 <0.01 <0.01

117

Table 3-6. Continued. Snook w/o Pike Killifish (N=431) Snook w/Pike Killifish (N=531) IRI Prey Item %O %N %W %IRI %O %N %W %IRI Level Caridea zoea/mysis 5 1.16 0.07 <0.01 <0.01 0.94 0.07 <0.01 <0.01 Upogebia larvae 5 0.00 0.00 0.00 0.00 0.19 0.04 0.01 <0.01 Order Decapoda (adults) 3 11.14 0.85 10.03 3.18 10.55 0.98 6.34 1.97 Family Palaemonidae 4 7.66 0.59 6.60 1.73 8.85 0.83 5.28 1.65 Palaemonetes 5 7.66 0.59 6.60 2.04 8.85 0.83 5.28 1.78 Family Penaeidae 4 1.62 0.11 1.06 0.06 0.00 0.00 0.00 0.00 Farfantepenaeus 5 1.62 0.11 1.06 0.07 0.00 0.00 0.00 0.00 duorarum Unidentified Shrimp 4 1.39 0.08 0.81 0.04 1.51 0.11 0.69 0.04 Shrimp 5 1.39 0.08 0.81 0.05 1.51 0.11 0.69 0.04 Family Ocypodidae 4 0.46 0.03 1.02 0.02 0.19 0.04 0.37 <0.01 Uca 5 0.46 0.03 1.02 0.02 0.19 0.04 0.37 <0.01 Family Portunidae 4 0.23 0.01 0.16 <0.01 0.00 0.00 0.00 0.00 Callinectes sapidus 5 0.23 0.01 0.16 <0.01 0.00 0.00 0.00 0.00 Superfamily Xanthoidea 4 0.23 0.01 0.13 <0.01 0.00 0.00 0.00 0.00 Xanthoidea 5 0.23 0.01 0.13 <0.01 0.00 0.00 0.00 0.00 Unidentified Brachyura 4 0.23 0.01 0.26 <0.01 0.00 0.00 0.00 0.00 Brachyura 5 0.23 0.01 0.26 <0.01 0.00 0.00 0.00 0.00 Class Actinopterygii 1 29.00 2.81 74.68 19.97 42.56 4.92 81.58 33.53 Infraclass Teleostei 2 29.00 2.81 74.68 33.68 42.56 4.92 81.58 52.70 Order Atheriniformes 3 0.46 0.04 2.11 0.03 0.00 0.00 0.00 0.00 Family Atherinopsidae 4 0.46 0.04 2.11 0.03 0.00 0.00 0.00 0.00 Menidia peninsulae/ 5 0.46 0.04 2.11 0.04 0.00 0.00 0.00 0.00 Menidia beryllina Order Clupeiformes 3 7.89 0.67 13.52 2.93 31.83 3.69 48.97 42.75 Family Engraulidae 4 7.89 0.67 13.52 3.52 31.83 3.69 48.97 51.03 Anchoa mitchilli 5 7.89 0.67 13.52 4.15 31.83 3.69 48.97 55.06 Order Cyprinodontiformes 3 10.44 0.96 25.68 7.29 0.94 0.08 1.40 0.04 Family Fundulidae 4 2.55 0.17 10.76 0.88 0.75 0.07 1.36 0.03 Fundulus grandis/ 5 1.86 0.13 7.59 0.53 0.00 0.00 0.00 0.00 Funduls confluentus Fundulus seminolis 5 0.23 0.01 0.37 <0.01 0.38 0.04 0.99 0.01 Fundulus xenicus 5 0.46 0.03 2.81 0.05 0.00 0.00 0.00 0.00 Lucania parva 5 0.00 0.00 0.00 0.00 0.38 0.03 0.36 <0.01 Family Poeciliidae 4 8.12 0.68 14.68 3.93 0.19 0.01 0.05 <0.01 Belonesox belizanus 5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Gambusia holbrooki 5 5.57 0.45 3.82 0.88 0.00 0.00 0.00 0.00 Heterandria formosa 5 0.70 0.04 0.32 <0.01 0.00 0.00 0.00 0.00 Poecilia latipinna 5 2.55 0.20 10.54 1.02 0.19 0.01 0.05 <0.01 Unidentified 4 1.16 0.11 0.23 0.01 0.00 0.00 0.00 0.00 Cyprinodontiformes Cyprinodontiformes 5 1.16 0.11 0.23 0.01 0.00 0.00 0.00 0.00 Order Perciformes 3 6.73 0.56 18.54 3.37 8.10 0.74 23.78 5.06 Family Centrachidae 4 0.70 0.04 0.25 <0.01 2.07 0.18 1.00 0.07 Lepomis macrochirus 5 0.70 0.04 0.25 <0.01 2.07 0.18 1.00 0.08 Family Centropomidae 4 0.23 0.01 0.06 <0.01 0.56 0.04 0.36 <0.01 Centropomus 5 0.23 0.01 0.06 <0.01 0.56 0.04 0.36 <0.01 undecimalis

118

Table 3-6. Continued. Snook w/o Pike Killifish (N=431) Snook w/Pike Killifish (N=531) IRI Prey Item %O %N %W %IRI %O %N %W %IRI Level Family Cichlidae 4 0.00 0.00 0.00 0.00 1.13 0.15 3.12 0.11 Hemichromis 5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 letourneuxi Oreochromis aureus 5 0.00 0.00 0.00 0.00 0.19 0.01 2.74 0.02 Sarotherodon 5 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 melanotheron O. aureus/ S. melanotheron 5 0.00 0.00 0.00 0.00 0.94 0.14 0.39 0.02 juveniles Family Gerreidae 4 1.16 0.08 8.99 0.33 1.51 0.14 13.68 0.63 Eucinostomus 5 1.16 0.08 8.99 0.39 1.51 0.14 13.68 0.68 Family Gobiidae 4 4.18 0.38 8.59 1.18 2.07 0.16 4.17 0.27 Gobiosoma 5 0.70 0.04 1.34 0.04 0.19 0.01 0.07 <0.01 Microgobius gulosus 5 3.48 0.34 7.26 0.98 1.88 0.15 4.10 0.26 Family Scianidae 4 0.23 0.01 0.54 <0.01 0.75 0.05 1.42 0.03 Cynoscion arenarius 5 0.00 0.00 0.00 0.00 0.38 0.03 1.06 0.01 Sciaenops ocellatus 5 0.23 0.01 0.54 <0.01 0.38 0.03 0.36 <0.01 Unidentified 4 0.46 0.03 0.11 <0.01 0.19 0.01 0.03 <0.01 Perciformes Perciformes 5 0.46 0.03 0.11 <0.01 0.19 0.01 0.03 <0.01 Unidentified_Teleostei 3 7.42 0.57 14.83 3.00 5.27 0.41 7.43 1.05 Teleostei 4 7.42 0.57 14.83 3.60 5.27 0.41 7.43 1.26 Teleostei 5 7.42 0.57 14.83 4.24 5.27 0.41 7.43 1.36 * The majority of the unidentified isopods were subsequenty identified as Munnidae; however, these had not been separated from other unidentified isopods upon initial analysis.

119

Table 3-7. Diet composition, based on prey functional groups, of early-juvenile Common Snook of different size classes from locations without Pike Killifish co-occurring. Percent frequency of occurrence (%O), percent numerical abundance (%N), percent weight (%W), and percent index of relative importance (%IRI) are reported for each size group. Snook w/o Pike Killifish All (N =431) ≤35 mm SL (N = 240) >35 mm SL (N = 191) Prey Functional Group %O %N %W %IRI %O %N %W %IRI %O %N %W %IRI

Planktonic Crustaceans 32.48 65.27 0.48 42.32 49.17 76.49 3.13 49.02 11.52 7.24 0.02 1.45 Small Benthic Crustaceans 58.93 16.03 10.10 30.52 58.75 8.55 26.82 26.02 59.16 54.69 7.19 63.76 Annelids 0.93 0.08 0.24 0.01 0.83 0.03 0.51 0.01 1.05 0.34 0.19 0.01 Aquatic Insects and Arachnids 35.50 14.88 4.92 13.93 44.17 14.11 26.49 22.46 24.61 18.86 1.11 8.56 Terrestrial/Unidentified Insects and Arachnids 1.39 0.08 0.21 0.01 1.67 0.07 1.08 0.02 1.05 0.17 0.06 <0.01 Shrimp 10.44 0.78 8.46 1.91 2.08 0.08 2.01 0.05 20.94 4.39 9.59 5.10

Crabs 0.93 0.07 1.57 0.03 0.00 0.00 0.00 0.00 2.09 0.43 1.84 0.08

Clupeiformes 7.89 0.67 13.52 2.22 2.92 0.12 4.23 0.16 14.14 3.53 15.14 4.60

Atheriniformes 0.46 0.04 2.11 0.02 0.42 0.02 0.18 0.00 0.52 0.17 2.44 0.02

Cyprinodontiformes 10.44 0.96 25.68 5.51 3.75 0.22 3.05 0.15 18.85 4.82 29.62 11.31 Order Perciformes excluding Gobiidae 2.55 0.18 9.95 0.51 1.67 0.07 2.35 0.05 3.66 0.78 11.28 0.77 Family Gobiidae 4.18 0.38 8.59 0.74 0.83 0.03 2.20 0.02 8.38 2.15 9.71 1.73

Unidentified Fish 7.42 0.57 14.83 2.27 5.00 0.22 32.17 2.03 10.47 2.41 11.86 2.60

120

Table 3-8. Diet composition, based on prey functional groups, of early-juvenile Common Snook of different size classes from locations with Pike Killifish co-occurring. Percent frequency of occurrence (%O), percent numerical abundance (%N), percent weight (%W), and percent index of relative importance (%IRI) are reported for each size group. Snook with Pike Killifish All (N = 531) ≤35 mm SL (N = 255) >35 mm SL (N = 276) Prey Functional Group %O %N %W %IRI %O %N %W %IRI %O %N %W %IRI

Planktonic Crustaceans 31.07 65.77 0.44 36.83 52.55 82.15 3.65 60.72 11.23 24.63 0.05 4.68 Small Benthic Crustaceans 49.34 21.41 8.49 26.40 55.69 12.33 23.81 27.10 43.48 44.22 6.46 37.23 Annelids 0.94 0.41 1.70 0.04 0.39 0.02 0.28 0.00 1.45 1.39 1.88 0.08 Aquatic Insects and Arachnids 16.76 6.33 1.44 2.33 18.43 3.73 4.42 2.02 15.22 12.87 1.08 3.59 Terrestrial/Unidentified Insects and Arachnids 2.26 0.18 0.18 0.01 2.75 0.15 0.97 0.04 1.81 0.24 0.08 0.01 Shrimp 10.36 0.94 5.97 1.28 7.06 0.42 9.94 0.99 13.41 2.26 5.50 1.76

Crabs 0.19 0.04 0.37 0.00 0.00 0.00 0.00 0.00 0.36 0.14 0.42 <0.01

Clupeiformes 31.83 3.69 48.97 30.00 16.08 0.82 38.25 8.46 46.38 10.90 50.37 48.01

Atheriniformes 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Cyprinodontiformes 0.94 0.08 1.40 0.03 0.78 0.04 0.79 0.01 1.09 0.19 1.48 0.03 Order Perciformes excluding Gobiidae 6.03 0.57 19.61 2.18 3.53 0.21 4.70 0.23 8.33 1.49 21.46 3.23 Family Gobiidae 2.07 0.16 4.17 0.16 0.39 0.02 1.42 0.01 3.62 0.53 4.51 0.31

Unidentified Fish 5.27 0.41 7.43 0.74 2.35 0.11 12.88 0.41 7.97 1.15 6.77 1.07

121

Table 3-9. Levin’s standardized index for early-juvenile Common Snook and Pike Killifish. Levin’s index was calculated for several size groupings (based on ontogenetic diet shifts) of early-juvenile Common Snook and Pike Killifish for all identified prey categories and prey functional groups. Levin’s standardized index ranges from 0 to 1 with value near zero being indicative of a more specialized diet and value near 1 being indicative of a more generalized diet. Early-juvenile Common Early-juvenile Snook Common Early-juvenile without Pike Snook with Common Common Common Killifish co- Pike Killifish Snook ≤35 Snook >35 Pike Killifish Pike Killifish Group Snook occurring co-occurring mm SL mm SL Pike Killifish ≤35 mm SL >35 mm SL N 962 431 531 495 467 160 14 146 Levin's standardized index: all prey categories 0.05 0.03 0.07 0.03 0.11 0.09 0.01 0.07

Levin's standardized index: prey functional groups 0.09 0.09 0.09 0.05 0.20 0.36 0.04 0.27

122

Table 3-10. Ivlev electivity index values for early-juvenile Common Snook and Pike Killifish based on prey collections from both bag seine and minnow seine hauls. Index values were calculated for the following groups for both sampling gears: Snook without Pike Killifish co- occurring (SNK w/o PK), Snook with Pike Killifish co-occurring (SNK w/PK), and Pike Killifish (PK). This index ranges from -1 to 1 with values near -1 being indicative of avoidance, values near 1 being indicative of selection, and values near 0 being indicative of non-

selective feeding on a particular group.

