Impacts of Urbanization and River Water Contaminants on Abundance, Locomotion and Aggression of a Local Freshwater Crustacean

Impacts of urbanization and river water contaminants on abundance, locomotion and aggression of a local freshwater crustacean

Thesis by

José L. Ortiz Lugo

In Partial Fulfillment of the Requirements for the Degree

Doctor in Philosophy (Ph.D.)

Thesis Committee:

Dr. María A. Sosa Lloréns - Dissertation Advisor, UPR Medical Sciences Campus Dr. Jennifer L. Barreto-Estrada - UPR Medical Sciences Campus Dr. Alberto M. Sabat - UPR Río Piedras Campus Dr. Carlos I. González - UPR Río Piedras Campus Dr. José L. Agosto - UPR Río Piedras Campus

University of Puerto Rico Río Piedras and Medical Sciences Campus Department of Biology and Department of Anatomy and Neurobiology Intercampus Doctoral Program 2015

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COPYRIGHT

In presenting this thesis dissertation in partial fulfillment of the requirements for a degree Doctor in Philosophy-Biology at the University of Puerto Rico-Río Piedras and Medical Sciences Campus. I agree that the library shall make its copies freely available for inspection. I further agree that extensive copying of this dissertation is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this dissertation for commercial purposes, or for financial gain, shall not be allowed without my written permission.

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Dedicatory

First at all, I thank God for giving me spiritual strength and hope to keep going on. I dedicate this thesis to my special family, to my mother and father, Nilda Lugo Espinosa and William Ortiz Solis for being with me always in this adventure. I also dedicate this thesis to my brothers and sisters, Joel Ortiz Lugo who passed away on 1998, William Ortiz Lugo, Elsa I. Ortiz Lugo, Javier Ortiz Lugo, and Wilma I. Ortiz Lugo for their important help, love, affection, and believe in me. Finally to my eight nephews Valerie Ortiz, Emely Ortiz, Carelys E. Soto Ortiz, Keiralys E. Soto Ortiz, Keliel J. Soto Ortiz, Kimberly E. Soto Ortiz, Jeilian I. Serrano Ortiz and Isander Y. Serrano Ortiz. All this work is for you, thanks.

Acknowledgements

During my graduate studies in the University of Puerto Rico several persons and institutions collaborated directly and indirectly with my research. I wish to dedicate this section to acknowledge their support. I would especially like to thank my thesis advisor Dr. Maria A. Sosa Lloréns for the trust and giving me the opportunity to develop as a science professional in her laboratory. I will be always grateful. Also to my thesis committee, Dr. Alberto M. Sabat, Dr. Jennifer L. Barreto-Estrada, Dr. Carlos I. González and Dr. José L. Agosto for dedicating time, guidance, and supervision during this research. I want to thank everyone who participated in this project especially to the members of my lab; without their support it would have been impossible to complete the research. I would also like to thank people from the lab of Dr. Alonso Ramírez at the UPR-Río Piedras for their collaboration and important advices in this project. This research was supported by the NSF HRD-1137725 (CREST) and the PRCEN Graduate Fellowship.

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Table of Contents

Research Summary………………………………………………………………………. 6 General Introduction………………………………………………………………………8 Chapter 1: The effects of urbanizing watersheds on population assemblages of freshwater macro-invertebrates…………………………………………………………11 Methods………………………………………………………………………16 Results………………………………………………………………………. 20 Discussion…………………………………………………………………... 23 Conclusions…………………………………………….…………………....28 Appendix……………………………………………………………...….…..30 Literature cited...……………………………………….…………………....91

Chapter 2: The influence of water pollutants from urban streams on agonistic behaviors in a Puerto Rican native adult omnivorous prawn……..…………………99 Methods………………………………………………………..……………103 Results………………………………………………………………………107 Discussion…………………………………………………………………..109 Conclusions…………………………………………….…………………..114 Appendix……………………………………………………………...….…117 Literature cited...……………………………………….…………………..166

Chapter 3: Locomotor activity as a measure of the effect of urban anthropogenic chemicals on freshwater prawns……………………..…………………..………...…171 Methods………………………………………………………..…………….174 Results………………………………………………………………………177 Discussion…………………………………………………………………..178 Conclusions…………………………………………….…………………..183 Appendix……………………………………………………………...….…186 Literature cited...……………………………………….…………………..225

General Discussion……………………………………………………………………231

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Summary

Humanity today is experiencing a dramatic shift to urban living. Local urban environmental stressors and global ones are combining to accelerate the rates of degradation of different marine, terrestrial and freshwater ecosystems worldwide. In the tropical regions, especially in the Caribbean natural habitats can be disturbed by human activities often more rapidly and persistently. As natural landscapes are altered by human disturbances, the health of streams and their fauna are increasingly at risk. Puerto Rico is a small island with a high population density and growing rate of urbanism that is driving a continual conversion of land to anthropogenic uses, increasing the accumulation of contaminants in water resources. An increasing number of chemicals liberated into the environment through human activities have demonstrated potential for disruption of biological processes critical to normal growth and development of wild species. So far, it is not known if the introduction of different types of contaminants into water bodies and urban development has the potential of altering the physiology, behavior and nervous system of aquatic animals. Organism responses vary by assemblage group, and aquatic macro-invertebrates often show the highest sensitivity to urbanization. Our study species Macrobrachium carcinus is the largest freshwater prawn that inhabits the rivers of Puerto Rico. Therefore, since it is known that macro-invertebrates are sentinel species, sensitive to contaminants, and environmental variables, it is thus a good candidate to explore if water contaminants from urban rivers can affect population rates and different aspects of neural function, including activity, and interactive behaviors. We hypothesized that anthropogenic activities near Puerto Rican urban watersheds are associated with changes on relative population density, and impairment on interactive behaviors, and general activity of freshwater prawn species. Effects of environmental contaminants can be studied at various levels of organization, including biochemical and cell levels, the level of individual organisms, and the population and community level. The results from this project will contribute towards the knowledge base of our understanding on behaviors, neural responses and management of Puerto Rico stream prawn´s communities and ecosystems. To achieve this goal three primary research objectives were postulated: (1) quantitatively determine differences in the relative abundance of individuals in four rivers with 6 | P a g e different levels of urbanism; (2) evaluate the effect of specific pollutants found in urban streams on the general activity; and (3) determine the effects of urban water chemicals on agonistic behaviors of adult Macrobrachium prawns.

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Introduction

All the freshwater prawns that have been cultured so far belong to the genus Macrobrachium, the largest genus of the family Palaemonidae. Macrobrachium species are distributed throughout the tropical and subtropical zones of the world. They are found in most inland freshwater areas including lakes, rivers, swamps, irrigation ditches, canals and ponds, as well as in estuarine areas (New and Singholka 1985). About 200 species have been described, almost all of which live in freshwater at least for part of their life. In the Western Hemisphere there are about 35 native species of this genus (Bowles 2000). In the rivers of Puerto Rico, large Decapod crustaceans are represented by members of the Macrobrachium genus of giant freshwater prawns. Local species of freshwater Macrobrachium include: M. acanthurus, M. carcinus, M. crenulatum, M. faustinum, and M. heterochirus (Fig ?). Most of them require brackish water for full larval development before the benthic post- larvae develop and begin to migrate considerable distances upstream to the rivers headwaters, where juveniles continue to grow into reproductive, omnivorous adults (Chace and Hobbs 1969; Crowl, Mc Dowell et al. 2001; Snyder, Anderson et al. 2011). Macrobrachium carcinus (Fig. 1A) is the largest and most prevalent of the prawn species found in Puerto Rico (Covich 2006); however, few studies have been conducted with this model (Pérez Reyes, Crowl et al. 2013). Other species such as the introduced giant Malaysian prawn M. rosenbergii is a classic example of a species that has become widespread because of its popularity in commercial aquaculture (Iketani, Pimentel et al. 2011). Expeditions by Holthuis were used to provide useful information on the distribution, local names, habitats and maximum sizes of commercial (fished and farmed) species of Macrobrachium (Holthuis 1950). Accessibility to local rivers and ease of handling in the lab make M. carcinus a good candidate to explore questions of how water contaminants from urban rivers can affect variables such as the number of individuals, levels of general activity, interactive behaviors, and their sensitiveness to pollutants. Crustaceans are major constituents of aquatic ecosystems, living in different habitats and having the ecological role of shredders and thus are important components of nutrient cycling (Chace and Hobbs 1969; Dorit, Walker et al. 1991; Crowl, Mc Dowell

8 | P a g e et al. 2001; Boxshall and Halsey 2004). They are frequently used as bio-indicators and bio-monitors in various aquatic systems (Pedersen and Perkins 1986; Sponseller, Benfield et al. 2001; Roy, Rosemond et al. 2003; Shah and Shah 2013) to integrate their total environment and their responses to complex sets of environmental conditions (Li, Zheng et al. 2010). Indicators of environmental stress in water include relative abundance and diversity, feeding activity, drifting, locomotion, molting, changes in metabolism and growth, immune functions, reproductive capacity, and abnormal behavior. (Resh and Unzicker 1975; Dorit, Walker et al. 1991; Weis, Cristini et al. 1992; Ruppert and Barnes 1994; Schindler 1997; Barton, Morgan et al. 2002 ). It has been suggested that Puerto Rico’s freshwater animal populations are influenced to varying degrees by the introduction of exotic species, constructions of dams, changes in stream flow patterns and water pollution (Kwak, Cooney et al. 2007). Nevertheless, not much information is available regarding the sensitivity to pollutants to population growth rates, interactive behaviors such as aggression, and nervous system function in crustaceans of tropical regions. Environmental contaminants can take a variety of forms including heavy metals, by-products of industry (e.g. polychlorinated biphenyls, PCBs), and chemicals derived from personal care products and pharmaceuticals (PPCPs) that are released or leach into waterways and may well impact the biology of freshwater species (Bailey, Elphick et al. 1999; Focazio, Kolpin et al. 2008; Veldhoen, Stevenson et al. 2013). Yet, urbanization and the resulting water contamination in our rivers are thought to constitute a significant problem in need of further study, particularly in what concerns the possible effects or damage they may have on the composition and health of aquatic fauna, and all other species that use it as a food source, including humans. The nervous system is the interface between an organism and its environment. Neuroscience, therefore, can provide critical and sensitive tools to help understand how organisms respond to change. Our project is designed as part of the Puerto Rico Center for Environmental Neuroscience (PRCEN) that focuses on local and global threats to ecosystems in Puerto Rico. This NSF proyect promotes integrative approaches that combine the ecology, biology and chemistry of four interconnected Puerto Rican ecosystems (marine, estuary, terrestrial, and freshwater) with approaches into the molecular/cellular neurobiology of organisms living within those

9 | P a g e habitats. Our island provides strategic freshwater waterways with a great diversity of organisms such as decapod crustaceans (Fig.2) that can serve as good ecological models for studying these variables.

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Chapter 1 The effects of urbanizing watersheds on population assemblages of freshwater macro-invertebrates

Introduction

Although ecosystems can be rapidly disturbed by the action of natural changes such as hurricanes, earthquakes or droughts, the major sources of damage to the environment are human (anthropogenic) activities. Different human stressors are contributing to this global situation and are receiving more attention in recent years. Urbanization is increasing rapidly in tropical regions and there are important links between human population size, its practices and activities, and environmental degradation (Schindler 1997; Laurance 2006; Baker, Cepa et al. 2012; Ali Khan, Ali et al. 2013). Human populations have been growing by approximately 78 million people per year (Jones 2000) and by the year 2025 almost two thirds of the world's population will live in urbanized locations (Pimentel, Cooperstein et al. 2007), a proportion predicted to reach 70% by 2050 (United Nations 2007). The generalized effects of urbanization on streams are collectively known as the “Urban Stream Syndrome” (USS), characterized by high human perturbations and identifier “symptoms” associated with urban development including changes in biotic communities, hydrology, water chemistry, and channel morphology (Wallace, Croft-White et al. 2013). The human transformation of the landscapes around the world over the last decades has been extensive and has greatly disrupted the underlying natural processes that have shaped aquatic ecosystems especially in the tropics. Puerto Rico is a small tropical island with a high level of population density. Excluding the two offshore municipalities of Culebra and Vieques, it covers 8,700 km2 and its dimensions are about 160 km east-west and 55 km north-south (Hunter and Arbona 1995). In the past decades as populations grow people tend to cluster near bodies of water (Comarazamy, González et al. 2013). High demand of water, construction in watersheds, agricultural practices, industrialization and sewage disposal can increase the probability of accumulation of many contaminants into the water flows. In addition to imperviousness, runoff from urbanized surfaces as well as municipal and industrial