Group

mes

Order Order Order Order

Shrimp

Gobiidae)

(excluding

Perciformes

Clupeiformes

Atheriniformers Cyprinodontifor Family Gobiidae Family

Bag Seine

SNK w/o 0.32 -0.20 -0.72 0.14 -0.75 0.12 PK

SNK w/PK 0.16 0.57 -1.00 -0.70 -0.43 -0.42

PK 0.56 -0.77 -0.54 0.65 -0.42 -0.72

Minnow

Seine SNK w/o -0.05 -0.18 -0.06 -0.26 0.44 0.62 PK

SNK w/PK -0.40 0.61 -1.00 -0.53 0.41 0.54

PK 0.05 -0.74 0.31 0.78 0.42 0.15

123

Table 3-11. Diet composition, based on prey functional groups, of Pike Killifish of different size classes. Percent frequency of occurrence (%O), percent numerical abundance (%N), percent weight (%W), and percent index of relative importance (%IRI) are reported for each size group. Snook with Pike Killifish All (N = 160) ≤30 mm SL (N = 14) >30 mm SL (N = 146) Prey Functional Group %O %N %W %IRI %O %N %W %IRI %O %N %W %IRI

Planktonic Crustaceans 3.13 17.91 0.00 1.09 35.71 80.00 0.58 52.31 0.00 0.00 0.00 0.00 Small Benthic Crustaceans 1.25 0.75 0.02 0.02 0.00 0.00 0.00 0.00 1.37 0.96 0.02 0.02 Annelids 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aquatic Insects and Arachnids 10.63 8.96 0.11 1.88 14.29 8.33 6.23 3.78 10.27 9.13 0.09 1.68 Terrestrial/Unidentified Insects and Arachnids 9.38 7.09 0.54 1.40 0.00 0.00 0.00 0.00 10.27 9.13 0.55 1.77 Shrimp 32.50 27.99 8.69 23.31 7.14 1.67 42.54 5.74 34.93 35.58 8.54 27.38

Crabs 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Clupeiformes 2.50 1.49 0.98 0.12 0.00 0.00 0.00 0.00 2.74 1.92 0.98 0.14

Atheriniformes 1.25 0.75 3.74 0.11 0.00 0.00 0.00 0.00 1.37 0.96 3.76 0.11

Cyprinodontiformes 35.63 24.63 74.86 69.30 35.71 8.33 49.99 37.86 35.62 29.33 74.98 66.01 Order Perciformes excluding Gobiidae 8.75 6.72 7.24 2.39 7.14 1.67 0.66 0.30 8.90 8.17 7.27 2.44 Family Gobiidae 1.25 0.75 1.52 0.06 0.00 0.00 0.00 0.00 1.37 0.96 1.53 0.06

Unidentified Fish 3.13 2.99 2.25 0.32 0.00 0.00 0.00 0.00 3.42 3.85 2.27 0.37

124

Table 3-12. Morisita’s index of similarity calculated for several pairings of Pike Killifish and early-juvenile Common Snook. Index values were calculated based on co-occurrence of Pike Killifish and ontogenetic diet shifts of both Snook and Pike Killifish for all identified prey categories and prey functional groups. This index ranges from 0 to 1 with values near 0 being indicative of little dietary overlap and values near 1 being indicative of complete diet overlap. Values greater than 0.6 are typically considered to be indicative of a high degree of dietary overlap. Early-juvenile Common Common Common Common Common Snook (≤35 Snook (>35 Common Pike Killifish Early-juvenile Snook (≤35 Snook (>35 Snook with mm SL) with mm SL) with Snook ≤ 35 ≤ 30 mm SL Common mm SL) and mm SL) and and without and without and without mm SL and and >30 mm Snook and Pike Killifish Pike Killifish Pike Killifish Pike Killifish Pike Killifish Comparison >35 mm SL SL Pike Killifish (≤30 mm SL) (>30 mm SL) co-occurring co-occurring co-occurring Morisita's index of similarity: all prey categories 0.33 0.03 0.13 0.22 0.11 0.80 0.80 0.73

Morisita's index of similarity: prey functional groups 0.45 0.09 0.40 0.99 0.14 0.99 0.99 0.92

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Table 3-13. Multivariate abundance tests comparing prey abundance, calculated based on total length (TL) size groups, between locations with and without Pike Killifish co-occurring. Prey abundances that are significantly altered by the presence of Pike Killifish are denoted in bold. NA represents a prey group that was not collected in the sampling. Prey abundance from bag seine samples based on TL size Prey abundance from minnow seine samples based on groups TL size groups Group Deviance p-value Group Deviance p-value Overall 12.40 0.02 Overall 8.59 0.05 Atheriniform ≤30 mm TL 0.00 1.00 Atheriniform ≤15 mm TL 0.00 1.00 Atheriniform >30/≤45 mm TL 0.00 1.00 Atheriniform >15/≤30 mm TL 0.00 1.00 Clupeiform ≤30 mm TL 0.00 1.00 Clupeiform ≤15 mm TL NA NA Clupeiform >30/≤45 mm TL 1.55 0.50 Clupeiform >15/≤30 mm TL 0.86 0.62 Cyprinodontiform ≤30 mm TL 3.10 0.01 Cyprinodontiform ≤15 mm TL 0.00 1.00 Cyprinodontiform >30/≤45 mm TL 1.55 0.50 Cyprinodontiform >15/≤30 mm TL 3.73 0.19 Gobiidae ≤30 mm TL 0.00 1.00 Gobiidae ≤15 mm TL 0.00 1.00 Gobiidae >30/≤45 mm TL 0.00 1.00 Gobiidae >15/≤30 mm TL 0.00 1.00 Perciform ≤30 mm TL 1.55 0.50 Perciform ≤15 mm TL 0.00 1.00 Perciform >30/≤45 mm TL 3.10 <0.01 Perciform >15/≤30 mm TL 0.00 1.00 Shrimp ≤30 mm TL 1.55 0.50 Shrimp ≤15 mm TL 0.00 1.00 Shrimp >30/≤45 mm TL 0.00 1.00 Shrimp >15/≤30 mm TL 4.00 0.19

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Table 3-14. Multivariate abundance tests comparing prey abundance, calculated based on prey maximum depth (MD) size groups, between locations with and without Pike Killifish co-occurring. Prey abundances that are significantly altered by the presence of Pike Killifish are denoted in bold. Prey abundance from bag seine samples based on MD size Prey abundance from minnow seine samples based on groups MD size groups Group Deviance p-value Group Deviance p-value Overall 12.40 0.01 Overall 3.16 0.66 Atheriniform ≤5 mm MD 0.00 1.00 Atheriniform ≤5 mm MD 0.00 1.00 Atheriniform >5/≤10 mm MD 0.00 1.00 Atheriniform >5/≤10 mm MD 0.00 1.00 Clupeiform ≤5 mm MD 1.55 0.49 Clupeiform ≤5 mm MD 0.74 0.59 Clupeiform >5/≤10 mm MD 1.55 0.49 Clupeiform >5/≤10 mm MD 0.00 1.00 Cyprinodontiform ≤5 mm MD 1.55 0.49 Cyprinodontiform ≤5 mm MD 2.36 0.58 Cyprinodontiform >5/≤10 mm MD 3.10 0.01 Cyprinodontiform >5/≤10 mm MD 0.00 1.00 Gobiidae ≤5 mm MD 0.00 1.00 Gobiidae ≤5 mm MD 0.00 1.00 Gobiidae >5/≤10 mm MD 0.00 1.00 Gobiidae >5/≤10 mm MD 0.00 1.00 Perciform ≤5 mm MD 0.00 1.00 Perciform ≤5 mm MD 0.00 1.00 Perciform >5/≤10 mm MD 3.10 <0.01 Perciform >5/≤10 mm MD 0.00 1.00 Shrimp ≤5 mm MD 1.55 0.49 Shrimp ≤5 mm MD 0.07 0.82 Shrimp >5/≤10 mm MD 0.00 1.00 Shrimp >5/≤10 mm MD 0.00 1.00

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Figure 3-1. Satellite image of Tampa Bay, FL with insets of standardized sampling locations for early-juvenile Common Snook without Pike Killifish co-occurring: un-named tributary of the Manatee River (2012) and Braden River (2012- 2013).

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Figure 3-2. Satellite image of Tampa Bay, FL with inset of standardized sampling locations for early-juvenile Common Snook without Pike Killifish co-occurring: Frog Creek (also known as Terra Ceia River) (2013).

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Figure 3-3. Satellite image of the un-named Manatee River tributary in Tampa Bay, FL, one of the standardized sampling locations for early-juvenile Common Snook without Pike Killifish co-occurring (sampled during 2012): The red lines represent the boundaries between the 12 fixed sampling sites (~100 m shoreline length). The yellow line is a 100 m scale bar.

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Figure 3-4. Satellite image of Frog Creek (Terra Ceia River) in Tampa Bay, FL, one of the standardized sampling locations for early-juvenile Common Snook without Pike Killifish co-occurring (sampled during 2013). A) Full extent of Frog Creek with sampling area (B) denoted by the red square. B) Inset of the sampling area in Frog Creek. The red lines represent the boundaries between the 12 fixed sampling sites (~100 m shoreline length). The yellow line is a 100 m scale bar.

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Figure 3-5. Satellite image of Braden River in Tampa Bay, FL, one of the standardized sampling locations for early-juvenile Common Snook without Pike Killifish co- occurring (sampled during 2012 and 2013). A) Full extent of the tidal portion of the Braden River with sampling areas denoted by the red squares. B-D) Insets of the sampling areas in the Braden River. The red lines represent the boundaries between the 12 fixed sampling sites (~100 m shoreline length). The yellow lines are 100 m scale bars.

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80 A 70 60 50 40 30

Unique Prey Items Prey Unique 20 10 0 0 200 400 600 800 1000 Stomachs Analyzed

50 B 40

10 Unique Prey Items Prey Unique

0 0 25 50 75 100 125 150 Stomachs Analyzed

Figure 3-6. Cumulative prey curves for early-juvenile Common Snook (A) and Pike Killifish (B) collected from Tampa Bay tidal tributaries.

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140 A 120 100 80 60

Prey TL (mm) Prey 40 20 0 0 20 40 60 80 100 120 140 Common Snook SL (mm)

140 B 120 100 80 60

Prey TL (mm) Prey 40 20 0 0 20 40 60 80 100 120 140 Pike Killifish SL (mm)

Figure 3-7. Total length (TL) of prey consumed by early-juvenile Common Snook (A) and Pike Killifish (B) as a function of standard length (SL). The solid line represents a 1:1 ratio between prey TL and predator SL.

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12 A 10

2 Prey Maximum Depth (mm) Depth Maximum Prey 0 0 2 4 6 8 10 12 Common Snook Gape Width (mm)

12 B 10

2 Prey Maximum Depth (mm) Depth Maximum Prey 0 0 2 4 6 8 10 12 Pike Killifish Gape Width (mm)

Figure 3-8. Maximum depth (MD) of prey consumed by early-juvenile Common Snook (A) and Pike Killifish (B) as a function of gape width (GW). The solid line represents a 1:1 ratio between prey MD and predator GW, which should approximate the maximum prey size that can be consumed based on gape limitations.

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A All

100

80 80

60 60

40 40

20 20

0 20 40 60 80 100 Prey Functional Groups

B ≤35 mm SL

100

80 80

60 60

40 40

20 20

0 20 40 60 80 100

C >35 mm SL

100

80 80

60 60

40 40

20 20

0 20 40 60 80 100 %N Figure 3-9. Three-dimensional representation of early-juvenile Common Snook diet composition in terms of percent frequency of occurrence (%O), percent numerical abundance (%N), and percent weight (%W) based on functional prey groups: A) All Snook, B) Snook ≤35 mm SL, and C) Snook >35 mm SL.

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4000 3500 3000 2500 2000 1500 1000

Crustaceans in in Diet Crustaceans 500 Number ofPlanktonicNumber

5-10 (3) 5-10

10-15 (7) 10-15 (9) 85-90

80-85 (14) 80-85 15-20 (47) 15-20 (67) 45-50 (55) 50-55 (42) 55-60 (35) 60-65 (26) 65-70 (20) 70-75 (20) 75-80 (10) 90-95 (7) 95-100

25-30 (157) 25-30 20-25 (172) 20-25 (196) 30-35 (128) 35-40 (105) 40-45 Standard Length (5 mm Bins)

Figure 3-10. Number of planktonic organisms consumed by early-juvenile Common Snook, from Tampa Bay tidal tributaries, based on size. Sample size for each size bin is in parenthesis.

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0.8 NPK BS prey/m2 NPK PS prey/m2 SNK w/o PK Diet 0.6 A

and%N 0.4 2 2

#/m 0.2

Shrimp

iformes

Atherin-

Gobiidae

iformes

Non-Goby

Perciformes Cyprinodont- Clupeiformes 0.8 PK BS prey/m2 PK PS prey/m2 SNK w/PK Diet 0.6 B

and%N 0.4 2

#/m 0.2

Shrimp

iformes

Atherin-

Gobiidae

iformes

Non-Goby

Perciformes

Cyprinodont- Clupeiformes

0.8 PK BS prey/m2 PK PS prey/m2 PK Diet 0.6 C

and%N 0.4 2

#/m 0.2

Shrimp

iformes

Atherin-

Gobiidae

iformes

Non-Goby

Perciformes Cyprinodont- Clupeiformes

Figure 3-11. Average relative abundance of fish and shrimp prey functional groups (≤45 mm TL) sampled with the bag seine (BS) and minnow seine (MS) with (PK) and without (NPK) Pike Killifish present and percent numerical abundance (%N) of these prey groups for: A) Common Snook without Pike Killifish present, B) Common Snook with Pike Killifish present, and C) Pike Killifish. Percent numerical abundance for each prey group was calculated only for the prey groups graphed.

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A All

100

80 80

60 60 %O

40 40

20 20

0 20 40 60 80 100 Prey Functional Groups

B ≤30 mm SL

100

80 80

60 60

40 40

20 20

0 20 40 60 80 100

C >30 mm SL

100

80 80

60 60 %O

40 40

20 20

0 20 40 60 80 100 %N Figure 3-12. Three-dimensional representation of Pike Killifish diet composition in terms of percent frequency of occurrence (%O), percent numerical abundance (%N), and percent weight (%W) based on functional prey groups: A) All Pike Killifish, B) Pike Killifish ≤30 mm SL, and C) Pike Killifish >30 mm SL.

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30 25 20 15 10 5

Number ofPlanktonicNumber

Crustaceans ConsumedCrustaceans

5-10 (0) 5-10

10-15 (2) 10-15 15-20 (2) 15-20 (4) 20-25 (8) 25-30 (2) 30-35

90-95 (16) 90-95 35-40 (10) 35-40 (16) 40-45 (13) 45-50 (17) 50-55 (38) 55-60 (36) 60-65 (58) 65-70 (50) 70-75 (42) 75-80 (40) 80-85 (23) 85-90

95-100 (10) 95-100 (4) 105-110 (3) 110-115 (5) 115-120 (2) 120-125 (2) 125-130 100-105 (19) 100-105 Standard Lenght (5 mm bins)

Figure 3-13. Number of planktonic organisms consumed by Pike Killifish, from Tampa Bay tidal tributaries, based on size. Sample size for each size bin is in parenthesis.

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CHAPTER 4 POTENTIAL INTERFERENCE COMPETITION AND SPACE RESOURCE COMPETITION BETWEEN PIKE KILLIFISH AND EARLY-JUVENILE COMMON SNOOK

Introduction

Early-juvenile Snook utilize shallow estuarine backwaters and tributaries that are typically lined by mangroves and emergent marsh vegetation, which not only provide a low-energy habitat with ample food, but also provide cover and therefore a reduced risk of predation. This species, which is important both ecologically and economically, has experienced long periods of being designated as overfished (Taylor and Muller 2012,

2013; Muller et al. 2015). In addition to actual fishing mortality, habitat loss, particularly juvenile habitat, and episodic large-scale natural mortality events, such as cold kills and red tides, are believed to contribute to periods of it being designated as “overfished”

(Taylor and Muller 2012, 2013; Muller et al. 2015). Additional stressors have the potential to further reduce populations or delay recovery after large natural mortality events.

Non-native Pike Killifish are found in relatively high densities in the same areas of

Tampa Bay, FL, where early-juvenile Snook densities are typically the highest

(McMichael et al. 1989; Peters et al. 1998 a; Stevens et al. 2007; MacDonald et al.

2010; Greenwood 2017) (Figure 1-3). Within these areas, Pike Killifish utilize the same backwaters and tidal tributaries that are the preferred habitat of early-juvenile Snook

(McMichael et al. 1989; Peters et al. 1998 a; Stevens et al. 2007; MacDonald et al.

2010; Greenwood 207), which has raised concerns about potential predation on and competition with early-juvenile Snook (MacDonald et al. 2010).

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Predation of Snook by Pike Killifish would result in a direct loss of individuals, while competition with Snook could lead to reduced growth and condition, as well as increased predation. Interspecific competition may take the form of exploitative competition or interference competition (Connell 1983; Schoener 1983; Mills et al.

2004). Exploitation competition involves individuals using (competing for) the same resources, such as food items, which are limiting in some way (e.g., reduced prey availability) (Connell 1983; Schoener 1983). Interference competition involves the production of toxins in sessile organisms, fighting, and other aggressive behaviors

(Connell 1983; Schoener 1983). Some cases of competition, such as for space, may involve components of both types of competition (i.e., space is a resource and thus exploitation competition is involved, but competition for space often involves aggression and thus interference competition is involved). The presence of two fish species that preferentially utilize a particular habitat can result in one species or the other utilizing a secondary and potentially less desirable habitat, this type of pattern has been observed to occur within two relatively common estuarine goby species found in Florida (Schofield

2003).

Pike Killifish are capable of consuming relatively large early-juvenile Snook but appear to rarely if ever consume them within Tampa Bay tidal tributaries (Chapter 2), and there is little evidence of food resource competition between these two species

(Chapter 3). However, exploitative competition for space and interference competition in the form of aggression (e.g., chasing Snook out of cover) could result in increased predation of juvenile Snook by other predators such as wading birds, larger juvenile and adult Common Snook, as well as other predaceous fishes. It could also lead to reduced

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growth or condition due to extra energy expenditures. The microhabitat usage of these two species has not been well-studied, particularly in relation to one another in the context of space resource competition.

Field studies investigating competition can be used to identify if competition is occurring and producing a prominent effect, but they may not be able to identify if the competition is exploitative or interference in nature (i.e., growth studies, such as those detailed in Chapter 5) or may only reveal one type of competition (i.e., diet studies, such as those detailed in Chapter 3). Lab studies, such as experimental aquarium habitats, may be useful in further identifying the type and magnitude of competition present. Both experimental tank studies and mesocosms have been used to investigate competitive effects between species with some focusing on measuring growth and condition over pre-defined durations (e.g., Chapter 5) and others using short term observations of behaviors and interactions (Mills et al. 2004; Brooks and Jordan 2010; Martin et al.

2010).

This study aims to investigate the potential for space resource competition and interference competition between Pike Killifish and early-juvenile Snook through the use of observations in an experimental aquarium habitat. Additionally, potential food resource competition, or the lack of, was further investigated by analyzing prey consumption of Snook and Pike Killifish in the aquarium habitat.

Methods

This study was conducted at the University of Florida’s FAS facility in Gainesville,

FL. Pike Killifish (59-95 mm SL) and early-juvenile Common Snook (52-90 mm SL) were collected from tidal tributaries on the eastern shore of Tampa Bay, FL (Figure 2-1) and

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prey items (Mosquitofish and grass shrimp) were collected on site at FAS ponds. Study organisms were acclimated and maintained under the same lab conditions described in

Chapter 2.

The experimental aquarium habitat consisted of a 150-gal (567.8 L) aquarium that was half-filled (i.e., a water depth of ~30 cm). There was ~50-75 mm of sand sediment distributed across the bottom. Cover (live Black Needlerush) was placed in the back 1/3 of the aquarium (~150-200 mm) while the remaining 2/3 of the aquarium had no cover (Figure 4-1). A set of mirrors attached to a wooden frame with a 45° angle was placed on top of the aquarium to allow for simultaneous viewing of fish position both vertically in the water column and along the horizontal plane (Figure 4-1). Two PVC frames holding 1.3 mm window screening and lined by weather stripping (to form a tight connection with the aquarium walls) were used to divide the aquarium into 3 equal sections along its length.

Treatments consisted of a control with 6 early-juvenile Common Snook and an experimental treatment with 3 Common Snook and 3 Pike Killifish. The control treatment served as a baseline for early-juvenile Snook habitat use and behavior in the absence of Pike Killifish but at the same fish density. Twelve potential prey items (6

Mosquitofish and 6 grass shrimp) were also present in each replicate. Each treatment was replicated 12 times. Pike Killifish and Snook were fed ad libitum during the first 1.5 hours of the light cycle. Fish were placed in the aquarium between 21:30 and 22:30

(after the end of the light cycle). Snook, Pike Killifish, and prey items were randomly selected from their holding tanks and placed directly into the aquarium. Prey were placed in the middle section, 3 Snook were placed in both the left and right sections for

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the control treatment, and each species (Snook and Pike Killifish) was placed separately on either the left or right section for the experimental treatment. The side in which each species was placed was alternated for each replicate. The following morning after the start of the light cycle the dividers were removed and the aquarium was recorded for 1 h with a digital camera. At the end of a replicate, Snook and Pike

Killifish were euthanized with buffered MS-222 (300 mg/L). Their stomachs were then removed and examined to determine what prey items they had consumed.

The first 2 minutes of each replicate was used as a settling period. The entire video for each replicate was viewed and any aggressive behaviors, both intra- and interspecific, were noted. A random 3600 points (10% of the video) was selected. The same set of points was used for each video after the initial random selection. At each of these points, each fish was designated as being in cover (in the cover or within 1 body length of it) or in the open, as well as being either near the surface (top half of the water column) or near the bottom (bottom half of the water column). The total proportion of time spent in cover, the proportion of time spent near the bottom, and the proportion of prey (as well as proportion of each individual prey type) consumed was calculated for

Snook and Pike Killifish for each trial. These data were arcsine square-root transformed and analyzed with an ANOVA with Tukey post-hoc tests (α = 0.05) to compare differences between Pike Killifish and Snook both with and without Pike Killifish present.

Results

No aggressive behaviors of Pike Killifish towards early-juvenile Common Snook were noted in any of the replicates or vice-versa. On several occasions, there were instances of intraspecific aggression among Pike Killifish but not Snook. These

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aggressive behaviors either involved a male aggressively courting a non-receptive female (chasing and bumping beyond typical courting) or larger males chasing and bumping/nipping smaller males. In the case of aggressively courting males, this occurred in three separate replicates. In two of the replicates there was more than one instance of aggressive courting, but in each case this only lasted for a few seconds. In the case of larger male Pike Killifish chasing smaller males, this behavior was only noted in 1 replicate and occurred on and off throughout the duration of that replicate.

Pike Killifish in the presence of Snook spent significantly less time near the bottom of the aquarium than Snook, both with and without Pike Killifish present (Table

4-1 and Figure 4-2). Snook without Pike Killifish present spent significantly more time near the bottom of the aquarium than Snook with Pike Killifish present; however, the actual difference in proportion of time near the bottom was relatively small (Figure 4-2).

Pike Killifish with Snook present and Snook with Pike Killifish present spent significantly less time in cover than Snook without Pike Killifish present, but the amount of time spent in cover between Pike Killifish with Snook present and Snook with Pike Killifish present was not significantly different (Table 4-1 and Figure 4-3).