11 | P a g e discharges contribute to increased loading of nutrients, metals, pesticides, and other contaminants to streams (Paul and Meyer 2008). Urban areas are hot spots that drive environmental change at multiple scales (Grimm, Faeth et al. 2008). At local scales, urbanization has been linked to changes in stream habitat (Roy, Rosemond et al. 2003; Chin 2006), water chemistry (Mahler, Van Metre et al. 2005; Gilliom, Barbash et al. 2006), and hydrology (Poff, Bledsoe et al. 2006). Altered physical and chemical characteristics of urban streams, in turn, can serve as stressors that affect assemblages of algae (Walker and Pan 2006), fish (Tate, Cuffney et al. 2005; Wenger, Peterson et al. 2008), macro-invertebrates (Cuffney, Brightbill et al. 2010; Chadwick, Thiele et al. 2012), and even plants (Serrano 1996). The global rise in human population is driving a continual conversion of land to anthropogenic uses (Cohen 2003), so there is a strong need for monitoring stream health. Indicators of stream health and stream stressors are important tools not only for assessing stream condition, but also for assessing the mechanisms of impacts, protocols for stream protection and water quality management. It is a well known fact that landscape use and pollution of a stream can reduce the number of various species of the system, while also creating an environment that is favorable to a few other species (Zimmerman 1993). For example, in streams and rivers polluted by organic matter (Metcalfe 1989; Whitehurst and Lindsey 1990) or heavy metals (Winner, Scott Van Dyke et al. 1975; Clements 1994; Hickey and Clements 1998), species richness and diversity of the macro-invertebrate community are strongly reduced as a result of the direct and indirect impact of contaminants. Habitat quantity, quality, and connectivity are among the most critical influences on community dynamics and the conservation of biodiversity, but anthropogenic activities have resulted in habitat loss, degradation, and fragmentation (Cooney 2013). Research has repeatedly demonstrated declines in assemblage richness, diversity, and biotic integrity of algae, invertebrates, and fishes with increasing urbanization (Paul and Meyer 2008). In addition, Walters and colleagues established that macro- invertebrate descriptors (land cover, geomorphology, and water quality variables) were linked negatively to changes in urban land cover, propagated through conductivity and sedimentology, and their results suggest that macro-invertebrates are more sensitive than fishes to urban effects in streams, at least in newly urbanizing systems (Walters,

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Roy et al. 2009). Moreover, streams macro-invertebrates appear to have important advantages for assessing the effects of urbanization on stream ecosystems because the response of their assemblages to a gradient of urbanization can be predictable (Brown 2009). There have been many old and recent studies on urbanization and its impact on stream systems (Klein 1979; Jones and Clark 1987; Hachmoller, Matthews et al. 1991; Bae and Lee 2001; Chadwick, Thiele et al. 2012; Johnson and Ringler 2014). A study evaluating the influences of urbanization in small urban cold water streams has indicated that the percentage of watershed urban land use is the most important environmental factor influencing macro-invertebrate assemblages (Wang and Kanehl 2003). Another study, measuring the aquatic health of urban streams in Australia, revealed that urban streams were significantly impaired compared with those that flowed through naturally vegetated non-urban catchments and urban streams had consistently lower family richness, and sensitive guilds were rare or missing (Davies, Wright et al. 2010). In North Carolina, forested streams contained less-tolerant communities with higher sensitive taxa richness than streams in the urban category and channel habitat complexity and watershed impervious surface cover, were the best predictors of sensitive taxa richness and biotic index at the reach and catchment scales (Violin, Cada et al. 2011). Results of a study that assessed how urbanization affects streams in Georgia, USA, indicated that for every 1% increase in impervious surface cover, macro-invertebrate biomass decline by 7% (Wood, Rosemond et al. 2013). Studies of macro-invertebrates in other countries have been focused on urbanization and population dynamics. For example, investigators in Costa Rica have suggested that prawn populations in protected stream reaches are resilient or resistant to certain land-use changes, while observing a decline of 87% in the relative abundance of M. olfersi in an unprotected stream reach (Snyder, Pringle et al. 2013). In the same region, M. digueti, M. occidentale, and M. tenellum comprised 90.1% of all palaemonid shrimps, and the first two species showed the widest distribution in the basin of the river Grande de Térraba (Rólier Lara and Wehrtmann 2011). In USA estuaries copepods exposed to sediments from urbanized sites have reduced molting efficiency, and reduced numbers of grass shrimps in a tidal creek at the urbanized site

13 | P a g e were found (Fulton, Chandler et al. 1996). Studies on reproductive aspects in the wild have also been conducted on populations of M. acanthurus and M. carcinus showing that reproduction was seasonal, and fertility has a large variation, but increase with female body size. (Lara and Wehrtmann 2009; Bertini and Baeza 2014). Different studies in tropical stream ecosystems have demonstrated the importance of macro-biota, such as fishes and shrimps, in controlling the structure of aquatic communities (Covich 1988; Pringle 1996; Pringle and Ramírez 1998). In Puerto Rico, Vélez made the first checklist of freshwater and terrestrial decapods and reported the presence of various species of the genus Macrobrachium and Atya (Vélez 1967). In 1989, after that Hurricane Hugo affected mountain streams in the Luquillo Experimental Forest, Atyid shrimp densities were greater in the headwaters, demonstrating that they are likely to be resilient after intermediate levels of disturbance (Covich, Crowl et al. 1991). In contrast, the lowest registered mean relative abundance of Macrobrachium occurred during the 1994 drought, the driest year in the Espiritu Santo drainage (Covich, Crowl et al. 2006). Responses by freshwater shrimps to a prolonged drought and their return to pre-drought densities were determined along an altitudinal gradient in a Puerto Rican headwater stream (Covich, Crowl et al. 2003). Perez-Reyes (1999) in his work regarding the relative abundance, diversity and life histories of freshwater decapods in Puerto Rico, concluded that there were no differences in species diversity, fecundity and density in rivers that flow through different ecological life zones (Pérez-Reyes 1999). In addition Pérez-Reyes reported new localities for Macrobrachium crenulatum in the Tanama, Cerrillos, Loco, and Camuy Rivers in Puerto Rico (Pérez Reyes, Crowl et al. 2013). The upstream migration of Xiphocaris elongata, Macrobrachium spp., and Atya spp. was studied by Kikkert (2009) at the Espiritu Santo River. He reported that Macrobrachium spp. migration was highly seasonal and massive migration occurs at night and when the water flow is minimal (Kikkert, Crowl et al. 2009). In another research study looking at the macro-invertebrates assemblage in 16 tributaries of the Río Piedras watershed, more macro-invertebrate families and pollution-sensitive taxa were found at sites where physico-chemistry reflected less urban cover. In the same study family richness and pollution-sensitive taxa were positively associated with greater % of forest cover (de Jesús-Crespo and Ramírez 2011). Other experiments on the ecology of the

14 | P a g e freshwater decapods from Puerto Rico have been done at the Luquillo Experimental Forest focusing on the effects of natural (Covich, 2003 and 2006) and anthropogenic disturbances on the populations of these species (Benstead, March et al. 1999; Benstead, March et al. 2000; March, Benstead et al. 2003; Greathouse, Pringle et al. 2006; Hein, Pike et al. 2011), and on the timing of migratory drift of larval shrimps (March, Benstead et al. 1998). However there is very little information on the relative abundance of M. carcinus in our island, and even less information is available on the effects of urbanism on their population, behavior and neural function. The main challenges for macro-invertebrates inhabiting impacted streams are related to a downstream gradient of increased habitat degradation for the tributaries. All human societies have dammed rivers for different purposes: water supply, land, irrigation, flood control, industrial use and energy generation (Martínez, Larrañaga et al. 2013). Dam building and channelization are two of the primary mechanisms by which humans alter freshwater ecosystems. Dams and water withdrawals have fragmented nearly all rivers in Puerto Rico. Contrary to most other tropical regions, over the period (1940-50) Puerto Rico exhibited an accelerated shift from an agricultural to an industrial based economy because of its association as a commonwealth of the United States (Grau, Aide et al. 2003). This rapid industrialization was accompanied by hydropower dam construction, which peaked in the 1950s. Today, Puerto Rico has more than three times the number of large dams per unit land area as the continental United States and more dams than any other

Caribbean island (Greathouse, Pringle et al. 2006). The Rio Piedras, Río La Plata and Río Bayamón are three representative urban rivers in the metropolitan area of Puerto Rico. Rio Piedras is a heavily urbanized watershed in Puerto Rico, reaching ~50% urban cover, but remaining largely free-flowing, with only one dam in the upper part of the watershed (de Jesús-Crespo and Ramírez 2011). Despite evident urban impacts such as channelization and water pollution, this urban watershed maintains large populations of native shrimps and fishes, along with several non-native species (Ramírez, De Jesús-Crespo et al. 2009 ). Shrimp assemblages are dominated by predatory palaemonids, and densities are similar to those in natural rivers (Pérez Reyes, Crowl et al. 2013). The watershed of the Rio La Plata is the third largest in Puerto Rico, with a catchment area of 241 mi2 and the

15 | P a g e longest on the island with 58.5 miles from its source to the mouth. The current population in the basin is estimated at more than 354,260 inhabitants, including municipal urban centers and its population density is one of the highest among major basins on the island with 1,470 people per square mile (DRNA 2004). It has water storage dams within the basin. The Bayamón River watershed includes a catchment area of about 89.9 mi2. The river was channeled in the 1970s to control flooding in the inner city of Bayamón and urban areas north of the city. The basin population in 2004 was about 348,400 inhabitants, including a large part of the urban area of this municipality (DRNA 2004). It has a dam on the elevated portion for storing water. We hypothesize that elevated levels of urbanization in rivers of Puerto Rico will cause changes in population distributions and relative abundance of the freshwater prawn M. carcinus. Our rationale for this hypothesis is that urbanization near bodies of water has been increasing during the past decades and prawns are sensitive organisms to highly urban perturbations near watersheds.

Methodology

Selection of the study sites and urbanism quantification The sampling sites to estimate prawn relative abundance were selected on the basis of accessibility and level of urbanization of the candidate rivers. The Rio Piedras, Río La Plata and Río Bayamón were selected as representative of urban rivers (Fig.5), and the Quebrada Los Azules tributary of the Rio Canóvanas, as representative of a non-urban river (Fig.6). Three sampling sites were assessed in each river. The sampling sites varied in elevation, and for each place we sampled a pool. The selection of pools was performed according to similarities in various ecological factors such as size, depth and type of substrate. The experimental design consisted of a sampling of three urban rivers and a reference river (control) with three elevations in each one. The level of urbanization in each river was determined by quantifying the number of structures and/or constructions near the selected water bodies using aerial photos from Google Earth. Five aerial photos were taken at each sampling point, using the specific coordinate of each site for the first standard picture and increasing five times the distance (Km) from the original point for the next four photos (+5, +10, +15,

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+20). Finally, the images were treated in the ImageJ platform to separate green areas from constructions.

Urban rivers

Rio Piedras, San Juan: The higher sampling point of the river is in latitude 18˚ 34' 38.9" N, longitude 66˚ 07' 03.4" W, at an elevation of 345 feet. This sampling point is near the Montehiedra Town Center Shopping Mall. The middle point is at latitude 18˚ 36' 71.0" N, longitude 66˚ 06' 31.7" W, at an elevation of 220 feet. This point is near the Winston Churchill Avenue, which is a busy and crowded avenue with food and clothing stores, schools, pharmacies and large buildings. Daily runoff in this place ends at this point in the river. The lower point of the Rio Piedras river is in latitude 18˚40' 24.3" N, longitude 66˚ 06' 32.0" O, at an elevation of 80 feet. This place is near to the Américo Miranda Avenue and Expreso Las Américas. Many urbanizations, condominiums and businesses are found near this point.

Rio La Plata, Dorado: The highest sampling point of the river was in latitude 18˚ 38' 36.2" N, longitude 66˚ 24' 94.9" W, at an elevation of 64 feet. This sampling point was near to the PR-86, PR-693 and PR-2. The middle point was at latitude 18˚ 39' 57.4" N, longitude 66˚ 25' 45.9" W, at an elevation of 32 feet. This point was near to the municipality of Toa Alta. The lower point of the river was situated in latitude 18˚45' 69.6" N, longitude 66˚ 25' 84.6" O, at an elevation of 15 feet. This place was near to a recreational fishing place in the PR-6165 and PR-301.

Río Bayamón: The higher point of the river was in latitude 18˚ 36' 32.2" N, longitude 66˚ 14' 04.9" W, at an elevation of 75 feet. This sampling point was near to the Paseo Lineal and Plaza del Sol Town Center Shopping Mall. The middle point was at latitude 18˚ 38' 88.2" N, longitude 66˚ 13' 70.8" W, at an elevation of 60 feet. This point was also near to the Paseo Lineal and the Main Avenue. The lower point of the river was situated in latitude 18˚40' 98.4" N, longitude 66˚ 15' 23.1" O, at an elevation of -2 feet. This place was near to the University of Puerto Rico Bayamón Campus in the PR-174.

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Non-urban river

Los Azules, tributary of Río Canóvanas: This river is situated in sector Los Castillo, in Campo Rico, Canóvanas. Only a single family home is found in the area. The higher point of the river is in latitude 18˚ 31' 41.8" N, longitude 65˚ 88' 14.4" W, at an elevation of 632 feet. The middle point is at latitude 18˚ 31' 58.3" N, longitude 65˚ 88' 40.2" W, at an elevation of 465 feet. This point is near to a bridge and is used for prawn fishing. The lower point is in latitude 18˚ 31' 81.4" N, longitude 65˚ 88' 64.4" O, at an elevation of 396 feet. This point is closer to road PR-185.

To achieve our objective we performed a monthly prawn population census of the species M. carcinus in two rivers with different levels of urbanization. Wire traps were placed in the above mentioned three different points along the watershed in each river. Caught prawns were measured, identified and returned again to the water. Other physico-chemical factors (sendimentation, pH, temperature, oxygenation and type of substrates) of the sampling pools in both rivers were measured. This sampling effort was extended for 16 months (1 year and 3 months).

The following parameters were measured at each of the sampling points (9 urban and 3 non-urban).