The proportion of prey consumed by Snook both with and without Pike Killifish present was significantly higher than the proportion consumed by Pike Killifish with

Snook present, and there was no significant difference in the proportion consumed by the two different Snook groups (Table 4-1 and Figure 4-4). The proportion of mosquitofish consumed mirrored the results for all prey items consumed (Table 4-1 and

Figure 4-5). The proportion of grass shrimp consumed by Snook without Pike Killifish present was significantly greater than the proportion of grass shrimp consumed by Pike

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Killifish with Snook present (Table 4-1 and Figure 4-6). The proportion of grass shrimp consumed by Snook with Pike Killifish present was not different from either the Pike

Killifish with Snook or the Snook without Pike Killifish. The same results were maintained after adjusting the proportion of prey (proportions halved) consumed for the

Snook treatment without Pike Killifish present (6 Snook in this group compared to the experimental treatment with 3 Snook and 3 Pike Killifish) (Table 4-1 and Figures 4-4, 4-

5, and 4-6). In general, after adjustment the two Snook groups had more similar proportions of prey consumed, with the Snook with Pike Killifish present group having a greater but not significantly different proportion of prey consumed for all prey and mosquitofish.

Discussion

This study points towards a lack of interference competition between Pike Killifish and early-juvenile Common Snook, as there were no incidences of fighting or other aggressive behaviors (chasing, nipping, etc.) between these two species. There was some evidence of interference competition among Pike Killifish, which most often took the form of males aggressively courting un-responsive females. This involved excessive chasing and occasional nipping beyond typical courting behaviors, which consist of males positioning themselves in front of and perpendicular to females, changing color slightly, and fanning their fins and gonopodium (Horth 2004). Beyond this aggressive courting, males often spent much of their time courting females. The amount of time devoted towards mating behaviors among the Pike Killifish could be one possible reason there were not any aggressive behaviors towards Snook noted in this study (i.e., too busy/concerned with mating rather than chasing/attacking Snook). If this is the case,

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it is probable that in the field mating behaviors also take up much of adult Pike Killifish’s time as they reproduce year round.

Intraspecific interference competition among Pike Killifish was also observed between larger and smaller males. In these interactions, larger males would repeatedly chase smaller males around the aquarium and occasionally nip at them. This occurred both when a larger male was courting a female, which has been noted in previous studies (Horth 2004), and when a large male was not actively courting a female. It has been noted in the past that captive Pike Killifish may set up a pecking order based on size that can occasionally lead to death due to excessive chasing and nipping (Belshe

1961). This chasing and nipping behavior among different-sized individuals was personally observed in the field on a few occasions during the collection of specimens for this study. Such behaviors have the potential to decrease the survivorship of the smaller individuals through increased energy expenditures and increased exposure to predation by wading birds and larger fish when the smaller individuals are chased into more open waters. This behavior could in part account for some of the cannibalism among sub-adult and adult Pike Killifish that has been observed (Chapters 2 and 3).

No intraspecific interference competition among juvenile Snook was noted during the course of this study and it has not been previously noted in other studies. A few minor incidences of one individual briefly chasing another have been personally observed both in captive and wild individuals, however, these behaviors were short and not repetitive as was the case with Pike Killifish. The general lack of interference competition among juvenile Snook likely occurs because they are juveniles rather than adults and do not need to defend potential mates or territories as is the case with adult

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Pike Killifish. There could still be interference competition for cover among juvenile

Snook, however, they tend to be gregarious, and school which may further lessen the predation on any one individual (Pitcher 1986; Magguran 1990).

This study also provides further evidence that Pike Killifish are not competing with juvenile Snook for food resources. The proportion of prey consumed by Snook was not significantly different between treatments with and without Pike Killifish present while both of these Snook treatments consumed significantly more prey than Pike

Killifish in the presence of Snook. If there were a negative impact based on food resource competition, it would appear that Snook may limit Pike Killifish’s food resources based on this study. However, a few aquarium habitat replicates with 6 Pike

Killifish and no Snook were conducted (a full analysis of these replicates was not completed due to low sample size), and in these replicates Pike Killifish also consumed very few prey items. There are several possible explanations for the low level of prey consumed by Pike Killifish in the aquarium habitats. Upon viewing the videos, Pike

Killifish often were unsuccessful in their attempts at consuming prey, but they still generally attacked prey less than the Snook. The Pike Killifish utilized in this study were all adults or sub-adults, while the Snook were juveniles. Diet analysis suggests that early-juvenile Common Snook fed often throughout the day as there was a low percentage of empty stomachs. Pike Killifish, on the other hand, had a high percentage of empty stomachs (>60%) and appeared to feed less often (Chapter 3). This likely relates to the greater energetic needs of the juvenile fish to grow quickly to reduce predation risks compared to adults. It may also be related to the reproductive focus of the adult Pike Killifish. Pike Killifish in the aquarium habitat would also often be

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“spooked” and dart to the bottom or into the cover when another fish (either a conspecific or a juvenile Snook) attacked a prey item on the surface. The juvenile

Snook displayed this reaction far less often. This likely relates to Pike Killifish’s surface dwelling habits and a need to avoid predation from wading birds and other predators.

In the wild, however, Snook consume a far wider number of prey resources and appear to select for the most abundant prey, which are often taxa that are rarely if ever consumed by Pike Killifish, which have a narrower prey base and appear to be more selective in their prey consumption (Chapter 3). This information would generally indicate that it is unlikely that early-juvenile Snook are negatively impacting Pike Killifish.

Some intraspecific prey resource competition among Snook likely exists and may reduce growth in some cases, as some minor but non-significant differences in growth of Snook at different densities has been noted in experimental enclosures (Chapter 5).

Relatively few of the grass shrimp prey were consumed by Snook during the course of this study and none by Pike Killifish. This prey item was regularly consumed by both species in Tampa Bay estuaries (Chapter 3). The lack of grass shrimp consumption by Pike Killifish may relate to the grass shrimp density in the aquarium habitat compared to the wild. In locations where Pike Killifish consumed grass shrimp, grass shrimp densities could be quite large over small spatial scales and were often observed near the surface on shoreline vegetation or among flotsam. In contrast, there were only 6 grass shrimp in each replicate, and these shrimp remained near the bottom most of the time. Grass shrimp may also have simply been a less desirable prey item for Pike Killifish (Belshe 1961; Miley 1978; Greven and Brenner 2008; Harms 2011;

Chapter 3). On a few occasions, Pike Killifish in the aquarium habitat did attempt to

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consume grass shrimp that were swimming near the surface (typically after the shrimp evaded a Snook attack) or among the Black Needlerush near the surface. Snook consumption of grass shrimp may have been relatively low for a number of reasons. It is possible that mosquitofish are a more desirable prey item for early-juvenile Snook, although diet composition and electivity indices do not appear to support this (Chapter

3). A combination of low shrimp density (compared to the wild) and blanching of the shrimp due to the clear water and sand bottom (e.g., hard to see) may have simply resulted in low encounter rates between the Snook and grass shrimp. This blanching could have also been a contributing factor to Pike Killifish not consuming grass shrimp in the aquarium habitats. Snook could have become satiated after consuming the more readily visible mosquitofish, or the grass shrimp may have been better at evading Snook attacks when they were encountered.

There also appears to be a lack of space resource competition between Pike

Killifish and early-juvenile Snook despite their shared preference for backwater tidal tributary areas within Tampa Bay (Greenwood et al. 2008; MacDonald et al. 2010;

Greenwood 2012, 2017). This study indicates that each species tends to occupy separate microhabitats. Pike Killifish with Snook present spent significantly less time near the bottom (i.e., more time near the surface) than Snook with and without Pike

Killifish present. The difference in amount of time spent near the bottom between the two Snook treatments does not appear to be a result of Pike Killifish presence as Pike

Killifish spent more time near the surface and would not displace Snook from the bottom to the surface. It is more likely that this arose in part from Snook following Pike Killifish to the surface (i.e., grouping with similar-sized fish despite not being the same species)

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and the greater number of Snook in the treatment without Pike Killifish consuming the majority of the mosquitofish prey (generally stayed near the surface) quicker than the

Snook in the treatment with Pike Killifish which had fewer individuals. There also appears to be separation in the use of cover between these species, with Snook utilizing cover significantly more than Pike Killifish when Pike Killifish did not co-occur.

Snook still spent a greater proportion of time in cover than Pike Killifish when the two species co-occurred in the aquarium, it was just not a significant difference. Again, the difference in the amount of time spent in cover by Snook with and without Pike Killifish co-occurring does not appear to have occurred because Pike Killifish displaced the

Snook. As described above, there were no aggressive behaviors of Pike Killifish towards Snook and Pike Killifish spent relatively little time in cover compared to Snook making displacement unlikely. Similar to the case with surface versus bottom time, it appears that Snook with Pike Killifish present may have spent more time in open water either because they were schooling with (following) the Pike Killifish or spent more time in the open chasing prey (i.e., took more time for three Snook to consume the majority of prey present compared to 6 Snook).

There may be some biases in this study as there were no large predators present to influence the distribution of these two species in the experimental aquarium habitat, and predation can be a driving factor in the distribution of small-bodied and juvenile fishes (Mittelback 1988; Werner and Hall 1988; Walter and Juanes 1993). The inclusion of larger predators would have necessitated a larger experimental aquarium or tank, which would have made it much more difficult to track the position of each individual

Snook and Pike Killifish throughout time, largely due to their relatively small size. The

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lack of large predators also simulates a low-tide situation in many of the backwater areas where these species co-exist in Tampa Bay, as the larger fish predators are often forced to leave the backwaters during low tides due to reduced area, depth, and dissolved oxygen, while early-juvenile Snook and Pike Killifish can remain due to their small size and tolerance of low dissolved oxygen levels (Hensley and Courtenay 1980;

Page and Burr 1991; Peterson and Gilmore 1991; Peterson et al. 1991; Brennan 2008).

Personal observations of wild Pike Killifish indicate that in low-tide scenarios they behave in a similar manner, with small groups located near the surface often along the edge of shoreline vegetation/cover but not actually within the cover. In many cases, they were observed a meter or more from the shoreline vegetation, especially when wading birds were not present. Whereas, on high tides, Pike Killifish are difficult to find and observe, likely because they have retreated deeper into the shoreline vegetation (cover) to avoid predation by larger piscivores, such as adult Snook and Ladyfish Elops saurus.

Observations and sampling with seine nets generally show the same pattern with juvenile Snook, where on low tides they remain near the bottom but tend to spread out with some individuals being found in cover and others in more open areas often forming schools with conspecifics and other similar-sized individuals of different species. On higher tides they tend to retreat into or near cover, whether it be in the form of shoreline vegetation or bottom cover such as dead branches. It is likely that even though during high tide or in the presence of predators these species appear to both be found in or near cover (based on personal observations), there would still be little to no interference competition or space resource competition as the vertical separation between the two species appears to remain. The focus during these periods is more likely on avoiding

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predation and consuming prey that may have previously been out of reach than defending a spatial territory.

This separation in microhabitat usage helps to explain the lack of food resource competition between Snook and Pike Killifish (Chapter 3). The surface-dwelling nature of Pike Killifish results in them feeding almost exclusively on surface dwelling organisms

(including terrestrial organisms falling onto the water’s surface) and benthic organisms that spend a large portion of their time near the surface, especially when they are present in large numbers (e.g. grass shrimp) (Chapter 3). Snook, with their superior mouth orientation, can ambush surface dwelling prey but also consume a wide variety of pelagic and benthic prey items. Both species also tend to be gregarious (both with themselves and other similar-sized fish) with aggressive behaviors generally being limited to adult male Pike Killifish aggression towards conspecifics. The lack of interference competition and space resource competition between these two species also points towards a lack of increased predation on early-juvenile Snook displaced from cover by Pike Killifish.

Interference competition, in the form of aggression, and space resource competition are often cited as negative impacts of non-native fishes, and there is increasing evidence that this does actually occur in many situations (Cucherousset and

Olden 2011; Almeida and Grossman 2012; Ricciardi et al. 2013). This often involves species that have been noted to be territorial both as juveniles and as adults that may defend a mating territory or nest, such as sunfishes and tilapias (Brooks and Jordon

2010; Martin et al. 2010; Almeida and Grossman 2012). Aquarium and tanks studies of tilapias have shown that they may displace native sunfishes from cover (Brooks and

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Jordon 2010; Martin et al.). However, in these artificial settings territoriality is often increased (Oldfield 2011). The fact that Pike Killifish showed no aggressive behaviors towards Snook in the confines of an aquarium setting where territoriality is often the greatest lends further support to the fact that it is highly unlikely that they would show aggression toward Snook in the wild. Species that are not typically thought of a defending a particular territory can also show a high degree of aggression that can result in displacement, direct mortality from attacks, and increased predation.

Mosquitofishes Gambusia spp. are a prime example and will repeatedly attack similar sized and often larger fishes of different species that occupy the same habitat.

Introduced populations of mosquitofish have been shown to have direct negative impacts on a variety of native small-bodied fishes and larval amphibians through predation and aggressive behaviors (Mills et al. 2004; Pyke 2008; Almeida and

Grossman 2012). Within their native range mosquitofish have actually been cited as a source of biotic resistance to small-bodied native fish introductions (Thompson et al.

2012; Tuckett et al. 2017). Pike Killifish are livebearers and thus do not make nests that need to be defended and they do not form mating territories. Despite their closest taxonomic relative being mosquitofish (Rosen and Bailey 1963), Pike Killifish do not appear to show the same high levels of territoriality and aggression. Based on the separation of microhabitat usage and general lack of aggression in Pike Killifish, it appears that there is little to no concern for interference competition and space resource competition with early-juvenile Snook. Furthermore, their separation in microhabitat usage helps explain the lack of food resource competition between these two species.

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Table 4-1. Pairwise comparisons of the proportion of time spent near the bottom, proportion of time spent in cover, and proportion of prey consumed by early-juvenile Common Snook without Pike Killifish present (SNK_NPK), early-juvenile Common Snook with Pike Killifish present (SNK_PK), and Pike Killifish with early-juvenile Common Snook present (PK_SNK) in an experimental aquarium habitat.

SNK_NPK SNK_NPK SNK_PK Proportion vs vs vs Compared Overall SNK_PK PK_SNK PK_SNK Time near <0.001 0.0340 <0.001 <0.001 Bottom

Time in Cover <0.001 <0.001 <0.001 0.210

Prey Consumed <0.001 0.217 <0.001 <0.001

Adjusted Prey <0.001 0.525 0.002 <0.001 Consumed

Mosquitofish <0.001 0.513 <0.001 0.004 Consumed Adjusted Mosquitofish 0.001 0.309 0.041 0.001 Consumed Grass Shrimp 0.003 0.063 0.002 0.394 Consumed Adjusted Grass Shrimp 0.013 0.300 0.009 0.236 Consumed

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45° Mirror Cover (Black Needlerush)

Water Depth ~30 cm Sediment Depth ~50-75 mm

Figure 4-1. Experimental aquarium habitat used to assess the potential interference competition and space resource competition between Pike Killifish and early- juvenile Common Snook. Photo taken by author.

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A B C

6 Snook Treatment 3 Snook/3 Pike Killifish Treatment Group

Figure 4-2. Average proportion (with 95% confidence intervals) of time spent near the bottom by early-juvenile Common Snook without Pike Killifish present (SNK_NPK), early-juvenile Common Snook with Pike Killifish present (SNK_PK), and Pike Killifish with early-juvenile Common Snook present (PK_SNK) in experimental aquarium habitat replicates. Significant differences are denoted by different letters.

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A B B

6 Snook Treatment 3 Snook/3 Pike Killifish Treatment

Group

Figure 4-3. Average proportion (with 95% confidence intervals) of time spent in cover by early-juvenile Common Snook without Pike Killifish present (SNK_NPK), early-juvenile Common Snook with Pike Killifish present (SNK_PK), and Pike Killifish with early-juvenile Common Snook present (PK_SNK) in experimental aquarium habitat replicates. Significant differences are denoted by different letters.

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AA AA BB

Mean ± 95%CI Adjusted Mean ± 95%CI

6 Snook Treatment 3 Snook/3 Pike Killifish Treatment

Group

Figure 4-4. Average proportion (with 95% confidence intervals) of prey consumed by early-juvenile Common Snook without Pike Killifish present (SNK_NPK), early-juvenile Common Snook with Pike Killifish present (SNK_PK), and Pike Killifish with early-juvenile Common Snook present (PK_SNK) in experimental aquarium habitat replicates. An adjusted mean was calculated by halving the proportion of prey consumed by the SNK_NPK group to account the greater number of Snook in that treatment (6 vs. 3). Significant differences are denoted by different letters.

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AA AA BB

Mean ± 95%CI Adjusted Mean ± 95%CI

6 Snook Treatment 3 Snook/3 Pike Killifish Treatment

Group

Figure 4-5. Average proportion (with 95% confidence intervals) of Eastern Mosquitofish consumed by early-juvenile Common Snook without Pike Killifish present (SNK_NPK), early-juvenile Common Snook with Pike Killifish present (SNK_PK), and Pike Killifish with early-juvenile Common Snook present (PK_SNK) in experimental aquarium habitat replicates. An adjusted mean was calculated by halving the proportion of mosquitofish consumed by the SNK_NPK group to account the greater number of Snook in that treatment (6 vs. 3). Significant differences are denoted by different letters.

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AA ABAB BB

Mean ± 95%CI Adjusted Mean ± 95%CI

6 Snook Treatment 3 Snook/3 Pike Killifish Treatment

Group

Figure 4-6. Average proportion (with 95% confidence intervals) of grass shrimp consumed by early-juvenile Common Snook without Pike Killifish present (SNK_NPK), early-juvenile Common Snook with Pike Killifish present (SNK_PK), and Pike Killifish with early-juvenile Common Snook present (PK_SNK) in experimental aquarium habitat replicates. An adjusted mean was calculated by halving the proportion of grass shrimp consumed by the SNK_NPK group to account the greater number of Snook in that treatment (6 vs. 3). Significant differences are denoted by different letters.

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CHAPTER 5 POTENTIAL IMPACTS OF NON-NATIVE PIKE KILLIFISH ON EARLY-JUVENILE COMMON SNOOK ABUNDANCE, GROWTH, AND CONDITION

Introduction

Larval and juvenile fish tend to experience relatively high levels of mortality compared to adults, with much of this mortality being attributed to episodic losses, predation, and food availability (Houde 1987, 1989; Lorenzen 1996). Episodic losses may consist of aberrant drift of larvae, abnormal weather conditions, acute toxic events, and other similar events. Predation typically accounts for the greatest losses in small juveniles, while starvation is typically more of a concern in larval stages than post- settlement stages (Currin et al. 1984; Houde 1987, 1989; Kneib 1993). However, competition and predation risk can reduce the amount or quality of food being consumed and lead to poorer condition and slower growth (Werner et al. 1983; Werner and Gilliam 1984; Werner and Hall 1988; Sogard 1992, 1994, 1997; Walters and

Juanes 1993; Dahlgren and Eggleston 2000; Halpin 2000). In cases where growth is reduced, natural mortality due to predation may increase, as it takes a greater period of time for individuals to grow large enough to reach a size refuge from many of the predators that are common within nursery areas (Post and Evans 1989; Sogard 1992,

1997; Sogard and Able 1992; Levin et al. 1997).

Snook are a relatively large estuarine species found throughout much of Florida’s coastal waters. They compose a large portion of the inshore recreational fishery

(Mueller et al. 2015) and are an important predator within the estuarine waters of

Florida. The Gulf coast stock of Snook in Florida has been classified as being overfished for long periods of its management in Florida (Muller et al. 2015), and it is thought that losses of juvenile habitat have likely been an important factor in the

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reduced population size of this stock (Taylor and Muller 2012, 2013; Muller et al. 2015).

Early-juveniles (≤100 mm SL) may be the most impacted by reductions of nursery habitat due to urbanization, as this life stage has the most restricted habitat usage

(Peters et al. 1998a; Stevens et al. 2007; MacDonald et al. 2010). Further stressors to this life stage that result in reduced growth and/or increased predation have the potential to lead to further population reductions and delay recovery rates after large natural mortality events, such as cold kills and red tides.

Non-native fishes have been linked to declines in native fish populations both as a result of predation and competition (Vitule et al. 2009; Cucherousset and Olden 2011;

Ricciardi et al. 2013). Pike Killifish, a native to the Gulf coast drainages of Central

America, were introduced to south Florida in 1957 and quickly became established

(Belshe 1961). A second population of Pike Killifish was first documented in the estuarine water of Tampa Bay in 1994 (Greenwood 2017). This species is the largest member of the Poeciliidae, reaching lengths of 150 mm SL or more, and are noted to be piscivorous from birth (Belshe 1961; Miley 1978; Turner and Snelson 1984; Greven and

Brenner 2008). Both decreases in abundance and altered size structure of several small-bodied fish native to Florida have been linked to the presence of Pike Killifish

(Miley 1978; Greenwood 2012). Within Tampa Bay, Pike Killifish distribution and habitat preference overlaps that of early-juvenile Snook and both species are found in greatest abundance in the tidal tributaries and backwaters along the western shore of Tampa

Bay (Figure 1.3) (McMichael et al. 1989; Stevens et al. 2007; Greenwood et al. 2008;

MacDonald et al. 2010; Greenwood 2017). This information has led to concerns about

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the potential impact of Pike Killifish on early-juvenile Snook in Tampa Bay due to predation and competition (MacDonald et al. 2010; Greenwood 2012, 2017).

The goals of this study were to 1) determine if Pike Killifish presence had a negative impact on the growth of early-juvenile Common Snook through the use of field enclosures; 2) determine if early-juvenile Snook abundance and condition was lower in locations where Pike Killifish co-occurred compared to locations without Pike Killifish present; and 3) model theoretical impacts to the production of late stage juvenile Snook

(>100 mm SL) as a result of both increased natural mortality on early-juvenile Snook due to Pike Killifish predation and reduced growth of early-juvenile Snook due to competition with Pike Killifish.

Methods

Assessment of Growth Impacts

Experimental field enclosures were used to assess the potential for reduced early-juvenile Common Snook growth in the presence of Pike Killifish. Experimental enclosures consisted of a PVC frame measuring 3 m long x 1.2 m wide x 1.2 m high that was enclosed on 4 sides and the top by 9.5 x 9.5 mm plastic mesh (Figures 5-1 and

5-2). This mesh was small enough to prevent the treatment fish from escaping and excluded avian and larger fish predators while allowing most potential prey items to enter the enclosure. The bottom edge of the enclosure was lined by 0.25 m of galvanized flashing that was sunk into the substrate (Figure 5-2). This was done to allow treatment fish access to the substrate while excluding predators, such as Blue

Crab Callinectes sapidus, that could otherwise easily burrowed into the enclosures.

Rebar poles measuring 1.5 m were hammered into the sediment approximately half-

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way at each of the enclosure corners and attached to the enclosure to prevent movement during storms and periods of high current velocity.

Enclosures were placed along a shoreline of mixed Black Needlerush and Red

Mangroves in a backwater tidal tributary in Mill Bayou within the Little Manatee River,

Tampa Bay, FL (Figure 5-3). This location was selected because it was comprised of the appropriate habitat for both species, was within a water body already invaded by

Pike Killifish (required to prevent an accidental introduction into an un-invaded water if any fish escaped from the enclosure) and was in close proximity to a boat ramp allowing for safe and easy transport of the enclosures. After placing the enclosure in the field, seine net and dips nets were used to remove any Snook, Pike Killifish, other larger fish

(potential competitors), and Blue Crabs (predators) from each enclosure. A small seine was pulled through the enclosure confining fish and crabs on one end of an enclosure

Dip nets were then used to remove the fish and crabs from the confined area. This was repeated until a minimum of 5 successful seine passes resulted in no fish or crab being found in the enclosure.

Treatments consisted of either 3, 6, or 12 Snook, or 6 Snook and 6 Pike Killifish together, in each enclosure. The 6 Snook treatment was intended to serve as a control, as this density of Snook fell within abundance estimates from standardized sampling within optimal early-juvenile Snook habitat (Greenwood et al. 2008; MacDonald et al

2010). The Snook with Pike Killifish treatment was used to examine the effect of Pike

Killifish on early-juvenile Snook growth. The 12 Snook treatment was used to examine the effect of increased Snook density on Snook growth in comparison to the Snook with

Pike Killifish treatment (i.e., are any potential changes in growth due to the presence of

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Pike Killifish or simply due to an increase in competitors regardless of species). The 3

Snook treatment was used to assess whether the densities within the other treatments were too high and resulted in decreased growth even within the control treatment.