Site coordinates and pool morphometry The exact coordinates of each location were obtained using a Geographical Positional System (GPS). The length and width of the selected pools were measured using a tape measure and a Ryobi Tek4 Pro Laser Distance Measurer. Physico-chemical parameters The physico-chemical parameters of interest were temperature, pH, salinity, conductivity, dissolved oxygen, pressure, sediment, and stream flow. Equipment available to measure these parameters includes an YSI 63 Portable pH & Conductivity Meter, an YSI ProODO Optical Dissolved Oxygen Meter, and a 705-

N10 Current Velocity Meter. The instruments were calibrated each time before being submerged. The multi-parameter meters simultaneously measured the factors with a single probe.

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Levels of sedimentation To measure suspended sediments 1-liter reusable plastic bottles were used to collect samples from the water column in each pool. Each bottle was identified with the river name, sampling point, number of sample replicate or control and date of collection. The procedure was done using gloves and samples were placed in ice immediately after collection. Experimental samples were made in triplicate, with collection of a control each time for monitoring of data consistency. A volume of 500 mL from of each water sample was filtered by suction using a reusable Millipore glass fiber filter system. The filtered samples were placed in aluminum containers and dried in an oven for 48 hours at 70 degrees Celsius. The filter membrane was weighed before and after the water filtration process to obtain the weight of dry sediment. The entire procedure was done once a month for each sampling site during the entire sampling. Substrate analysis To obtain a geomorphologic characterization and physical habitat assessment of the selected rivers, two transect lines of equal length were drawn in each pool. The transect lines were marked using nylon ropes transverse to the streams. Each transect line was further divided into equal lengths to obtain multiple points on each one. The type of substrate at the edges and the bottom of each pool were determined by walking along the pre-defined transect lines and registering the information detected through touch by the feet, to discern substrate type and size of component elements. We used the substrate categories described by Thompson and colleagues, such as silt, sand, gravel, pebbles, boulders or for a more specific characterization (Thomson, Taylor et al. 2001). Prawn collection Inverted funnel traps (Fig.8) were used to capture local prawns (Covich, Crowl et al. 2006). Six coconut-baited wire traps with enlarged 5-cm entry openings were used in each river, (2 per pool). The traps were identified with the institution´s credentials and were placed before sunset, approximately between 5:00 and 6:00 PM, and collected early the next morning, between 8:00 and 9:00 AM. Prawns collected were counted, measured with a caliper, classified by sex and released again into the water to maintain the relative number of individuals. The entire procedure was done in the four rivers the same week and successively once a

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month during 16 months in the reference river, 8 months in Río Piedras, 6 months in La Plata and 6 months in Bayamón. The differences in the sampling period were due to the failure in capture prawns in an urban river for more than six months (Río Piedras), so we decided to move the sampling to two other urban rivers during 6 months. The whole sampling included regular, reproductive, rainy and dry seasons. Data analysis Differences in the number of collected prawns and stream pool parameters were analyzed using GraphPad Prism 6, and SigmaPlot 12.3, two scientific graphing and statistical analysis platforms. The data obtained from field experiments was subjected to a statistical One Way or Two Way ANOVA tests. These tests allowed us to analyze the variance in the mean values of the prawn's relative abundance, sex distribution, elevation, and physico-chemical parameters. Finally, a correlation analysis was conducted to indicate the linear relationship and proportionality between the relative abundance of captured prawns and physico-chemical parameters measured during the population census in the non-urban river.

Results

In our attempt to determine changes in urbanizing riparian zones, we chose several areas with different characteristics, and with the required ecological and geographical conditions necessary for the presence of M. carcinus species. The analysis of the quantification of green areas and constructions around each sampling point in each river revealed a higher percentage of green areas than constructions in the non-urban river in Canóvanas (Fig. 9). In the case of Río Piedras we found more constructions instead of green areas, and in the Río Bayamón there was a similar scenario (Figs. 10 and 12). In contrast, the Río La Plata had much more green areas than constructions (Fig. 11), which was surprising due to the high anthropogenic activity near their sampling points. However, in terms of our analysis, the river was ranked as a semi- urban due to its gradual progress towards a higher degree of urbanization. The three measures of pool morphometry at the beginning, middle and the end of the census showed that length, width, and depth did not varied much in each sampling point (Fig.13). Also, different levels of elevation across the rivers did not significantly affected the relative abundance of prawns in the selected pools (Fig.14).

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Our results in the monthly prawn census showed that population relative abundance of M. carcinus individuals in the non-urban stream was significantly higher than that in the urban and semi-urban streams (Fig.15). The relative number of prawns collected by wire traps was 172 individuals in the non-urban river, compared with 0, 0, and 4 prawns in comparable areas of the three other rivers. Regarding prawn’s population abundances in the entire census, we have found an average of 12prawns/month (57prawns/pool) in the non-urban river Los Azules in Canóvanas. During six months of sampling we found an average of 0.6 prawns/month (1.3prawns/pool) in the Río Bayamón and we have no record of captured prawns in the other two rivers Río Piedras and La Plata. The largest number of prawns caught during sampling was in January with 16 individuals in the Quebrada Los Azules, Canóvanas. The lower number of prawns caught was in the month of December with only 3 individuals. Males of this species molt during this period so perhaps is the reason for the lower number, and also we observed during this month fishermen near the area of study. In the sampling effort, comparing both sexes (Fig.16) we found more males (89%) than females (11%). The analysis of physico-chemical parameters revealed important differences between the four rivers. In the three selected urban rivers, water termperature was significantly higher than that in the non-urban river (Fig.17). Interestingly the highest temperatures were recorded in the Rio La Plata, followed by Rio Bayamon and finally the Rio Piedras. Temperatures in these urban rivers ranged from 25-30 degrees Celsius compared to 22 to 23 degrees in the non-urban river. In our study water temperature was negatively correlated (R= -0.23) with the relative abundance of prawns in the non- urban river (Fig.23). The Río Bayamón had the higher levels of oxygen in the middle and higher elevation compared to the same points in the other rivers (Fig.18). The comparison between sampling points within each river revealed that in the low point in both the Rio La Plata and Rio Bayamon had significantly less dissolved oxygen than the middle and higher sampling points. When there is insufficient dissolved oxygen in a stream, shrimps, fish and other aquatic organisms can die. A healthy stream, according to various USA states water- quality standards, has a dissolved oxygen level of 5.0 mg/L. Interestingly, a negative association (R= -0.13) between oxygen and abundance of prawns was found in the

21 | P a g e non-urban stream (Fig.24). In terms of sediment, La Plata and Bayamón had the highest reported levels of suspended particles (Fig.19). Within the Rio La Plata and Río Bayamón, the middle and lower elevations respectively had significantly more sediment than in the other elevations. However, in the lower point of Río Bayamón there was significantly more suspended sediment concentration compared to the same areas in the other urban rivers including the non-urban river. Correlation coefficient analysis revealed that there was a negative association (R= -0.30) between high levels of sediment and captured prawns (Fig.25), and a negative relationship (R= -0.30) between sediment levels and water temperature (Fig.27) in the non-urban river. The Río La Plata had the highest conductivity, specifically the lower point where there is a huge anthropogenic activity, followed by Río Bayamón and Río Piedras. Comparison of conductivity within the rivers showed that only the concentration of the lower elevation in the Río La Plata was significantly higher than the middle and higher elevations (Fig.20). In the non-urban stream we found a negative correlation (R= - 0.37) between conductivity and suspended sediment (Fig.30) in the same months where increased water temperatures and there was a decreased in captured prawns. When there is little water flow in the pools and the sediment begins to concentrate, the charged particles tend to do the same and they not be distribute. Drought seasons aggravate this scenario, since there are no strong currents of water to wash out ions from river pools. Measuring the levels of salts during the biomonitoring, we found a negative association between M. carcinus prawn populations, and a combination of high temperature and high salinity in the urban rivers. Higher levels of salinity were recorded in La Plata, followed by Río Bayamon and Río Piedras (Fig.21). Comparison of salinity within the rivers showed that only the concentration of the lower elevation in the Rio La Plata was significantly higher than the middle and higher elevations. The high concentration of salts in the lower elevations of La Plata and Bayamón could be explained by the proximity of these points to the river mouth and tide changes. Although we captured a total of 4 prawns at this point in the Río Bayamón, water temperatures in this region were among the warmer identified, and in combination with salt levels could be a factors that explain the few animals captured in six attempts.

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The geomorphology of the sampled river pools showed that each river had a variety of substrates, including boulders, pebbles and gravel. In the non-urban basin and in the Río Piedras there was a higher frequency of occurrence of boulders, pebbles and gravel. Substrates such as sand and sediment were much more common in urban rivers (Fig.22). Discussion For biomonitor species, the studies of their population dynamics have become increasingly important because investigations of mechanisms that allow populations to resist extinction risks (resiliency), and to respond to declines and collapses are particularly important in the face of global and local environmental stressors. The effective conservation of freshwater prawn populations and their habitats requires the collection of detailed information on their distribution and understanding the factors influencing the abundance of each species. Monthly occurrence and distribution patterns of the freshwater prawn M. carcinus along watersheds with different urban intensity levels were investigated in four representative rivers in the northern area of Puerto Rico. In the literature there are evidences of the existence of M. carcinus species in urban rivers such as Río Piedras (Garcia Vazquez 2008) (Kwak, Cooney et al. 2007), Rio Bayamón and Río La Plata (Pérez Reyes, Crowl et al. 2013) according to previous investigations, and we were interested in gaining knowledge about how prawn populations have responded to levels of urbanization in Puerto Rico. Total distribution of the sampling period was 16 months for the non-urban river Los Azules in Canóvanas, 8 months for the Río Piedras, 6 months for the Río La Plata and 6 months for the Río Bayamón. Over time, the structure of a drainage network responds to variations in stream flow, sediment load, and human interventions. These changes can alter upstream migrations and access to headwater pools. However, during the season of sampling in each river, slight variations were recorded in terms of the spatial structure of the pools (Fig.13). Freshwater species require specific environmental conditions in their habitat. Benthic macro-invertebrates, such as freshwater prawns, are organisms primarily living in a varied streambed. In contrast, the addition of fine substrates to streams can result in significant changes to stream macro-invertebrate assemblages (Richards and Bacon 1994). Various studies about this aspect found that, when comparing four

23 | P a g e rivers, the streambed stability (gravels and cobbles) was the main factor that influenced macro-invertebrate assemblages (Zhao, Wang et al. 2014), and river morphology influences the longitudinal distribution of the shrimp species of the family Palaemonidae (Yatsuya, Ueno et al. 2012). River geomorphic structures, especially pebbles and boulders, may have an important effect on abundance and the spatial distribution of prawns. Rocks provide the main habitat for freshwater macro- crustaceans including small shrimps, crabs and large prawns. These organisms build caves for shelter during the day usually in rivers with clear water. In rivers containing pools with little variety of rocks, crustaceans depend on the level of sediment and visibility. In Puerto Rico there are rivers of extremely turbid/blue waters, but have a lot of crustaceans because them have adapted to this environment for survival. Although the sand and sediment are not the most associated with the presence of prawns in Puerto Rico compared to the elevated rocky regions, these animals could use these types of substrates for camouflage and not be seen by bird hunters in shallow pools or predatory fishes, such as mountain mullet (Agonostomus monticola) and eels (Anguilla rostrata) characteristic of the lower parts of the rivers. The relative abundance sampling showed that levels of urbanization are affecting native prawn populations, and possibly there is an association with other anthropogenic factors. In the sampling effort we captured more males than females because these months of sampling are not belonging to the mating season in Puerto Rico (May-July), where more females would be expected in the river pools. Likewise, there is a possibility that, in this species, territorial males are the ones that have the greatest access to resources within the river pools. Thus the number of females is less in the sample, which may mean that the proportion of males versus females obtained is not reflecting the reality of the natural environment. Although most individuals were captured in the lower sampling point of the reference river, there were no significant changes in abundance among the three sampling points, suggesting that these migratory species can reach the raised portions of natural rivers without difficulty, especially in its early stages of growth. Our findings are validated with previous investigations on urban rivers, and impairment in the relative abundance of prawns may be strongly related to multiple environmental factors. Our work relates urbanism with the abundance of prawns, but this is not

24 | P a g e enough to establish this phenomenon as the direct cause of water impacts. Therefore, we measure other variables that may be associated with the processes of urbanization in order to have more information on human impacts in urban rivers. An study confirmed that shrimp species diversity and abundance (including Macrobrachium) were lower in rivers receiving urban effluents in their catchment area, and environmental variables showed that conductivity, water temperature, dissolved oxygen, percentage of mud, and pH were the environmental factors that strongly influenced spatial variation in shrimp abundance (Djiriéoulou, Konan et al. 2014). Physico-chemical factors of river pools such as mentioned were measured each month during the entire sampling and it was found that water temperature, dissolved oxygen, and suspended sediment were the best predictors of the prawn’s abundance. Elevated temperatures can result in temperature stress, and are considered a ‘pollutant’ under the US Clean Water Act of 1972 (Dietrich, Van Gaest et al. 2014). A number of studies have demonstrated that the temperatures of rivers have increased (Webb and Nobilis 2007; Durance and Ormerod 2009), and is known that affects the overall biological and chemical composition of a stream (Poole and Berman 2001; Paul and Meyer 2008). Water temperature was cited as one of the most important environmental factors that affect assemblages of cold water macro-invertebrates in urbanizing watersheds (Wang and Kanehl 2003). At elevated temperature, embryos of the northern shrimp, Pandalus borealis hatched 3 days earlier, but experienced 2–4 % reduced survival (Arnberg, Calosi et al. 2013). In our study water temperature was negatively correlated with the relative abundance of prawns in the non-urban river. The amounts of captured prawns in the control river were less and were in a relative decrease in the months comprising the dry period between April and August 2014. Increases in freshwater temperatures can arise from the impediment of flow due to flood control, and development within the watershed (Goniea, Keefer et al. 2006; Karvonen, Rintamäki et al. 2010), as well as due to global climate change (Hardiman and Mesa 2014; Luce, Staab et al. 2014). The anthropogenic thermal degradation associated with land development has been found to permanently alter water temperature regimes (Nelson and Palmer 2007). For example, Galli 1990 suggests that runoff originating from urbanized areas during summer rainfall events provides a large thermal energy flux to receiving waters and temperatures increase approximately