Each treatment was replicated five times, and there 5 separate enclosures.

Replicates of each treatment were placed into the enclosures using a completely randomized block design (i.e., each treatment was randomly assigned to an enclosure for each replicate without the same treatment being assigned to the same enclosure more than once). Pike Killifish and early-juvenile Common Snook utilized for this study were collected from locations outside of the standardized sampling areas described in

Chapters 2 and 3 (Figure 5-4) with cast nets (1.5 m radius with 4.8 mm mesh and 2.1 m radius with 6.4 mm mesh). These fish were held in aerated concrete tanks that measured 230 cm X 76 cm X 70 cm (l X w X h) and had a holding capacity of approximately 1080 L (located at the UF Tropical Aquaculture Lab in Ruskin, FL). These tanks were filled approximately 1/3 full of water (~360 L) and held at ambient air temperature and 5-10 ppt salinity. Fish were held in these tanks for a period of <72 hours prior to being placed in enclosures. During holding, Snook and Pike Killifish were fed daily ab libitum. Prey consisted of small native Poeciliidae captured on-site. For each replicate, treatment fish were randomly assigned to a treatment and measured with Vernier calipers (0.05 accuracy). Each fish was tagged with a coded wire tag in their caudal peduncle to identify each individual upon recapture (Brennan et al. 2005,

2007). Fish were then transported to the field enclosures in aerated holding containers and placed in their assigned enclosure. Fish remained in the enclosure for 10 days. This time period would be long enough for measurable growth increases based on previous

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studies of daily growth (McMichael et al. 1989; Aliaume et al. 2000) while preventing excess growth on the enclosure or prey depletion within them. After 10 days the Snook and Pike Killifish were removed and placed in an ice slurry to be euthanized (UF IFAS

Non-regulatory Animal Research Protocol 006-12FAS). At the end of a replicate, each fish was measured, had its stomach removed, and had its coded wire tag removed and read to identify each individual. Algal growth and detrital accumulation was removed from the enclosures between replicates by lightly brushing them.

Daily growth was calculated as the change in length from the beginning to the end of a replicate by the number of days a fish was in the enclosures. The average daily growth of fish from each replicate for each treatment was compared with a two-way analysis of variance (ANOVA) with the treatment and enclosure (the blocking factor) as factors. The presence of stomach contents was also noted for each fish. The average proportion of non-empty Snook stomachs (arcsine square-root transformed) was compared with a two-way ANOVA with treatment and enclosure as factors. Statistical significance was determined at the α = 0.05 level.

Assessment of Abundance and Condition Impacts

To assess the potential for reduced abundance and condition of early-juvenile

Common Snook from locations with Pike Killifish co-occurring, standardized sampling was conducted in two locations both with and without Pike Killifish co-occurring with

Snook. The same standardized sampling (locations, methods, captured fish) detailed in

Chapters 2 and 3 were utilized for this study. The abundance of early-juvenile Snook and Pike Killifish for each standardized seine was calculated both in terms of area (m2)

(for comparison with other studies) and shoreline sampled (m). Abundance based on

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the length of shoreline sampled was calculated because it may be a better metric for

Snook and Pike Killifish as they tend to orient to shore and not open water. Additionally, multiple shorelines may be sampled with one seine haul in the small tidal tributaries and backwaters sampled in this study. Due to violations in normality and homogeneity of variance that were not resolved with commonly used data transformations, a Kruskal-

Wallis (nonparametric ANOVA) was used to compare early-juvenile Snook abundances between each sampling location (α = 0.05). Dunn’s test was applied for post-hoc pairwise comparisons. Pike Killifish abundances were also compared between the two locations where they occur using a t-test (α = 0.05) as the data did not violate normality and homogeneity of variance assumptions. Pike Killifish were noted to regularly jump out of the seine net upon retrieval and occasionally swim between net floats on the surface. Individuals that were seen escaping the net were enumerated, and an adjusted abundance was calculated in addition to the number actually captured.

To compare the condition of early-juvenile Snook from locations with and without

Pike Killifish co-occurring, an analysis of covariance (ANCOVA) was performed. Snook weights between locations with and without Pike Killifish were compared with SL as a covariate. Weight and SL data were log transformed to linearize their relationship and correct for heteroscedasticity prior to the ANCOVA analyses. Weight, with SL as a covariate, of Snook from each separate location were also compared with an ANCOVA and Tukey HSD post-hoc test to determine if a particular location accounted for any observed differences.

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Modelling Theoretical Impacts

The production of late stage juvenile Snook (>100 mm SL) within a cohort was used to assess the potential impacts of Pike Killifish on early-juvenile Common Snook through increased natural mortality (predation) and decreased growth (competition).

This was a theoretical exercise exploring the potential for reduced Snook production if

Pike Killifish were to have negative impacts on juvenile Snook, as current data from this study and others (Chapters 2 and 3), indicate that Pike Killifish do not appear to prey upon or compete with juvenile Snook. A simple population model was created in R

(Appendix) that utilized daily growth and natural mortality to estimate the number of early-juvenile Snook remaining at each daily time step until their length exceeded 100 mm SL (i.e., were no longer early-juveniles).

An initial arbitrary value of R0, the initial size of the cohort recruiting to a tidal tributary after settlement from the larval stage, was set to 5000. Common Snook typically recruit to nursery areas after approximately 2.5 weeks (Peters et al. 1998a).

Based on this information, the initial age used in the model was 19 days. Standard length at age (in days) was determined from an equation modified for early-juvenile

Snook growth in Tampa Bay (McMichael et al. 1989). The equation in this study was only valid for ages up to approximately 220 days, after which the equation actually predicted negative growth as it was only fit to data for fish younger than this. The equation utilized in the model needed to predict length at older ages, as the modeled decreases in daily growth rate result in a greater number of days for individuals to exceed 100 mm SL. The equation from McMichael et al. (1989) was nearly linear for ages of 40-200 days. A linear approximation of this part of the equation was developed as SL = 0.89338*A – 26.916 (R2 = 0.999), where SL is the predicted standard length

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and A is a fish’s age in days (Figure 5-5A). Predicted length values using the previous equation for ages 195-250 days (in 5-day increments) were added to the predicted values from McMichael et al. (1989) for ages 20-190 (in 5-day increments). A polynomial equation, similar to the approach taken by McMichael et al. (1989), was then fit to these data yielding the equation used to predict standard length in the model: SL =

0.0000001*A4 – 0.00006*A3 + 0.0125*A2 – 0.1621*A + 2.084 (R2 = 0.999) (Figure 5-5B).

There are no direct measures of natural mortality in juvenile Snook, however, general estimates based on adult mortality and fish size have been developed. Natural mortality at size (M) was estimated as: M = ((Mmat * Lmat)/L (Taylor et al. 2013), where

Mmat is the natural mortality at maturity, Lmat is the length at maturity, and L is a length at a particular time, in this case the SL at each daily time step. Values of Mmat and Lmat were set at 0.25 yr-1 and 200 mm SL, respectively (Taylor et al. 2000; Mueller et al.

2015). The value from this equation was divided by 365 to estimate the daily natural mortality at each age based on the estimated size at age (MD,A). To simulate Pike

Killifish predation at age, daily natural mortality was adjusted to incorporate a Pike

Killifish “exploitation rate” (U) on juvenile Snook: Mpk,A = (MD,A + (MD,A * U * Usize,A), where Usize,A is a vector incorporating the probability that a Snook could be consumed by a Pike Killifish at a particular age based on its size as determined from the logistic regressions in Chapter 2 (Table 5-1). Pike Killifish “exploitation rates” ranged from 0 to 1 in 0.5 increments. Survival at age (SA) from one day to the next was calculated as: SA =

푒−푀푝푘,퐴.

To calculate the number of individuals remaining at age (NA), the number from the previous time step was multiplied by the survival for that age: NA = NA-1 * SA-1. To

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simulate reduced growth due to the presence of Pike Killifish, the SL equation was multiplied by a scalar representing percent decreased in daily growth of 0-50% in 5% increments. For each simulation of altered growth, the maximum age and Usize,A were adjusted appropriately. The number of fish reaching 100 mm SL for each combination of increased natural mortality and decreased growth was determined and expressed as a percent reduction compared to the base scenario with no simulated Pike Killifish impacts. These values were visualized with a contour plot to demonstrate the relative reduction of late stage juvenile production under each model scenario.

Results

Growth Impacts

Snook utilized in the enclosure study ranged in size from 60 to 103 mm SL and

Pike Killifish ranged in size from 71 to 122 mm SL upon initial placement in the enclosures. Daily growth of Snook recovered from the enclosures was 0.00-0.62 mm/day. Three individuals were not recovered for growth analysis: one was dropped in the water in transferring it from the enclosure to the holding container; one escaped through a small hole in a depression made in an enclosure corner while removing the treatment fish; and one was not found. These three fish all came from different replicates (2 from 12 Snook replicates and one from a 3 Snook replicate). Another

Snook (from a 6 Snook replicate) was not initially recovered but recovered at a later date and excluded from the growth analysis. No significant differences were detected in

Snook growth between the different enclosures (F = 0.466, p = 0.969) indicating that the blocking treatment worked as intended (i.e., no single enclosure had higher or lower

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growth based on its location). There were also no significant differences in the average daily growth of early-juvenile Snook between the different treatments (F = 0.128, p =

0.711). Average daily growth of Snook in the presence of Pike Killifish was most similar to the 3 Snook and 6 Snook treatments (Figure 5-6). In general, Snook growth decreased, though not significantly, as Snook density increased (Figure 5-6).

As with the growth data, there was no significant difference in the mean proportion of Snook containing stomach contents between the different enclosures (F =

0.334, p = 0.850) (i.e., no effect of the blocking factor). No significant difference in the mean percentage of Snook containing stomach contents by treatment (F = 1.029, p =

0.414) was detected. All fish contained stomach contents in the 3 Snook treatment, while this value ranged between 80% and 100% for the replicates in all the other treatments with one exception (Figure 5-7). One of the 6 Snook/6 Pike Killifish treatments only had 2 fish (33%) containing stomach contents (Figure 5-7).

Abundance Impacts

There were no significant differences between locations that were sampled in both 2012 and 2013, allowing these samples to be pooled for abundance analysis.

Mean Snook abundance by location based on area ranged from 0.02 individuals/m2 to

0.18 individuals/m2, and mean abundance based on shore length ranged from 0.10 individuals/m of shoreline to 0.94 individuals/m of shoreline (Table 5-2). One seine haul from the Alafia River in 2012, was excluded from the analysis because of an exceptionally large number of early-juvenile Snook (284) being captured due to an extremely low tide that had drained a large portion of a marsh into a deeper hole along the edge of the mainstem river (result of the statistical tests were the same both with

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and without this outlier included). The Kruskal-Wallis test indicated that there was a significant difference in the abundance of early-juvenile Snook based both on area (H =

17.735, p = 0.001) and shore length (H = 17.223, p =0.002) between locations. Dunn’s post-hoc tests revealed that the abundance of early-juvenile Snook captured in the un- named tributary of the Manatee River were significantly lower than all of the other sampling locations (Table 5-3 and Figure 5-8). There were no other significant differences in the mean early-juvenile Snook abundance between any of the other sampling locations.

As with the Snook data, abundance in 2012 and 2013 did not differ significantly for Pike Killifish, and the data were pooled for abundance analysis. Mean Pike Killifish abundance by area was 0.03 and 0.04 individuals/m2 in Wildcat Creek (Little Manatee

River) and Alafia River backwaters, respectively (Table 5-4). Mean abundance by shore length was 0.17 individuals/m of shoreline in Wildcat Creek and 0.23 individuals/m of shoreline in the backwaters of the Alafia River (Table 5-4). Pike Killifish abundance did not differ significantly between Wildcat Creek and the Alafia River backwaters for area (t

= 0.474, p = 0.638) or shore length (t = 0.819, p = 0.418) (Table 5-4 and Figure 5-9).

Mean adjusted Pike Killifish abundance based on area sampled did not differ significantly (t = 0.289, p = 0.774) between locations, with means of 0.05 individuals/m2 for both locations (Table 5-4 and Figure 5-9A). This was also true for abundance based on shoreline (t = 0.732, p = 0.469). Wildcat Creek had a mean abundance of 0.24 individuals/m of shoreline and the Alafia River had a mean of 0.31 individuals/m of shoreline sampled (Table 5-4 and Figure 5-9B).