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0.08°C for each 1% increase in overall watershed imperviousness (Galli 1990). Temperature also can affect the ability of water to hold oxygen as well as the ability of organisms to resist certain pollutants. The species M. carcinus commonly inhabits in relatively cold waters in the rivers of Puerto Rico, making a dramatic change in water temperature due to high human activity could cause danger to populations of native prawns. Oxygen availability is a widely recognized factor influencing the composition of freshwater communities because it critically affects the distribution of many species. Rapidly moving water, such as in a mountain stream or large river, tends to contain a lot of dissolved oxygen, whereas stagnant water contains less. Is known that caridean shrimp Palaemonetes subjected to water from 10 to 30°C, increased the rate of oxygen consumption (Oliphant, Thatje et al. 2011), therefore, this may be affecting prawn populations that living in warmer water, because oxygen levels may be affected by the demand of dissolved O2. Interestingly, in our study, no prawns were captured in the urban river La Plata during six months of sampling, and this was the one that had recorded the warmer temperatures and the lower levels of dissolved oxygen. However, oxygen was above the accepted levels in all the sampled rivers (at least 5mg/L and 85% saturation), allowing the viability of M. carcinus. Except the lower point of the Rio La Plata (which was sampled only twice due to trap's robbery) the other sites reached acceptable levels of dissolved oxygen, and all the other 3 rivers had levels above 100% of oxygen saturation. In each sampling area the critical point of oxygen consumption varies with the temperature, salinity and the prawn body size, but anthropogenic impacts have increased the frequency, duration, and intensity of activities in many aquatic systems, resulting in changes in community composition and often a loss of diversity. We found that levels of oxygen were negatively associated with elevated water degrees in the months belonging to the dry season (Fig.28). The dramatic changes in the oxygen levels at the Río La Plata may be due to the occurrence of anthropogenically induced hypoxia that is common in urban streams. In months of low rainfall, the level of water in the rivers decreases considerably and sediment tend to concentrate in the center of the pools, causing a not uniform distribution. Fast-moving water can pick up, suspend, and move larger particles more easily than slow-moving waters. This is why rivers are more muddy-looking during

26 | P a g e heavy rain periods, because they are carrying more sediment than they carry during a low-flow period. In turbid rivers, where macro-crustaceans used the sediment particles to avoid being seen by predators, this may be a factor for not leave their caves. Similarly, changes in these parameters (high temperatures and few suspended sediment) prevent upstream and downstream migration of individuals between river pools. This can be explained by a negative association seen between stream water discharge and captured prawns in Canóvanas (Fig.26). These associations in the non- urban river could explain why no prawns were captured in the Río La Plata and the minimum relative abundance reported in Río Bayamón. Major inorganic anions and cations are ubiquitous in freshwater. The proportion of major ions present in freshwater rivers is primarily linked to human activities such as the discharge of industrial wastewaters and inappropriate land management practices. Freshwater biota occurs within an optimal range of ionic strength. An increase or decrease in ionic strength from their preferred range requires biota to balance their internal ion concentrations against an external gradient, placing them under osmotic stress (Dunlop 2013). Is known that conductivity associated with metal ions is an abiotic descriptor that negatively impacts richness and structure of macro-invertebrate assemblages (Moya, Hughes et al. 2011; Gardham, Chariton et al. 2014). For example scientist found that a mixture containing the ions Ca+, Mg+, HCO , and SO , as measured by conductivity, is a common cause of extirpation of aquatic macro- invertebrates (Cormier, Suter et al. 2013). In our research we found that the concentration of metal ions was significantly higher in the two urban and semi-urban rivers compared with the non-urban river (Fig.20), suggesting a strong association with anthropogenic sources. Salinity alone as a parameter is apparently not a factor affecting prawn stocks in the wild. Actually, these brackish habitats act as mediators between freshwater and estuarine species and serve a nursery function for estuarine-dependent decapods. However, the salinity in combination with factors such as water pH and temperature can adversely affect freshwater organisms. Combined effect of temperature and salinity cause a prolongation of the pre-hatching period in shrimp species as both increased (Atashbar, Agh et al. 2014) and low temperature and high salinity significantly decrease weight gain and reduce growth of crayfish (Prymaczok, Chaulet

27 | P a g e et al. 2012). Study of abundance indicated that there was a significant negative relationship between salinity and turbidity and freshwater species (Peterson and Ross 1991). Non-urban rivers in Puerto Rico are characterized by a low concentration of salts (0-0.1 ppt) that is essential for the survival of adult M. carcinus. The same salinity concentration of 0.1ppt was recorded during a year in the control watershed Quebrada Los Azules in Canóvanas. The community dynamics along a gradient of urban land cover and altitude, distribution and abundance of prawns varies spatially and seasonally along the gradient in concert with varying physical-chemical conditions. The presence or absence of a species, community or population reflects environmental conditions. Absence of a species give us information, but is not as meaningful as it might seem as there may be reasons, other than the expected one, that result in its absence (e.g. pollution, predation, competition, or geographic barriers which prevented it from ever being at the site). However, this study shows how river morphology, water temperature, oxygen levels, salinity, conductivity and riverbed gradients, are important factors determining the relative abundance of prawns. The data suggest that M. carcinus populations in urban rivers of Puerto Rico may indeed be affected by higher levels of urbanism and the resulting stream changes characteristic of the urban stream syndrome. Conclusions

According to the results, it can be concluded that high levels of urbanization have the potential to affect population dynamics and assemblages of Puerto Rican native prawns, influencing the quality of streams. This is the first research that uses the Puerto Rican omnivorous prawn M. carcinus as a biomonitor in several urban rivers. Before this, only aspects of feeding and reproductive behavior of this species from studies in Central and South America and in the Caribbean were known. Contamination of urban water resources including increased organic compounds, sediment, and heavy metals may result from growing populations, land conversion and management practices, and impervious surfaces. The daily human activities can increase the probability that many pollutants can reach waterways, and in conjunction with urbanism aggravate the scenario for our aquatic ecosystems. Additionally, we can conclude that the low rate of relative abundance in urban and semi-urban rivers 28 | P a g e responds not only to the levels of constructions near the basin, but also to a range of external factors affecting rivers that are clearly associated to human alterations in the natural habitat. Finally, this study gives us a good idea of the low tolerance and high sensitivity of freshwater decapod macro-invertebrates to environmental disturbances. In this way, M. carcinus can be proposed as a sentinel species to determine the health of freshwater ecosystems. This biological species and its population status can reveal what degree of ecosystem or environmental integrity is present and may also provide quantitative information on the quality of the environment around it. For high levels of urbanization, and for many parameters, our hypothesis that the influence of anthropogenic activities alters freshwater ecosystems was supported. Watershed development alters hydrology and delivers anthropogenic stressors to streams via pathways affected by impervious cover. Land and riparian uses affect hydrologic connectivity, water quality, oxygen levels, sediment regimes, salinity and conductivity, which then can affect prawn populations, and other sensitive taxa.

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Appendix

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Figure 1: Species of Macrobrachium that occur in Puerto Rico. (A) Macrobrachium carcinus, (B) Macrobrachium crenulatum, (C) Macrobrachium heterochirus, (D) Macrobrachium faustinum and (E) Macrobrachium acantharus.

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Figure 2: List of 14 decapod species found in the rivers of Puerto Rico (Holmquist, Schmidt‐Gengenbach et al. 1998). Within the list, the genus Macrobrachium is highlighted with 5 species.

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Figure 3: Conceptual illustration of how land uses modifies hydrologic and geomorphic processes on streams and, thus, induces ecological responses on population dynamics.

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Figure 4: Puerto Rico is driving a continual conversion of ecological zones to anthropogenic uses, and urban development requires the alteration of natural habitats.

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Dam at Río La Plata Channelization at Río Bayamón

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Figure 5: Selected urban river watersheds for monitoring prawn populations in the Metropolitan area of Puerto Rico (DRNA 2004). Río Piedras Watershed (A), Rio La Plata Watershed (B), and Río Bayamón Watershed (C)

.

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Figure 6: Selected non-urban river for monitoring prawn populations in the wild (Lugo and García Martinó 2011). The Rio Canóvanas watershed has the stream basin Rio Los Azules.

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Figure 7: Summary of research logistic for conducting a prawn census in the wild. Various physico-chemical parameters (pH, temperature, oxygen, sediment, conductivity, salinity, water flow) were measured at each of the sampling points (9 urban and 3 non-urban).

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Figure 8: Inverted funnel wire traps with enlarged entries were used to capture freshwater prawns (A). Different prawns can enter to the trap, but it is almost impossible for them to leave (B).

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Figure 9: Levels of urbanization in the non-urban stream Los Azules, Canóvanas. Quantification of google earth images at different distances in Km (default, +5, +10, +15, +20) showed a high level of green areas than constructions in the three sampling points.

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Los Azules, Canóvanas

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Figure 10: Levels of urbanization in the urban river Rio Piedras, San Juan. Quantification of goggle earth images at different distances demonstrated a high level of constructions near watershed in the three sampling points.

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Río Piedras

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Figure 11: Levels of urbanization in the semi-urban river La Plata, Dorado/Toa Alta. Quantification of goggle earth images at different distances demonstrated a high perecentage of green areas than constructions. However, urban intensity is increasing gradually in lower elevations.

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Río La Plata

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Figure 12: Levels of urbanization in the urban river Río Bayamón. Quantification of goggle earth images at different distances demonstrated a high level of constructions than green areas in the three sampling points.

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Río Bayamón

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Figure 13: Pool morphometric changes during the census of abundance. Along the entire prawn monitoring for each river, morphometric changes in the pools (length, width, depth) were minimal.

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Figure 14: Elevation and population distribution in the urban and non-urban streams. The number of captured prawns is not altered by the levels of elevation within the rivers. ANOVA One Way (p<0.05).

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Figure 15: Population relative abundance in the non-urban stream Quebrada Los Azules, Canóvanas and in the urban streams Río Piedras, Río La Plata and Río Bayamón. Periodic census during 15 months revealed that there are significantly more prawns in the non-urban stream compared with the three other urban rivers. Relative abundances appear to be altered, since there are significantly lower in the 3 sampling elevations at the urban streams compared with the non-urban stream.

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Figure 16: Sex distribution of M. carcinus between urban and non-urban streams. Even in the breeding season, male individuals were more frequent than females during the entire monitoring.

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Figure 17: Registered temperatures in the sampling points between urban and non-urban streams. During monitoring, water temperatures were significantly higher in the urban rivers compared with those recorded in the non-urban river. ANOVA One Way (p<0.05).

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Figure 18: Average of oxygen levels registered in the sampling points between urban and non-urban streams. During monitoring, dissolved oxygen (DO) varies according to the river. The Río La Plata had the lowest levels of DO. ANOVA Two Way (p<0.05).

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Figure 19: Levels of suspended sediment in urban and non- urban streams. Urban rivers La Plata and Río Bayamón have concentrations of particles significantly higher than non-urban river. ANOVA Two Way (p<0.05).

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Figure 20: Levels of conductivity in urban and non-urban streams. Representative northern urban rivers of Puerto Rico have a concentration of metal ions significantly higher than non-urban river. ANOVA One Way (p<0.05).

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Figure 21: Salinity concentration in urban and non-urban streams. Salt particles were detected in higher amounts in urban rivers compared with those recorded in the control non-urban river. ANOVA One Way (p<0.05).

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Figure 22: Types of substrate between urban and non- urban streams. Transects in sampled river pools show a variety of substrates typical of Puerto Rican watersheds including boulders, pebbles, gravel, sand and sediment.

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Figure 23: Correlation coefficient between water temperature and capture prawns in 3 pools along the non-urban stream Quebrada Los Azules in Canóvanas. There was a negative association between warmer temperatures and the number of collected prawns.

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Figure 24: Correlation coefficient between dissolved oxygen (DO) and capture prawns in 3 pools along the non-urban stream Quebrada Los Azules in Canóvanas. We found a negative association between oxygen levels and the numbers of collected prawns in the months belonging to the dry season.

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Figure 25: Correlation coefficient between suspended sediment and capture prawns in 3 pools along the non-urban stream Quebrada Los Azules in Canóvanas. A negative correlation was observed between levels of sediment particles and the numbers of collected prawns in the months belonging to the dry season.

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Figure 26: Correlation coefficient between river water discharge and capture prawns in 3 pools along the non-urban stream Quebrada Los Azules in Canóvanas. The volume rate of water flow was negatively associated with the number of collected prawns in the months belonging to the dry season.

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Figure 27: Correlation coefficient between water temperature and suspended sediment in 3 pools along the non-urban stream Quebrada Los Azules in Canóvanas. The amount of sediment that is moved in the water itself was negatively associated with the water temperature in the months belonging to the dry season.

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Figure 28: Correlation coefficient between dissolved oxygen (DO) and water temperature in 3 pools along the non-urban stream Quebrada Los Azules in Canóvanas. The levels of oxygen were negatively associated with elevated water degrees in the months belonging to the dry season.

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Figure 29: Correlation coefficient between river water discharge and water temperature in 3 pools along the non-urban stream Quebrada Los Azules in Canóvanas. The volume rate of streamwater flow was negatively associated with high water temperatures in the months belonging to the dry season.

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Figure 30: Correlation coefficient between river conductivity and suspended sedement in 3 pools along the non-urban stream Quebrada Los Azules in Canóvanas. The concentration of metal ions was negatively associated with amounts of sediment particles especially in the months belonging to the dry season.

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Chapter 2 The influence of water pollutants from urban streams on agonistic behaviors in a Puerto Rican native adult omnivorous prawn

Introduction

When two animals share a space and limited resources, conflicts of interest on which one takes precedence in their control and access are inevitable. Most of the time, these conflicts are resolved through struggles, fights, threats and many other aggressive behaviors. Social dominance is a form of a social relationship in which individuals aggressively interact repeatedly. The interaction between individuals is a well-studied sequential series of interactions, with each individual having the option of terminating or continuing the interaction/contest at any time (Bierbower, Nadolski et al. 2013). Agonistic interactions between conspecifics often result in dominance where one individual wins the encounter by driving off the opponent and the other loses the encounter by retreating. Animals that engage in subsequent agonistic interactions are subject to winner and loser effects (Dugatkin and Druen 2004; Martin III 2007). Animals that “win” an agonistic encounter become dominants, and those that “lose” a confrontation become subordinates. The influence of these interactions increases the likelihood of winning and losing in subsequent encounters (Dugatkin 1997; Hsu and Wolf 2001; Rutte, Taborsky et al. 2006). Animals need aggressive behavior to establish dominance positions within their territory. Crustacean species have a marked territorial behavior (Flórez-Brand 2011) and the level of dominance is well marked on the phenotype and physiology of each sex (Moore, Haynes et al. 2002). Other studies of foraging competence (Khoury, Powers et al. 2009) and alpha-dominance (M Vannini 1971) in crustaceans have also been conducted. For example, lobsters and crayfish exhibit stereotyped patterns of agonistic interactions to establish social status. Previous studies have shown that in lobsters subordinates tend to avoid engaging dominants and if interactions do take place, their duration and intensity are reduced (Huber and Kravitz 1995; Rutishauser, Basu et al. 2004). Crayfish males form and maintain social stable hierarchical interactions since their first encounter, as we know also happens in other freshwater decapods including the freshwater prawn M. rosenbergii, where social status is

99 | P a g e relatively fixed (Sosa and Baro 2002). Freshwater prawns such as Macrobrachium rosenbergii and Macrobrachium faustinum, common invertebrates used to measure aggression, maintain their social status using their big sized chelae to win the encounters. M. rosenbergii has different colors on its chelae as it grows and this change in colors and morphotypic differentiation establish the dominance level or status of each male. Our model of study M. carcinus is considered a fully territorial species that travels at night for food and in the day remains in caves to avoid confrontations with other prawns (Benítez Mandujano 2012). Nevertheless, little is known about the characterization of its behavior of aggression in laboratory conditions and its sensitivity to water pollutants. Water pollution is a major negative impact of urbanization to stream ecosystems everywhere and stream impairment arising from land use is multifaceted. Urban sources of contaminants result in water quality degradation, changes in ecosystem function, and negative impacts on aquatic organisms (Walsh 2004). Agricultural and urban runoff leads to eutrophication and pollutant loading (Lenat and Crawford 1994, Carpenter et al. 1998, Bernhardt et al. 2008), creating a dangerous scenario in the stream health. More specifically, water pollution is related to changes in toxins, ionic concentrations, available nutrients, temperature, pH, dissolved oxygen, and suspended sediments, among others, that have diverse impacts on stream ecosystems (Paul and Meyer 2008). In tropical countries, stream pollution can be a severe problem and the magnitude and urgency of dealing with it was recognized by the United Nations in 2008 (Ramírez, Engman et al. 2012). Globally, aquatic ecosystems are highly polluted with organic and inorganic chemicals, heavy metals and derivatives from anthropogenic daily uses. According to the United States Geological Survey (USGS), the most frequently compounds found in water streams are nonprescription and prescription drugs, insect repellents, detergent metabolites, plasticizers, fire retardants, human and veterinary antibiotics, herbicides, hormones, and solvents (Barnes 2008; Focazio 2008). Several of these compounds are used intensively, in large volumes, are persistent, bioactive and exhibit bioaccumulation and endocrine disrupting activity (Caliman and Gavrilescu 2009). Compounds in personal care products (PCP’s) are continuously released into the environment. For example in Minnesota, concentrations of triclosan (TCS) and

100 | P a g e triclocarban (TCC), antimicrobial agents for daily use, were significantly greater downstream in 58% of the sampling sites (Venkatesan, Pycke et al. 2012). A water survey in Canada showed that PCP’s were always present, in nanogram and sometimes microgram per liter concentrations downstream, and included antibiotics, analgesics, anti-inflammatories, lipid regulators, metabolites of caffeine, cocaine and nicotine, and insect repellent (Waiser, Humphries et al. 2011). Additionally, pharmaceutical compounds have been identified as ubiquitous contaminants in water resources (Klosterhaus, Grace et al. 2013; Nilsen, Furlong et al. 2014). Neuro-active pharmaceuticals such as fluoxetine are used by great part of the population worldwide and are frequently detected in surface waters (Schultz, Furlong et al. 2010; Schultz, Painter et al. 2011). In Brazil, scientists noted that in tributaries that receive large amounts of untreated sewage, in addition to cocaine and its metabolite, ten human pharmaceuticals were all detected in two urban tributaries at concentrations similar to those typically found in urban surface waters (Thomas, da Silva et al. 2014). Moreover, in a study relating macro-invertebrate richness to carbamazepine, pharmaceutical concentrations may alter freshwater species composition, which could have significant consequences to ecosystem processes (Jarvis, Bernot et al. 2014). Runoff from urbanized catchments has been identified as a major source of contaminants, especially metals, impairing receiving waters. In a study on eight tributaries of New York multivariate regression models indicated that urban development was the most powerful predictor for the same eleven measured parameters (conductivity, TN, TP, NO−3, Cl−, HCO−3, SO2−4, Na+, K+, Ca2+, and Mg2+) (Halstead, Kliman et al. 2014). Another approach in China showed that watershed urbanization levels significantly correlated with increased values of conductivity, total nitrogen, ammonia, phosphate, calcium, magnesium, and chemical oxygen demand in the streams. Farm land and urban land were positively, and forest and green land were negatively associated with metal loadings Cu, Zn, Pb, Cr, Cd, and Mn (except Cr) in stream water (Yu, Wu et al. 2014). Other study state that differences in macro-invertebrate community structure by urban non-point source pollution were highly correlated with stream-water specific conductivity and dissolved inorganic phosphorus (DIP) concentrations. In the same study macro-invertebrate richness, the Shannon diversity index and the Shannon evenness index were all

101 | P a g e negatively correlated with specific concentrations of metal ions (Johnson, Jin et al. 2013). Investigating the pollution of channelized stream, investigators found that lead, cadmium and zinc were the major sources of pollution in the sediment (Sekabira, Origa et al. 2010). Moreover, among the various sources of water quality impairment , agricultural practices are ranked as the most important factor for rivers and lakes, and third in importance for estuaries (Puckett 1995). During the 20 years from 1992 to 2011, pesticides were found at concentrations that exceeded aquatic-life benchmarks in many rivers and streams that drain agricultural, urban, and mixed-land use watersheds across USA (Stone, Gilliom et al. 2014). Agricultural pesticides are common in urban waterways at concentrations posing a risk to a wide variety of stream invertebrates. For example it was found that insecticides in urban areas are more toxic to invertebrates, and shrimps are one of the most sensitive species (Weston and Lydy 2014). Other compounds can alter crustacean normal behaviors and development. A study analyzing the effects of injecting the neuropeptide Hyperglycemic Hormone (cHH) on the agonistic behaviour of crayfish demonstrated its role in enhancing aggressive behavior (Aquiloni, Giulianini et al. 2012). In contrast, higher supplemental dietary tryptophan levels caused a significant decrease in the aggressive behavior, but an increase in the calmness of crayfish (Harlıoğlu, Harlıoğlu et al. 2014). In lobsters individuals, 5,7-dihydroxytryptamine (a neurotoxin) was injected in order to deplete the animals of serotonin in their nervous tissue, but they found that the treated animals showed an increased tendency to engage in agonistic encounters. Therefore, the authors concluded that either high or low levels of serotonin increased the tendency of lobsters to engage in fights (Fong and Ford 2014). In crustaceans, antidepressants affect freshwater amphipod activity patterns, marine amphipod photo and geotactic behavior, crayfish aggression, and daphnia reproduction and development (Fong and Ford 2014). Nonylphenol, a xenoestrogen ubiquitously found in aquatic ecosystems, clearly affected locomotor activity and aggressive behavior of the male zebrafish at 100 μg/L (Xia, Niu et al. 2010). In a research using juveniles crabs collected from an impacted estuary were found to attacked a threatening stimulus significantly more often (70%) than conspecifics from a less impacted estuary (Reichmuth, MacDonald et

102 | P a g e al. 2011). The chemical composition of water should be strongly related with animal’s behavior, because that interaction may cause the specific behavioral outcomes. In the case of Puerto Rico there are some studies that highlight the importance of stream urban development and pollution. Studies by local investigators have shown that the Río Piedras urban river has high concentrations of phosphates, potassium, and magnesium (Ramírez, De Jesús-Crespo et al. 2009 ). Also the influence of built urban infrastructure on stream chemistry was quantified throughout the drainage network of the tropical Río Piedras watershed. Mean base flow concentrations of chloride (Cl), ammonium (NH4), dissolved organic carbon (DOC), dissolved organic nitrogen (DON), and phosphate (PO4) all increased with urban infrastructure, while nitrate (NO3) and dissolved oxygen (DO) decreased (Potter, McDowell et al. 2014). Interestingly, few studies are focusing in the effect of sub-lethal concentrations of emergent water pollutants on aggression and to our knowledge, there are no other studies using Puerto Rico´s most abundant freshwater prawn, M. carcinus, as a model to assess the impact of water pollution on agonistic behaviors. Our hypothesis for this aim was that phthalates and heavy metals would increase dominant/aggressive behaviors of M. carcinus freshwater prawns. Our rationale for this aim was that a small increase in the concentration of certain pollutants in water may lead to significant changes in the interactive behaviors of affected crustaceans.

Methodology

Experimental animals For this assessment we worked with pairs of male prawns extracted from non-urban rivers and exposed in the lab to specific pollutants. Adult prawns (M. carcinus), in the intermolt state, with cephalothorax-abdomen length of 7-15cm, were obtained from local non-urban streams located in Yabucoa and Rio Grande, Puerto Rico. Three weeks prior to the experiment, animals with all appendages intact were separated into individual containers providing visual and tactile isolation. All containers and experimental observation chambers were maintained under controlled environmental conditions at a holding facility in the Department of Anatomy and Neurobiology, Medical Sciences Campus-University of Puerto Rico, under 12h light:12h dark. Tap water was supplied from a central tank, where it was treated with an anti-clorox

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(AquaSafe Plus) and beneficial bacteria. In addition the water was partially recirculated, filtered, aerated, and held at temperatures between 24-26°C and the pH adjusted to 7.4 (safe range is 6.9–8.5). Animals were fed every other day ad libitum with marine protein-based diet pelleted food. All procedures involving the use of animals were approved by the University of Puerto Rico Medical Sciences Campus Institutional Animal Care and Use Committee (IACUC) prior to the start of the experiments.