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Condition Impacts

The interaction term of SL and Pike Killifish presence (F = 65.9, p = <0.001), as well as the Pike Killifish presence factor in the ANCOVA were both significant (F = 22.8, p = <0.001); this prevented the main effects being examined independently. In general, however, weight-at-length was greater for smaller individuals from locations without

Pike Killifish co-occurring, while larger individuals tender to have greater weight from locations with Pike Killifish present. However, when the actual data points and linear models were plotted they were nearly indistinguishable from one another (Figure 5-10).

A similar pattern was seen in the ANCOVA with location as a covariate (F = 26.1, p =

<0.001) with a significant interaction (F = 13.9, p = <0.001). The significant interaction factor makes post-hoc tests unreliable. Visual inspection of the data points and their linear models for each location showed that they were nearly indistinguishable with no clear patterns (Figure 5-11).

Theoretical Impacts

The number of days to exceed 100 mm SL increased steadily with each 5% reduction in daily growth rate. Starting with an increase of 5 days at a 5% reduction and exceeding a 75% increase in days (111 more than initial value) at a 50% reduction in daily growth (Table 5-5). Moderate levels of Pike Killifish predation (up to 0.4 exploitation rate) on early-juvenile Snook produced relatively small reductions (<10%) in late stage juvenile Snook production (number of individuals). And even at the greatest levels of predation modeled, the reduction in late stage juveniles remained below 50% as long as growth was also not reduced (Figure 5-12). A similar pattern occurred with reduced growth wherein even at relatively large percentage reduction in growth (up to

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35%), late stage juvenile production would not be reduced by more than 50% as long as there were no impacts of Pike Killifish predation. However, smaller reductions in Snook growth yielded a greater impact than modeled Pike Killifish predation (e.g., a 10% reduction in Snook growth resulted in a >10% reduction in individuals that survived to become late stage juveniles (Figure 5-12). A combination of modeled Pike Killifish predation and reduced growth from competition resulted in greater reductions than either negative impact on its own. At the extremes of the scenarios modeled for both predation and reduced daily growth, production of late stage juveniles could be reduced by nearly 100% (Figure 5-12); however, based on the other findings in this study such extreme scenarios would appear to be highly unlikely.

Discussion

In general, it would appear that Pike Killifish have little effect on the abundance, growth, or condition of early-juvenile Common Snook within tidal tributaries of Tampa

Bay. Snook growth within the field enclosures was relatively variable, and the mean growth rates were, in general, lower than expected based on previous studies of early- juvenile Snook growth (McMichael et. 1989; Aliaume et al. 2000). This may have been a result of there being an initial period of slow growth as fish adapted to the enclosure setting, with some individuals adapting quicker than others as some individual’s growth rates fell within the previously reported range of 0.4 – 1.0 mm/day.

It is possible that there were also enclosure artifacts present, which resulted in different growth than wild fish (Sogard and Able 1992; Kneib 1993). The height of the shoreline vegetation along with its dense root system, coupled with the extensive size of an enclosure that would be needed to enclose the shoreline while remaining in the

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water during low tides prevented direct shoreline access by the study fish. The enclosures were placed as close as possible to the shoreline vegetation. This, combined with the size of mesh required to keep the experimental fish in the enclosure, may have prevented Snook access to some prey items. Grass shrimp and cyprinodontiform fish were observed entering the enclosure, and small early-juvenile

Snook actually settled in the enclosures on several occasions; but schooling pelagic prey items such as Bay Anchovy may have avoided the enclosures. There were also few places to rest from the current during ebbing and flooding tides without access to the shoreline vegetation, although the currents in the enclosure locations were generally slow and fish could utilize the flashing along the bottom edge of the enclosure as a current break. If such enclosure artefacts were present (even if they were restricted to only some of the enclosures), they would have been experienced by all of the treatments due to the completely randomized block structure of the experiment. It would be expected that if Pike Killifish were to have an impact on Snook growth, it would have still been present with the Snook-Pike Killifish treatment having a lower average growth rate, which was not the case.

Although it was not significant, the mean growth of Snook from the enclosure study tended to decrease as Snook density increase, which would indicate that a greater number of conspecifics may have a greater impact on juvenile Snook than Pike

Killifish. In a review of competition studies, it was found that in most cases where inter- and intraspecific competition occurred, the intraspecific competition was greater than the interspecific competition (Connell 1983).

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There were no apparent differences in the abundance of early-juvenile Snook in any of the study locations with the exception of the un-named tributary of the Manatee

River. This site had lower abundances of early-juvenile Snook than all of the other sampling locations. The low catches in this tributary could have been related to being located too far upstream, relatively strong currents in several areas of this tributary, a greater abundance of larger juvenile Snook, poor recruitment to this particular area, or some other unknown reason. These low catches prompted this site to be replaced with

Frog Creek in the second year of the study (as detailed in Chapter 3).

The lack of difference in Snook abundance among locations with and without

Pike Killifish co-occurring would point towards a lack of Pike Killifish predation on early- juvenile Snook. This was also supported by the diet analyses of Chapters 2 and 3 that found no Snook in the diet of Pike Killifish. Some of the seine hauls with the greatest number of early-juvenile Snook, including a large outlier that was removed from analysis, came from locations where Pike Killifish co-occurred. Similar results have been noted in previous studies as well (Greenwood et al. 2008; MacDonald et al. 2010).

Early-juvenile Snook weight-at-length (condition) had a significant interaction term between length and Pike Killifish presence, which prevents the main effects from being examined independently. It appears that weight-at-length was slightly greater for smaller individuals from locations without Pike Killifish co-occurring while larger individuals tender to have greater weights from locations with Pike Killifish. However, the weight-at-length relationship overlapped between both groups (with and without

Pike Killifish) over the entire size range examined. The results from the ANCOVA analysis for each separate sampling location also had a significant interaction term and

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there was no clear pattern of difference in the weight-at-length among the different locations with overlap across the entire size range analyzed. This would generally suggest that Pike Killifish are not having a significant impact on early-juvenile Snook condition and the minor differences in the models may merely be due to the large sample sizes (over 500 fish both from locations with and without Pike Killifish present) involved in the analysis.

The theoretical modelling of Pike Killifish impacts on late stage juvenile Snook production due to predation (increased mortality) and competition (reduced growth) showed that small to moderate changes in either of these parameters will result in moderate reductions in production. Larger changes, especially if larger changes in both parameters occur, can result in substantial losses to the production of larger juvenile

Snook. Reduced growth, especially with larger changes, had a greater effect than the increased natural mortality due to Pike Killifish. This likely occurred because Snook essentially escape possible Pike Killifish predation when they reach 60 mm SL (too large for adult Pike Killifish to consume), while reduced growth exposes fish to a higher level of natural mortality (including that of Pike Killifish when it is applied) for a greater period of time. Given the results of this study as well as the data from Chapter 2 and 3, it appears that Pike Killifish currently exert essentially no negative impacts on early- juvenile Snook. For such impacts to become realized, it is likely that Pike Killifish abundance would have to increase substantially to reduce their prey base enough that they started regularly consuming juvenile Snook. It is questionable whether increased

Pike Killifish abundance would result in reduced Snook growth, as Chapter 3 revealed that although Snook regularly consume the same prey items as Pike Killifish, they also

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feed heavily on other organisms that are not consumed by Pike Killifish. Even if Pike

Killifish abundance increased, it is likely that they would still not consume many of these prey items due to their distribution (e.g., benthic invertebrates and pelagic schooling fish). It would generally appear that Pike Killifish are also more likely to be cannibalistic than to consume Snook (Chapters 2 and 3). Other studies have also noted that cannibalism in Pike Killifish occurs on occasion (Belshe 1961; Miley 1978; Anderson

1980; Greenwood et al. 2008).

Overall, there currently seem to be negligible impacts of Pike Killifish on early- juvenile Snook abundance, condition, and growth in Tampa Bay tidal tributaries, and the potential for impacts to occur in the future seems minimal.

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Table 5-1. Estimated percent consumption of juvenile Common Snook by Pike Killifish based on standard length. Reported length is the maximum size that would experience a particular percentage of consumption. % Consumption Standard Length 0.9 13.60 0.8 21.06 0.7 26.04 0.6 30.05 0.5 37.51 0.4 41.52 0.3 46.50 0.1 53.88 0.0 100.00

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Table 5-2. Mean abundance of early-juvenile Common Snook with 95% confidence intervals from tidal tributaries of Tampa Bay, FL. Individuals/m2 Individuals/m shoreline Location Lower Mean Upper Lower Mean Upper Alafia River 0.08 0.17 0.27 0.39 0.82 1.24 Braden River 0.12 0.18 0.24 0.59 0.94 1.30 Frog Creek 0.06 0.18 0.30 0.22 0.71 1.21 Wildcat Creek 0.08 0.13 0.18 0.36 0.58 0.80 Un-named Tributary 0.01 0.02 0.04 0.05 0.10 0.15 (Manatee River)

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Table 5-3. Mean abundance of early-juvenile Common Snook from different locations based on area and shore length sampled in Tampa Bay, FL. Locations without Pike Killifish co-occurring: BR = Braden River, FC = Frog Creek, and MR = un-named Manatee River tributary. Locations with Pike Killifish co-occurring: AR = Alafia River, WC = Wildcat Creek. Bold indicates a significant difference using Dunn’s post-hoc tests. Individuals/m2 Individuals/m shoreline

AR BR FC WC AR BR FC WC BR 1.000 - - - BR 1.000 - - - FC 1.000 1.000 - - FC 1.000 1.000 - - WC 1.000 1.000 1.000 - WC 1.000 1.000 1 - MR 0.013 0.001 0.011 0.011 MR 0.012 0.001 0.032 0.014

Table 5-4. Mean abundance of early-juvenile Pike Killifish with 95% confidence intervals from tidal tributaries of Tampa Bay, FL. Individuals/m2 Individuals/m shoreline Location Lower Mean Upper Lower Mean Upper Alafia River 0.02 0.04 0.05 0.09 0.23 0.37 Wildcat Creek 0.02 0.03 0.05 0.08 0.17 0.26

Adjusted Individuals/m 2 Adjusted Individuals/m shoreline Location Lower Mean Upper Lower Mean Upper Alafia River 0.03 0.05 0.07 0.14 0.31 0.47 Wildcat Creek 0.03 0.05 0.07 0.13 0.24 0.34

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Table 5-5. Estimated number of days for Common Snook to exceed 100 mm standard length under varying degrees of reduced daily growth. % Decrease Days 0 143 5 148 10 155 15 162 20 171 25 181 30 192 35 205 40 220 45 237 50 254

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Figure 5-1. Experimental enclosure placed in a small tributary of Mill Bayou in the Little Manatee River, Tampa Bay, FL. Photo taken by author.

PVC Frame

9mm x 9mm Plastic Mesh

Galvanized Flashing

Figure 5-2. Experimental enclosure design as viewed from the top. Photo taken by author.

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Figure 5-3. Location of experimental enclosures within Mill Bayou in the Little Manatee River, FL.

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Figure 5-4. Collection locations in Tampa Bay, FL of early-juvenile Common Snook and Pike Killifish used in the enclosure study.

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200 y = 0.89338x - 26.916 180 R² = 0.999 160 140 120 100 80 McMichael et al. 1989 60

Standard Length (mm) LengthStandard 40 Linear approximation 20 of McMichael et al. 1989 0 0 50 100 150 200 250 Age (Days)

200 180 y = 1E-07x4 - 6E-05x3 + 0.0125x2 - 0.1621x + 2.084 R² = 0.999 160 140 120 100 McMichael et al. 1989 80

60 Linear approximation of McMichael et al. 1989 Standard Length (mm) LengthStandard 40 20 Polynomial regression used for model 0 0 50 100 150 200 250 Age (Days)

Figure 5-5. Development of the equation used for daily growth of early-juvenile Common Snook in the population model: A) estimated values from McMichael et al. 1989 for 40-200 days old (in 5 day increments) and linear approximation of these data and B) estimated values from McMichael et al. 1989 for 20-190 days old (in 5 day increments), estimated values from the linear approximation of McMichael et al. 1989 for 195-250 days old (in 5 day increments), and polynomial regression used in the population model.