Behavioral observations and quantitative assessment of aggressive behavior Body/claws of prawns were measured before experiments began, and pairs were matched according to body/claws ratios, and assigned randomly to one of seven groups (1 control and 6 experimental). Since the dominance hierarchy of this species of prawn is not predetermined on the basis of the animal’s morphotype, specific pairs of prawns of similar sizes were placed together before experiments to establish dominance. Asymmetries in size, sex, molt stage, physical condition serve as predictors of success in a contest, and decapods confrontations are more likely to escalate if combatants are closely matched in these factors (Huber and Kravitz 1995). These animals are nocturnal and the light cycle is set at our lab so that at the time of the experiments (usually in the morning between 8:00 AM and 12:00 PM) they´ll be near to their peak of activity. Each pair was placed in a specialized water tank prepared for recording of agonistic interactions (Fig.1), where they were initially be separated by a divisor during a period of ten minutes of habituation prior to the start of video recording. After the habituation period, we removed the divisor and recorded for 30 minutes, after which the animals were placed back into their respective isolated home tanks. The observation chamber (76.2cm length × 30.5cm width x 30.5cm deep × 25.4cm water level) was constructed from glass and black rounded corners. The bottom surface of the arena was spatially homogeneous. Water filters, heaters, and aeration tubes were temporarily removed during observation sessions to minimize sources of distraction for the animals, and sessions were performed in dark to minimize disturbances during filming. All interactions were recorded using a video camera (Sony JVC HD Everio GZ-EX210) mounted centrally in front of the tank. This protocol was followed daily, at the same time each day, for a minimum of 3 days to

104 | P a g e establish control levels of aggression or dominance. Aggression/dominance was quantified as a dominance index, defined below. Once a stable level of aggression/dominance during the control period is established, the contaminant of choice, or saline (Ringer) solution, was injected in the hemolymph sinus of the submissive prawn of each pair at concentrations equal to the limits established by the EPA for drinking water. The specific contaminants tested were those identified, through another project in our lab, to be present in urban rivers of Puerto Rico. For the injections, animals were anesthetized/immobilized on ice for 2-3 minutes. The effects of handling and injecting the animals were assessed with injections of prawn Ringer solution. Injection volume was always 0.1 cc (100ul). After injection, the animal was placed in an observation tank (20-L glass tank 16 x 8 x 10 in ~ [40.6 x 20.3 x 25.4 cm]) until the effects of exposure to ice fade away. The animal was not placed in contact with the same animal it had been tested with during the control sessions until its mobility level was reestablished to a level comparable to that observed before the injection. After the 30-min recovery period, the animal was placed in the behavioral observation 75-L glass tank in contact with the other prawn, and their interactions were recorded and compared with those of the control session. Data from animals that molted during the control period were discarded. Besides the recording following the injection, the pair´s dominance index (DI) was measured for a minimum of three consecutive days, to determine any persistency, delay or reversibility in the level of aggression/dominance. This was done to determine whether any changes induced by the injection of a contaminant are reversible or not. In addition to the basic measure of the DI, we also measured the number and duration of interactions and the number of interactions started by the dominant and the submissive prawns of each pair.

Video and data analysis Agonistic behavior is a set of patterns adjusted to situations of conflicts among conspecifics. According to studies by previous investigators, aggression includes threats, submission, chases, and physical combat, and there are two distinguished categories of agonistic behavior: (1) approach-oriented behavior – direct an animal towards an opponent, and (2) avoidance-oriented behavior – steer an animal away

105 | P a g e from a opponent (Huber and Kravitz 1995). The analysis to quantify dominance/aggressiveness was rigorously performed through an ethogram, and a dominance index template used to quantify how dominant and/or submissive each prawn is relative to its partner. The selected ethogram was used in previous investigations to quantify aggressive behavior in lobsters and crayfish and validated in our laboratory measuring M. rosenbergii aggressiveness (Vázquez-Acevedo, Rivera et al. 2009). The ethogram has different behavioral parameters that serve as indicators of dominance/subordination in each prawn pair. These parameters were the same as those used for prawns by Barki et al. (1992) and similar to those used for crayfish by Huber and Kravitz (1995), with some additions and modifications based on our own observations (Fig.2). The values in the Level column designate the weight of the parameter as an indicator of dominance/subordination, 6 indicating the most highly dominant behavior and 0 indicating complete or total subordination [based on (Barki, Karplus et al. 1992)]. Dominance index of each prawn was obtained using a validated formula based on this ethogram, where different parameters of the animal´s posture, movements and fight intensity were taken into account. The total observation period, monitored separately for each animal, was dived into 30s segments. During each segment the occurrence of behavioral variables was recorded, and we assigned a number that ranged from zero to six in each category depending on behaviors displayed by the animals. These values were used to calculate a dominance index (DI) for each individual tested in the paired interactions, calculated as the sum of the weights of all the instances recorded during the observation period, divided by the total number of instances. To determine whether specific parameters could be differentially affected by the injections in comparison to the others, the mean level of aggression reached for each parameter was also calculated for each animal during the observation periods. To determine whether specific agonistic parameters could be differentially affected by the contaminant injections, the mean level of aggression reached for each parameter was also calculated for each animal during the observation periods. Analysis of video recordings was performed both by me and by a person that was blind regarding the experimental condition under which the interactions being analyzed were carried out, nor to the classification of each prawn as dominant or submissive. In

106 | P a g e the event that resulting values of DI differ by more than 5% for these two persons, a third (or more) person(s) that was (were) also blind regarding the experimental conditions under which the videos were obtained was (were) asked to conduct the analysis, until the standard of variance of less than 5% amongst observers was achieved. All data was analyzed using GraphPad Prism 6, and SigmaPlot 12.3, two scientific graphing and statistical analysis platforms. For the analysis of DIs and other agonistic parameters obtained from the assessments of aggression, a statistical Two or One Way ANOVA test was used. These tests allowed us to analyze the dominance status of the prawns as well as the effect of the contaminants in aggressive behavior. Levels of significance (P) was reported as P<0.05.

Results

In our effort to determine the sensitivity of prawns to pollutants, we have thus far found that some contaminants found in urban rivers of Puerto Rico, including phthalates, esters of phthalic acid that are widely used in the manufacture of plastics as nonreactive plasticizers that increase the flexibility and workability of high molecular weight polymers, and ubiquitous heavy metals, affect aggressive behavior of the freshwater prawn M. carcinus. An initial quantitative view of the data summarized the number and behavioural characteristics of agonistic interactions across the seven groups (Figs. 3-16). In the research, to assess the effects of handling and injecting the animals, we carry out injections with prawn Ringer in a control group, and we found that there is no effect of saline solution on the aggressive behavior of prawns (Figs.3-4). In contrast, injection of a low concentrations of dibutyl phthalate (DBP; 0.006 mg/L), significantly increases the dominance index (DI) of submissive adult male prawns (Fig.5), and the effect of the contaminant still happens until the next days after the exposure [(mean DI for subordinates was 1.59 ± 0.08 control, 1.99 ± 0.07 injection, and 1.97 ± 0.06 post- injection (n = 5)]. The same contaminant also decreases the interactions, duration and the number of fights initiated by dominant prawns (Fig.18). Detailed statistical analysis of each parameter of aggression separately, revealed that DBP changed prawn's abdomen posture and movement and position of claws on submissive animals. In the same way, the contaminant significantly reduced the DI of dominant prawns regarding

107 | P a g e the movement and position of claws, and an effect of suppressing alpha's dominance status was observed after DBP treatment compared to the controls (Fig.6). Injected animals had a tendency to increase the DI in moving their chelae, but the data was not significant. In the case of diethyl phthalate (DEP; 0.006 mg/L), no effect in the mean DI was observed on submissive injected prawns, but the dominance status of dominant prawns was abolished immediately after the treatment (Fig.7). Independent parameters showed no DEP effect on submissive prawns. In contrast, there was a contaminant effect on alphas which reduced their DIs in both the position of the body/legs as in the movement/ position of claws. In these parameters also the alphas lost their fight status (Fig.8). Interestingly, when we analyzed video recordings for parameters of interactions, there was a significant effect more noticeable. Low concentrations of DEP reduce the number and duration of close interactions and significantly decrease the number of fights started by dominant prawns (Fig.19). This means that although the dominant prawns abandoned their dominance status, submissive injected prawns possibly showed aggressive behavioral responses that did not allow the initiation of intense battles by dominants as they did in controls. Few days after DEP administration to subordinates, dominant prawns regained their dominance status in the subsequent encounters. Benzylbutyl phthalate tended to increases the aggressive behavior of submissive prawns (Fig.9), but there was no statistical differences (p=0.272). Although in average we did not see any significant change in the aggression of subordinates due to BzBP, separate parameters analysis revealed a DI increased in the abdomen posture of these prawns (Fig.10), and abolishment of the dominance status of alphas in the moving and position of claws. This common plasticizer also tends to decrease the number of interactions between pairs, and the number of attacks initiated by alphas (Fig.20), but data is not significant (p=0.110, p=0.129). Our results using metal toxicants reveal that injection of a low concentrations of chromium, cadmium, and manganese (Cr+3; 0.100 mg/L, Cd+2, 0.005 mg/L, Mn+2; 0.05 mg/L) causes a significant increase in the DI of submissive prawns. The effect of chromium was observed immediately the day of the injections (Fig.11), and persisted in the post-injection days of exposure [(mean DI for subordinates was 1.71 ± 0.08 control, 2.04 ± 0.1 injection, and 2.0 ± 0.1 post-injection (n = 5)]. The movement of the

108 | P a g e chelae was the primary parameter that significantly increased the dominance of these animals (Fig.12). With cadmium, as one of the most toxic heavy metals, we found that submissive individuals reverse their dominance status (Fig.13), increasing their DI following the injection [(mean DI for subordinates was 1.59 ± 0.08 control, 1.93 ± 0.1 injection, and 1.69 ± 0.1 post-injection (n = 5)]. The DI of the abdomen posture was the major contributor to increase the aggressiveness of subordinates, including also a drastic reduction in the dominance of alphas both in motion/position of claws and moving towards the other prawn. After Cd+2 administrations, the status gained by alphas in control sessions was repressed in three agonistic parameters (Fig.14). Similarly, small amounts of cadmium significantly reduced the number and duration of attacks, and also caused a decrease in the fights started by dominant prawns (Fig.22). Using manganese, a potentially toxic mineral element, we saw that there was an increase in the DI of injected prawns during the post-injection sessions (Fig.15). The mean DI for subordinates was 1.62 ± 0.04 control, 1.84 ± 0.08 injection, and 2.15 ± 0.1 post-injection (n = 5). Of the parameters of dominance, the most contributors to the overall aggressiveness of subordinate prawns were the movement of the chelae, including a significant decrease in aggressive behavior of alphas (Fig.16). Such a reduction in aggression resulted in the abolishment of their dominance statuses in many occasions. The heavy metals excluding Cr+3 significantly decreased the number and duration of fights, and interactions initiated by dominant prawns (Figs.21-23). In general, our results show that low concentrations of DBP, Cr+3, Cd+2, and Mn+2 increase levels of aggression of submissive prawns in a reversible and repeatable manner, and it is possible that these contaminants may have an effect in the mechanisms and neural circuitry underlying aggression in the freshwater prawn.

Discussion Environmental pollutants such as metals, pesticides, and other organics pose serious risks to many aquatic organisms. A great deal of research has therefore been conducted to understand the effects of toxicants on the physiology and survival of many animals (Wood 2001). Physiological effects of toxicants include disruption of sensory, hormonal, neurological, and metabolic systems, which are likely to have profound implications for many animal behaviors. One aspect that may be affected in

109 | P a g e organisms by water toxicity is their sociability, how to behave with conspecifics. Perhaps the most simple and frequently measured indicator of altered social relations in animals exposed to toxicants is an altered frequency of agonistic acts, such as threats, nips, or chases. The fighting behaviour of clawed decapod crustaceans has attracted considerable interest due to conspicuous visual displays and potentially lethal weaponry (Sastry and Ehinger 1980). Endocrine disrupters such as phthalates are anthropogenic chemicals released into the environment that can affect animal physiology. Endocrine disrupters have been found to affect sexual behavior in mammals (Carbone, Ponzo et al. 2013; Barrett, Parlett et al. 2014), induce reproductive malfunction (Kumar, Srivastava et al. 2014), and territorial marking behavior in mice (Palanza, Morellini et al. 1999). In Puerto Rico an study suggests a possible association between phthalates with known estrogenic and anti-androgenic activity and the cause of premature breast development in the female population (Colón, Caro et al. 2000). However, only a few studies have looked at the effects of endocrine disrupters on aggressive behavior. Our present data indicate that these compounds can be detrimental to the normal behavior of aquatic organisms at low concentrations. Thus, these compounds should be considered as environmental pollutants, and a more detailed evaluation of toxicological effects of phthalic acid esters is needed to elucidate their impact on aquatic ecosystems. Specific phthalates may affect in various ways the behavior of invertebrate crustaceans, and future research has to see if any effect occurs in higher animals including humans. In fact, it is known that prenatal phthalate exposure was associated with childhood social impairment in a multiethnic urban population (Miodovnik, Engel et al. 2011), and it would be interesting to measure aggressiveness and other effects of endocrine disrupters in humans. Metal contamination in aquatic environments has received much concern due to its toxicity, abundance and persistence in the environment, and subsequent accumulation in aquatic habitats. The metals generally referred to as heavy metals include mainly lead, mercury, copper, cadmium, nickel, cobalt, chromium, manganese, zinc, and selenium, being lead, mercury, and cadmium the most dangerous to flora and fauna (Fingerman, Devi et al. 1996). Therefore, a continuous monitoring of rivers is required and knowledge of the possible effects of these chemicals on aquatic organisms.

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The action of heavy metals in the aggressive behavior of crustaceans is not well understood. So far is known that agonistic encounters between pairs of fish are altered by cadmium, reducing the total number of attacks (Sloman, Baker et al. 2003), and cadmium was detected differentially distributed in the brain of aggressive rats after treatment (Terçariol, Almeida et al. 2011). Cadmium is a calcium antagonist, its mechanism of toxicity probably related to disruption of calcium homeostasis because is well appreciated that Cd2+ participates in a number of Ca2+-dependent pathways, attributable to its actions as a Ca2+ mimetic (Choong, Liu et al. 2014). Behavioral effects of cadmium are likely caused by neurological disturbances during chronic exposure, but low levels are enough to cause significant changes at the behavioral level in prawns. When cadmium exists as the Cd2+ ion in biological systems can exerts a broad range of adverse actions such as Cd2+-induced neuron cell apoptosis and ROS, impaired neurogenesis, altered gene expression and epigenetic effect and has estrogen-like effect, which can induce endocrine disruption (Wang and Du 2013). Chromium can exert a range of biological effects in the central nervous system at different doses, but until now there is no association between chromium administration and aggression in crustaceans. Amaldoss and Mary (1992) studied the effect of chromium on the neurosecretory cells in the brain and thoracic ganglia of the shrimp, Penaeus monodon and found that acute exposure produced an increase in the amount of stored neurosecretory material and shrinkage of the neurosecretory cells (Amaldoss and Mary 1992). In addition an increase of brain chromium levels in rats enhanced metabolism and serotonin and decreased cortisol levels (Komorowski, Tuzcu et al. 2012). In the case of manganese, many studies have shown that brain tissue and neurotransmitter activity can be altered via Mn+2 exposure (Babadi, Sadeghi et al. 2013; Daoust, Saoudi et al. 2014; Fordahl and Erikson 2014; O’Neal, Lee et al. 2014; Schmitz, de Oliveira et al. 2014). Adverse mood effects of Mn+2 overexposure have been described including anxiety, nervousness, irritability, psychotic experiences, emotional disturbance, impulsive/compulsive behavior and aggression/hostility (Bowler, Mergler et al. 1998). Other studies in vertebrates showed that Mn+2-treated group of quails had significant age-related increases in aggressive behavior and depression (Laskey and Edens 1985), rats fed diets that were high in Mn+2 showed increased aggressive behavior compared to controls (Penland 1997),

111 | P a g e and rats exposed simultaneously to manganese through drinking water and lead had marked increased aggressive behavior and this was associated with a serious derangements in the behavioral pattern and levels of biogenic amines in the brain (Chandra, Ali et al. 1981). The individual behaviors of shrimps used in their aggressive motivation, including escape, postural changes, forward walking, backward walking, and defense, have been shown to result from activation of discrete neural circuits (Edwards, Issa et al. 2003), that can be excited by specific sensory stimuli or command systems of central neurons. However, little is known about how these circuits are excited by water pollutants in a social/agonistic context to produce patterns of aggressive behaviour. Activation of the different neural circuits and patterns of behaviours are highly coordinated, but the pattern of coordination can change dramatically as when an animal is exposed to a specific compound. In the experiments of dominance hierarchy using the model M. carcinus, we note that between pairs of males the dominance status was established during the first confrontation, where finally appear one dominant and one submissive. Interestingly, throughout the control sessions dominant offensive behaviours were frequent and submissive defensive behaviours were rare before the point of chemical administration, whereas afterward the reverse was true for the dominant prawns and the new subordinates. The behaviour in the laboratory differs from that observed in the wild, where encounters are of brief duration, usually decided by body/chelae size disparity. The consequence of winning an encounter is that animals are more likely to win their next encounters, whereas losing animals are unwilling to fight against alphas of previous encounters for periods of up to several days (control sessions). Such animals appear to develop a loser memory which can be reversed either for short or more prolonged periods of time by specific contaminant injections. Changes in attack responses by submissives suggest that urban water chemicals increase the willingness of animals to engage in fights (enhance their ‘aggressive motivation’). This suggests that low concentrations of organic and heavy metals can modulate the excitability of these defensive circuits suddenly changed from being low before the subordinate’s decision to attack to very high afterwards, while the excitability of circuits that mediate offensive action (approaches, aggressive postures, fights) increased significantly. In crayfish during agonistic encounters the excitability of

112 | P a g e the lateral giant reflex falls, substantially in subordinates, whereas at rest excitability seems to be independent of social status (Krasne, Shamsian et al. 1997). This mechanism that involves escape responses mediated by the lateral giant axons could be affected by contaminants administered to prawns. Aggressive behavior observed in subordinates might be reversed due to increased reflex excitability used to protect them from an unexpected attack. Dominant animals always tried to remain alphas, but in some assessments they lost their status as the aggression index increased in the subordinates, and the excitability of circuits that evoke offensive behaviour remained relatively equal or low. It is interesting that when only the subordinates received a contaminant injection, the behavior of alphas was also modified. Under these circumstances, a significant decrease was observed in the levels of aggression of the normally dominant subject. This change in the behavior of alphas could be explained as an effect of observation of the unexpected change in behavior of its rival or by influence from water-borne agents produced by the injected submissive. The results observed in our experiments using M. carcinus were similar to those found in our lab injecting Gly-SIFamide in a BC/BY M. rosenbergii pair (Vázquez-Acevedo, Rivera et al. 2009) . Following injection of certain pollutants in a subordinate suggest a partial or full reversal in status of both the injected and non-injected animals of a fighting pair. However, how the underlying mechanisms of these and other circuits are orchestrated to produce these complex behaviours remains unclear. Although water chemicals might account for the immediate shift in the excitability of the affected circuits in M. carcinus, it is likely that longer-lasting mechanisms activated by those compounds can influence neuromodulators as well. Neuromodulators that have been identified as affecting the social/aggressive behaviour of decapod crustaceans include the monoamines serotonin and octopamine (Kravitz 2000), steroid hormones (Bolingbroke and Kass-Simon 2001) and peptide stress hormones (Chang, Chang et al. 1999). In decapods crustaceans injections of biogenic amines induce specific types of postures linked to social status, such as serotonin-flexed abdomen and octopamine-hyperextended abdomen. We showed previously that injections of serotonin and octopamine can modulate the levels of aggression in the freshwater prawn (Sosa and Baro, 2002). We have observed in our lab how aggression in the

113 | P a g e giant freshwater prawn M. rosenbergii increases in response to injection of the biogenic amine serotonin (5-HT) or the neuropeptide SIFamide, and decreases in response to injection of the biogenic amine octopamine (OA). This evidence suggests that biogenic amines play a role in modulating aggression in these animals, and concentrations of water contaminants administered to prawns may be affecting the levels of these aggressive behavioral neuromodulators in the nervous system. Serotonin has received the most attention in studies with decapod crustaceans, but other transmitters and molecules play key roles in social behaviour, but the detailed experiments on the effects of this and other substances on the nervous system of M. carcinus are just beginning. Short-term changes in social behaviour may require corresponding short-term changes in the neuromodulatory systems and circuits that mediate the different components of aggressive behaviour. This could be a clue to understand the mechanism used by pollutants at physiological level to produce an effect in the behavior of M. carcinus and other crustaceans.

Conclusions The determination of an individual's behaviour often relies upon complex signals that convey messages about the environment it inhabits. In aquatic environments such signals take varied forms including chemical cues indicating a potentially danger. Despite ever increasing knowledge of the chemical compounds, little is known of how the environment affects an animal's neural response towards such toxicants. This is the first investigation to our knowledge that is trying to characterize the aggressive behavioural responses of M. carcinus species and its sensitivity to water pollutants. Our behavioural characterization uncovered evidence for the existence of winner and looser M. carcinus prawns in agonistic encounters, and possible effects of urban stream pollutants on aggressive behaviour. In the present study, we performed behavioral experiments in which the interactions between prawn pairs were recorded and quantified before and after injecting urban river pollutants directly into the circulating hemolymph of the living animal. Our data shows that levels of aggression escalated in subordinate prawns by the injection of specific phthalates and heavy metals (Table 1), suggesting a function in the modulation of aggression in this species. According to the results, we consider that changes identified in the aggressive

114 | P a g e behavior of the species M. carcinus may be associated to an effect of pollutants at systemic level likely to affect areas associated with normal behavior. Prawns exposed to DBP, Cr+3, Cd+2, and Mn+2 showed great hyperactivity and interestingly another group of prawns exposed to the same pollutants showed significant increases in their dominance indexes. Among the changes, we observed that not all chemicals acted simultaneously. Some contaminants can affect social behavior depending on how quickly they are processed and if they reach target areas within the Nervous System. This may suggest that the mechanisms of action of contaminants that affect the overall level of activity and modulate the aggressiveness of these crustaceans may be the same or closely related, although the effects will occur at different times. Male prawns compete among themselves for establishing and holding a territory and achieving dominance. Because resources are largely confined to dominant, territorial males, aggression plays a crucial role in the accessibility and survival according to ecological conditions. The work presented here is consistent with a dynamic view of prawn hierarchies, where fighting success, aggressive state, and social status are represented by alphas, generally characterized by more pronounced bodies and claws. Interestingly, our hypothesis coincided with the results, since the agonistic dynamics and hierarchy in these prawns may be affected by a slight exposure to toxic chemicals characteristic of urban rivers. Taking this in advance, we can suggest that the range of phenotypic variation in M. carcinus species may be correlated to specific environmental conditions. Observations that water pollutant injections into subordinate animals triggered the appearance of postures and attacks, resembling those seen in dominants. If exposure to water chemicals creates that phenotype, leading to a different range of traits, such as an altered level of aggressions, an impact on the individuals in the population is likely to occur, and changes in population dynamics could follow. Contaminant-dependent alteration of conspecific competitive strategies and the modification in social behaviors may disrupt individually genetic responses, thus resulting in disruption of the nervous system, with profound impact on the behavioral ecology of the species. Our results demonstrate, for the first time, that water contaminants enhance individual aggression, up to reverse, although transitorily, the dominance status rank. Because

115 | P a g e behavior links physiological function with ecological processes, behavioral indicators of toxicity appear ideal for assessing the effects of aquatic pollutants on prawn behavior and population dynamics. Because in the natural environment these animals can be exposed to sudden changes in its ecosystem, as periodic exposure to low and high concentrations of contaminants and physical changes of water that could be detrimental, a major question for now is whether exposure to mixtures of low-dose or high concentrations of phthalates and heavy metals, having different action mechanisms, affects the behaviour and neural function differently than exposure to individually compounds. The challenge ahead lies in linking environmental neuroscience to the observed changes in aggression within a framework of dynamic behavioural mechanisms. This study give us a better understanding of the mechanisms involved in mediating and/or modulating aggressive behaviour in simple model systems such as crustaceans will ultimately help enable the identification of novel sources for treatments of a wide variety of neurological diseases in higher organisms associated with hyperactivity, hostility, and aggression.

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Appendix

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Figure 1: Agonistic confrontations of control and experimental groups were recorded in a specialized tank.

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Figure 2: Ethogram of dominance index parameters used to quantify aggressive behavior in M. carcinus. Panels show specific postures and movements of each prawn of a pair in agonistic encounters during the 30 minutes video recordings.

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Figure 3: Effect on the mean dominance index (DI) of M. carcinus prawns before (control), during (injection) and after (post-injection) with 0.1cc of saline (n=5). Injection of vehicle solution alone did not change the DI of the injected submissive prawns within each pair. ANOVA Two Way (p<0.05).

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Figure 4: Individualized measures of dominance index (DI) for each agonistic parameter. Exposure to 100uL (0.1cc) of saline solution does not causes significant changes in the DI of submissive prawns in any of the parameters. The dominant prawns always had a well marked dominance status even after the injection. ANOVA Two Way (p<0.05).

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Figure 5: Effect on the mean dominance index (DI) of M. carcinus prawns before (control), during (injection) and after (post-injection) with 0.006ppm of DBP (n =5). Dibutyl phthalate solution, increased the DI of the injected submissive prawns within each pair, and the effect of the contaminant remains until the post-injection sessions. ANOVA Two Way (p<0.05).

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Figure 6: Individualized measures of dominance index (DI) for each agonistic parameter. Prawn exposure to 0.006ppm of DBP causes significant changes in the DI of the abdomen posture and movement/position of claws. Dominance status of prawns also was affected by the contaminant. ANOVA Two Way (p<0.05).

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Figure 7: Effect on the mean dominance index (DI) of M. carcinus prawns before (control), during (injection) and after (post-injection) with 0.006ppm of DEP (n =8). Diethyl phthalate solution does not increased the mean DI of the injected submissive prawns within each pair. In contrast, dominant status of alphas was abolished in the injection session compared to the control, even though the animals did not receive an injection. ANOVA Two Way (p<0.05).).

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Figure 8: Individualized measures of dominance index (DI) for each agonistic parameter. Prawn exposure to 0.006ppm of DEP causes significant changes in the DI of the body/legs posture and movement/position of claws. Dominance status of prawns also was affected by the contaminant. ANOVA Two Way (p<0.05).

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Figure 9: Effect on the mean dominance index (DI) of M. carcinus prawns before (control), during (injection) and after (post-injection) with 0.006ppm of BzBP (n =6). Injected animals with the phthalate tend to increase their aggressive behavior, but data does not reach significance. Alphas were dominant during the control session, but they reduced their DI in the injection session and their dominance statuses were repressed in response to the unexpected change in behavior among the submissive animals. ANOVA Two Way (p<0.05).

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Figure 10: Individualized measures of dominance index (DI) for each agonistic parameter. Prawn exposure to 0.006ppm of BzBP increases the DI of the abdomen posture and abolished the dominance status of alphas in the movement/position of claws parameter. ANOVA Two Way (p<0.05).

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Figure 11: Effect on the mean dominance index (DI) of M. carcinus prawns before (control), during (injection) and after (post-injection) with 0.1ppm of Cr3+ (n =5). The chromium solution increased the DI of the injected submissive prawns within each pair. The effect of the contaminant remains until the post-injection sessions. ANOVA Two Way (p<0.05).

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Figure 12: Individualized measures of dominance index (DI) for each agonistic parameter. Prawn exposure to 0.1ppm of chromium increases the DI of submissive prawns in moving their chelae. ANOVA Two Way (p<0.05).