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Figure 5-6. Mean daily growth rate (with 95% confidence intervals) of early-juvenile Common Snook under different treatments in experimental enclosures.

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Figure 5-7. Boxplot of the mean proportion of early-juvenile Common Snook containing stomach contents under different treatments in experimental enclosures. The solid black line represent the median, the upper and lower edges of the box represent the 25th and 75th percentiles, the whisker represent 1.5 times the interquartile range, and open dots represent outliers.

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Pike Killifish No Pike Killifish

Figure 5-8. Mean abundance of early-juvenile Common Snook from different sampling locations in Tampa Bay, FL: A) based on area and B) based on length of shoreline. AR = Alafia River, WC = Wildcat Creek, BR = Braden River, FC = Frog Creek, and MR = un-named Manatee River tributary.

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Figure 5-9. Mean abundance and adjusted abundance of Pike Killifish from different sampling locations in Tampa Bay, FL: A) based on area and B) based on shore length. AR = Alafia River, AR-adj = Alafia River adjusted for escapes, WC = Wildcat Creek, and WC = Wildcat Creek adjusted for escapes.

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Figure 5-10. Weight-at-length relationship for early-juvenile Common Snook from locations with (PK) an without (NPK) Pike Killifish co-occurring: A) Weight- length data, B) linear model of weight-length data, and C) linear model of weight length data without data points.

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Figure 5-11. Weight-at-length relationship for early-juvenile Common Snook based on sampling location: A) linear model of weight-length data and B) linear model without data points. Locations with Pike Killifish: AR = Alafia River and WC = Wildcat Creek, locations without Pike Killifish: BR = Braden River, FC = Frog Creek, and MR = un-named Manatee River tributary.

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0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05

0.00 in Growth Daily ReductionProportion

0.15 1.00 0.95 0.90 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.10 0.05 0.00 Pike Killifish "Explotation Rate" (U)

% Reduction in Number of Late Stage Juvenile Common Snook 0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 90-100

Figure 5-12. Contour plot of late stage juvenile Common Snook (exceeding 100 mm SL) production (# of fish) from a single cohort within a tidal tributary under varying reductions in daily growth and simulated Pike Killifish predation.

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CHAPTER 6 CONCLUSION

The studies conducted in this dissertation have revealed that Pike Killifish in the tidal tributaries of Tampa Bay pose little to no impact on early-juvenile Common

Snook based on Pike Killifish predation trials and diet; diet analysis and overlap of Pike

Killifish and Snook; assessment of interference and space resource competition between Pike Killifish and Snook in aquarium habitats; and assessment of growth, abundance, and condition of Snook with and without Pike Killifish present.

Pike Killifish are capable of consuming Snook up to 60 mm in total length, but rarely if ever consume them in the wild. There is relatively little diet overlap between these two species. This is attributable to the relatively narrow diet of Pike Killifish, which feeds almost exclusively on surface dwelling organisms (especially cyprinodontiform fish), terrestrial organisms that fall onto the water’s surface, or organisms that are regularly found near the water’s surface when they occur in large abundances, such as grass shrimp. In comparison, early-juvenile Snook feed on a wide variety of organisms throughout the water column, and although their diet is dominated by a few taxa, their diet appears to largely be determined by the abundance of those prey items. There was a notable reduction in the abundance on cyprinodontiform fish from locations where

Pike Killifish were present compared to locations without Pike Killifish. The reduction was reflected in the diet of early-juvenile Snook; however, the overall overlap of Snook diet from locations with and without Pike Killifish co-occurring was quite high. Snook will readily consume cyprinodontiform fish when they are present, and they have been noted as a large part of juvenile Snook diet in a number of previous studies (Fore and

Schmidt 1973; McMichael et al. 1989; Aliaume et al. 1997; Peters et al. 1998 a; Adams

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et al. 2009; Rock 2009). However, it appears that when cyprinodontiforms are not present Snook will readily consume other common fish species such as Bay Anchovy,

Clown Goby, and mojarra, which also represent high energy prey items. This information points towards a low likelihood of reduced cyprinodontiform abundance due to Pike Killifish predation resulting in reduced Snook condition or growth in most cases.

Analysis of aquarium habitat videos has revealed that Pike Killifish do not appear to show aggressive behaviors toward early-juvenile Common Snook and that these two species general occupy different microhabitats. Snook were typically more associated with the bottom and available cover and Pike Killifish were located near the surface and quite often in open water adjacent to available cover. This separation in microhabitat usage helps explain the differences noted their diets and the lack of competition between these species.

The abundance and growth of early-juvenile Snook was not found to be reduced in the presence of Pike Killifish. The weight-at-length models for Snook from locations were significantly different; however, this difference was not discernable upon visual inspection and likely arose simply due to the large sample sizes in each group. As such this difference has no biological significance. Population modelling of simulated Pike

Killifish impacts on early-juvenile Snook through predation (increased natural mortality) and competition (reduced growth) showed that large reductions (>50%) in the production of late stage juvenile Snook (>100 mm SL) required relatively large simulated impacts from Pike Killifish. Such large impacts would seem highly unlikely given the lack of negative impacts of Pike Killifish on Snook found in Chapters 2-5. More moderate impacts (10-50% reductions) were observed at lower levels of impact,

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especially for a reduction in early-juvenile Snook growth. However, even these more moderate impacts are not likely to occur in most circumstances given the findings of

Chapters 2-5, particularly within Tampa Bay.

In their native range, the Atlantic drainages of Central America, Pike Killifish coexist with Snook in estuarine waters, with no apparent impact of Pike Killifish on

Snook, although no one has studied their interactions in these locations. It is quite possible that the long co-evolutionary history of these species together in Central

America may have long ago sorted out any major competition between them (i.e., there may have been competition in the past but each species co-evolved to occupy slightly different ecological “niches”. Connell (1980) argued that such a “ghost of competition past” likely does not exist and that species likely diverged separately long ago on their on evolutionary paths without the influence of interspecific competitors. While more recent research has found at least some evidence of such previous interactions shaping current ecological patterns of co-existing species (Crowder 1984; Pritchard and Schluter

2001). Regardless, Snook from Florida, would have been naïve to Pike Killifish and there could have been some type of negative impacts since Snook in Florida had existed for such a long period without the presence of Pike Killifish or a similar species.

Taking this evidence as a whole, Pike Killifish do not appear to impact early- juvenile Snook in Tampa Bay tidal tributaries. This is not to say that there may not be small isolated areas within Tampa Bay where there could be negative impacts of Pike

Killifish on early-juvenile Snook, such as small ponds that are only tidally connected for brief periods of time throughout the year. In such areas, the number of available prey items is likely lower and there is less replenishment from outside sources making the

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potential for prey depletion and competition or forced predation more likely. However, habitats such as these are relatively uncommon in Tampa Bay with most of them being located outside of the current range of Pike Killifish within Tampa Bay. These habitats are more common and often utilized by early-juvenile Snook in Charlotte Harbor

(Stevens et al. 2007) and likely other locations throughout the state, and the potential for another future introduction to a currently uninvaded estuary is always a possibility.

Pike Killifish populations in Tampa Bay, both on the western shore and the

Pinellas peninsula, have deep hardened shorelines on the edges of their current ranges. This appears to be preventing further spread of Pike Killifish along the coastal margin of Tampa Bay (Greenwood 2017). However, the potential for human assisted movement (bait bucket release) or a new introduction (aquarium release) has the potential to spread this species to new areas of the bay and is the likely avenue of introduction to the invaded tidal tributaries of the Pinellas peninsula (Figure 1-3). Spread across drainage basins via flooded headwaters during years of high rainfall or following tropical storms is also a potential vector for future spread around the bay.

Despite the lack of impacts on early-juvenile Snook, Pike Killifish do still cause ecological impacts within the tidal tributaries of Tampa Bay. This is most notable in the reduction of small to medium sized cyprinodontiform fish, Mosquitofish in particular.

Even if reductions in these fishes do not appear to impact juvenile Snook directly there are still possible impacts that could arise. Mosquitofish are noted to be highly aggressive and are thought to potentially serve as a biological control preventing the establishment of many small bodied non-native fish through predation and harassment

(Thompson et al. 2012; Tuckett et al. 2017). They also have the potential to reduce the

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establishment of larger non-native fish by preying upon eggs and juveniles and harassing juveniles. The lack of mosquitofish in Tampa Bay tidal tributaries invaded by

Pike Killifish could lead to the establishment or increased abundance of other non- native fishes that could potentially have impacts on early-juvenile Snook or other fishes that utilize these areas throughout different times of the year.

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APPENDIX R CODE FOR POPULATION MODEL

#Late Stage Juvenile Common Snook Production Model with Base Parameterization rm(list=ls(all=TRUE))

Amax=143 Mmat=0.25 U=0.1 Ro=5000

iso=function(U){ Usize=vector(length=(125));Usize[1:24]=(U*0.9);Usize[25:35]=(U*0.8);Usize[36:42]=(U* 0.7); Usize[43:47]=(U*0.6);Usize[48:51]=(U*0.5);Usize[52:56]=(U*0.4);Usize[57:60]=(U*0.3); Usize[61:66]=(U*0.2);Usize[67:74]=(U*0.1);Usize[75:125]=0 Age = seq(19,Amax) SL=((0.0000001*Age^4)-(0.00006*Age^3)+(0.0125*Age^2)-(0.1621*Age)+2.084)*1 M=((Mmat*200/SL)/365) Mpk=(M+(U*M*Usize)) S=exp(-Mpk)

Number=vector(length=(125));Number[1]=Ro;for(i in 2:125) {Number[i]=Number[i-1]*S[i-1]}

FinalN=Number[125]}

Uvec=seq(0,1,length=21) Umatrix=matrix(NA,length(1),length(Uvec)) for(i in 1:length(Uvec)) { tmp = iso(Uvec[i]) Umatrix[,i]=max(0,tmp)}

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BIOGRAPHICAL SKETCH

Geoffrey H. Smith Jr. was born in Sarasota, Florida. He grew up in this same city and spent much of his younger years at sea on his parent’s commercial grouper fishing vessel. The time spent with his parents on their vessel is what started a lifelong love of the sea. His interest with the ocean and all of its organisms grew as he did. Geoffrey became an avid fisherman and aquarist, who collected most of his own specimens. He attended Sarasota High School and graduated 2002. While in high school he spent the majority of his summer vacation volunteering for the Stock Enhancement Program at

Mote Marine Laboratory. He continued this work for two summers after his high school graduation as a sub-contractor.

In the fall of 2002 Geoffrey began his undergraduate studies at New College of

Florida. While at New College he worked as a lab assistant at the Pritzker Marine

Biology Research Center. He also acted as a teaching assistant for a number of classes and co-taught a field course on local marine fauna. Geoffrey’s studies at New College culminated in a thesis focusing on spatial mapping in an intertidal fish species. In 2006 he earned his B.A. in marine biology. Upon graduating he continued to work at New

College as a lab assistant at the Pritzker lab and for the chemistry department.

In 2008, Geoffrey began work as a lab technician in both the Phlips lab and

Murie/Parkyn lab in the Program of Fisheries and Aquatic Sciences at the University of

Florida. His work in the Murie/Parkyn lab lead to a position as a M.S. graduate assistant in the fall of 2008. His thesis was concentrated on non-lethal sexing and sex ratio impacts on population dynamics in greater amberjack. In 2010, Geoffrey begin working on his Ph.D. in fisheries and aquatic sciences at the University of Florida, and he received his M.S. degree in 2011. His dissertation work was focused on the potential

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negative impacts of a non-native piscivore, the Pike Killifish, on early-juvenile Common

Snook. While working completing his doctoral degree, Geoffrey worked as a research assistant and late as an OPS biologist in the Murie lab. He worked on a variety of projects investigating migration and release mortality in Greater Amberjack; trophic relationships of fish between coral reefs, seagrasses, and mangrove shorelines in the

U.S. Virgin Islands; release mortality in Gag and Red Grouper; impacts of the

Deepwater Horizon spill on the growth of various commercially and recreationally important fish species; and most recently an assessment of ecological impacts of dredging sand banks off the east coast of Florida for beach re-nourishment.

After graduation, Geoffrey plans to pursue a teaching and research career at a small to moderate sized university where he will be able to work one on one with students while continuing to pursue his own research interests.

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