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Figure 13: Effect on the mean dominance index (DI) of M. carcinus prawns before (control), during (injection) and after +2 (post -injection) with 0.005ppm of Cd (n =5). Cadmium solution increases the DI of the injected submissive prawns within each pair. Interestingly, this increase in aggression in the normally submissive prawn was accompanied by a statistically significant decrease in the levels of aggression of the normally dominant prawns when compared to control conditions, even though the animal s did not receive an injection. ANOVA Two Way (p<0.05).

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Figure 14: Individualized measures of dominance index (DI) for each agonistic parameter. Prawn exposure to 0.005ppm of cadmium increases the DI of submissive prawns in the abdomen posture and decreases the DI of alphas in their movement/position of claws and their movement towards the submissive prawns. After been injected, submissive prawns reverted their dominance status in some parameters in comparison to the control session. ANOVA Two Way (p<0.05).

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Figure 15: Effect on the mean dominance index (DI) of M. carcinus prawns before (control), during (injection) and after (post-injection) with 0.005ppm of Mn+2 (n =7). Manganese solution increased the DI of the injected submissive prawns within each pair. Dominant status of alphas was abolished in the injection session and the levels of aggression were reduced compared to control conditions, even though the animals did not receive an injection. ANOVA Two Way (p<0.05).

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Figure 16: Individualized measures of dominance index (DI) for each agonistic parameter. Prawn exposure to 0.05 ppm of manganese increases the DI of submissive prawns in the movement of chelae and decreases the DI of alphas in their body/legs position, movement/position of claws and movement of chelae. Dominance statuses of alphas were repressed in some parameters after submissive prawns been injected. ANOVA Two Way (p<0.05).

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Figure 17: Detailed observations of close interactions in aggressive behavioral assessments. No statistically significant effects were observed on the mean number of attacks, attacks duration, and the number of fights initiated by the dominant prawns (n=5), before (control), during (injection) and after (post-injection) physiological saline exposure. ANOVA One Way (p<0.05).

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Figure 18: Detailed observations of close interactions in aggressive behavioral assessments. Low concentrations of DBP significantly reduced the mean number of attacks, attacks duration, and the number of fights initiated by the dominant prawns during injection (n=8). ANOVA One Way (p<0.05).

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Figure 19: Detailed observations of close interactions in aggressive behavioral assessments. Injections of low concentrations of DEP significantly affect the mean number of attacks, attacks duration, and the number of fights initiated by the dominant prawns (n=8) in the injection session compared to the control. ANOVA One Way (p<0.05).

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Figure 20: Detailed observations of close interactions in aggressive behavioral assessments. No significant effects were observed on the mean number of attacks, attacks duration, and the number of fights initiated by the dominant prawns before (control), during (injection) and after (post- injection) BzBP administration in submissive prawns (n=6). ANOVA One Way (p<0.05).

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Figure 21: Detailed observations of close interactions in aggressive behavioral assessments. No significant changes were observed on the mean number of attacks, attacks duration, and the number of fights initiated by the dominant prawns before (control), during (injection) and after (post- injection) chromium injection on subordinates (n=5). ANOVA One Way (p<0.05).

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Figure 22: Detailed observations of close interactions in aggressive behavioral assessments. Injections of low concentrations of Cd+2 significantly affect the mean number of attacks, attacks duration, and the number of fights initiated by the dominant prawns (n=5). ANOVA One Way (p<0.05).

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Figure 23: Detailed observations of close interactions in aggressive behavioral assessments. Injections of low concentrations of Mn+2 significantly reduced the number of fights, duration, and the number of interactions initiated by the dominant prawns (n=7). ANOVA One Way (p<0.05).

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Table 1: Summary of the obtained results in the experiments measuring aggressive behavior using pollutants simulating those found in urban rivers.

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Experiment Dominance Index Agonistic Encounters prawn Ringer No effects on subordinates No effects on number of fights (saline) No effects on dominants No effects on duration of fights

No effects on fights initiates by dominants

DI of subordinates Number of fights DBP Dominants lost dominance Duration of fights status Fights initiated by dominants No effects on subordinates Number of fights DEP DI of dominants and abolished dominance status Duration of fights of alphas Fights initiated by dominants No effects on subordinates No effects on number of fights

BzBP Dominants lost dominance No effects on duration of fights status No effects on fights initiates by dominants No effects on number of fights DI of subordinates Cr+3 No effects on duration of fights Dominants lost dominance status No effects on fights initiated by dominants

DI of subordinates Number of fights Cd+2 DI of dominants and Duration of fights abolished dominance status of alphas Fights initiated by dominants

DI of subordinates Number of fights Mn+2 DI of dominants and Duration of fights abolished dominance status of alphas Fights initiated by dominants

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References

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Chapter 3 Locomotor activity as a measure of the effect of urban anthropogenic chemicals on freshwater prawns

Introduction

Increased industrialization, urbanization and human’s motivation to over exploit nature has led to imbalance of various intrinsic and extrinsic elements of environment causing global problems in the form of different type of chemical pollutants. Effects of environmental contaminants can be studied at various levels of organization, including biochemical and cell levels (genetics) , the level of individual organisms (behavior), and the population and community level (ecology) (Weis, Smith et al. 2001). Environmental chemicals have the potential to alter the nervous system functions of an animal including shifts in locomotor activities. Locomotion is at the basis of practically all other complex behaviors, due to its link with physiological, metabolic and neurological processes of the animal and their anatomical condition, being at the same time important for the environmental pressures (Amiard-Triquet 2009; Baatrup 2009; Oliveira, Almeida et al. 2013). Among the movements made by an animal, there are vital movements, such as breathing and cardiac movements, locomotive movements for prey finding and predator escaping, and behavioral movements, such as courtship and copulation (Negro, Senkman et al. 2012). Every movement, even the simplest, depends on the harmony of every single movement to complete a desired action. Movements are transmitted through the nervous system and the synaptic cleft by neurotransmitters, but their function can be affected by molecules, such as water pollutants, which can inhibits the nerve impulse or synaptic transmission. The impairment of locomotion by contaminants may have effects immediately on other behaviors involving movement, like social relationships, migration, and foraging which may induce changes in the population fitness, and possibly at community and ecosystem levels (García-de La Parra, Bautista-Covarrubias et al. 2006; Nørum, Friberg et al. 2010; Morillo-Velarde, Lloret et al. 2011). In some cases behavioral impairments of pollutants are manifested years after the exposure and there have been reports of delayed effects in aquatic organisms (Weis 2014). Different bioassays have demonstrated the sensitivity of potential water pollutants on animal specific behaviors, anatomy and physiology (Lorenzon, Francese et al. 2000;

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Lorenzon, Francese et al. 2001; Revathi, Arockia Vasanthi et al. 2011; Götze, Bose et al. 2014; Oulton, Taylor et al. 2014). In lobsters, scientists conclude that alkylphenols are endocrine disruptors to larvae at metamorphosis, delay molting, reduce growth, and are toxic at relatively low concentrations (Laufer, Baclaski et al. 2012). Various studies have been conducted recently on the bioaccumulation, toxicity and organ malformation in different species of the genus Macrobrachium (Revathi and Munuswamy 2010; Ogunsanya, Durborow et al. 2011; Revathi, Arockia Vasanthi et al. 2011; Shuhaimi-Othman, Yakub et al. 2011; Barbieri, Moreira et al. 2013; Soegianto, Winarni et al. 2013; Cresswell, Smith et al. 2014). Arsenic exposures in prawns produced significant effect in terms of length, weight, growth and deteriorated the animal condition (Rajkumar 2013). Study of toxicity of copper at different life stages in the freshwater prawn Macrobrachium rosenbergii, noted that after copper exposure, swelling of lamellae, multiple hyperplasia and necrosis were observed in gill, resulting in abnormal gill tips (Asih, Irawan et al. 2013). Sub-lethal concentrations (0.6 ppm) of water-soluble fraction of hydrocarbons have been shown to induce changes on protein expression level in the prawn Macrobrachium borellii (Pasquevich, Dreon et al. 2013) and also cause changes in the antioxidant and oxidative stress responses (Lavarías, Heras et al. 2011). The metabolic response of the freshwater prawn Macrobrachium nipponense changes with nitrate toxicity (Jiang, Dilixiati et al. 2014), and survival, productivity, weight gain and larval stage index of Macrobrachium amazonicum larvae decreased linearly with increasing ambient nitrite concentration (Hayd, Lemos et al. 2014). Changes in activity and locomotion patterns are other measures of the effect of water pollutants on different vertebrates and invertebrate species. For example, on invertebrates, locomotor ability of earthworms exposed to a range of soil lead (Pb), showed a general decrease with increasing Pb concentrations (Zheng and Li 2009). In a study with a freshwater oligochaete determined that copper produced time and concentration dependent reductions in the ability of tactile stimulation to evoke body reversal and helical swimming, and electrophysiological testing indicated that contaminant reduced conduction velocities throughout the giant nerve fibers (O’Gara, Bohannon et al. 2004). Acute exposure to lead inhibited several behavioral activities including locomotion, feeding, tentacle extension and emergence from the shell in a

172 | P a g e freshwater snail (Pyatt, Pyatt et al. 2002). Examining the modulation of Di (2- ethylhexyl) phthalate (DEHP) in the nervous system of D. melanogaster, results showed that the acid ester modulated the cholinergic mini-synaptic transmission of projection neurons in the antennal lobe decreasing significantly the frequency of action potentials, the frequency and amplitude of mini excitatory postsynaptic currents and the peak current amplitude of calcium channels (Ran, Cai et al. 2012). Exposure to acetylcholinesterase inhibitors in honeybees demonstrated that motor functions such as grooming activity decreased after the presence of the neurotoxic pesticides (Williamson, Moffat et al. 2013). Studies in vertebrates showed that in rodents perinatal exposure to low-dose BDE-47, an emergent environmental contaminant, causes hyperactivity in rat offspring (Suvorov, Girard et al. 2008). Moreover, the spontaneous locomotion activity of rats exposed to uranium was increased one day post exposure and the spatial working memory was less efficient six days post exposure, compared with control rats (Monleau, Bussy et al. 2005). In the same study scientists found that the contaminant was bioacummulated in the olfactory bulb, hippocampus, frontal cortex, and cerebellum. These data suggest that the action of chemical toxicants is evolutionarily conserved in mammals, and compounds are able to enter the brain after exposure, producing behavioral changes. This also has relevance to humans, because bioaccumulation can alter the interactions of the food web. Locomotor studies in crustaceans for example showed that isopods revealed average velocity and path length as the principle components separating the polluted site and control animals, and also specified that animals from the polluted site where significantly hyperactive when compared to all controls (Bayley, Baatrup et al. 1997). In contrast, behavioral responses, such as locomotor and ventilatory activities, were also significantly reduced in silver (Ag) exposed amphipods (Arce Funck, Danger et al. 2013), and copepods exposed to heavy metals and pesticide manifested higher ability to escape than controls, and cladocerans reduced their escape ability in response to the pesticide (Gutierrez, Paggi et al. 2012). Abnormal foraging behavior was observed in shrimps exposed to Zn+2 at untreated storm water concentrations compared to treated water (Oulton, Taylor et al. 2014). Studies confirm that heavy metals such as copper (Asiha, Irawana et al. 2013), nickel (Shanker 2011), cadmium (Kaoud and

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Rezk 2011) and other pollutants have the potential to alter general activity of freshwater prawns. A study that measures the impact of cadmium on carbonic anhydrase activity reveals that in vivo exposure causes inhibitory effects in crabs (Vitale, Monserrat et al. 1999).Assessing the effects of the pharmaceutical fluoxetine, the results showed that locomotion was significantly increased in crabs, with animals spending more time moving, and walking longer distances than controls (Mesquita, Guilhermino et al. 2011). An study evaluating the effect of continuous exposure to sub- lethal ammonia concentrations on locomotor activity rhythms showed that in control groups, organisms have tidal activity onset mainly in low tide and no response to light cycles while ammonia exposed crabs shifted their onset of activity to high tide, and a general increasing activity was observed along the experiment (Azpeitia, Vanegas- Pérez et al. 2013). Exposure to sub-lethal concentrations of Cd+2 did not change the nocturnal activity patterns of amphipods, although their swimming activity during the night was significantly decreased by exposure to concentrations of 0.24 and 0.28 mg/L of Cd+2 (Morillo-Velarde, Lloret et al. 2011). Results in a research using Palaemon serratus, showed that prawns exposed to the sub-lethal concentrations of fenitrothion (Oliveira, Almeida et al. 2013) and deltamethrin (Oliveira, Almeida et al. 2012) exhibited a significant inhibition of swimming velocity, (Oliveira, Almeida et al. 2013). Crayfish exposed to low and medium concentrations of an herbicide had significantly faster walking speeds than that of the controls, and exposure to low levels resulted in the highest walking speed across all treatment groups. so as concentration of the herbicide increased, the walking speeds of the crayfish decreased (Browne and Moore 2014). These results suggest that general activity of animals exposed to a certain chemical can be affected differentially, either by up or downregulation. To examine the possible effects of phthalates and heavy metals on prawn’s physiology our hypothesis claimed that locomotor behavior of M. carcinus would be affected by sub-lethal concentrations of common chemicals found in Puerto Rican watersheds.

Methodology

Animal model

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Male M. carcinus prawn on intermolt state measuring 7–15 cm in length from eyestalk to telson were obtained from non-urban rivers in Yabucoa, Río Grande and Canóvana’s, Puerto Rico. They were maintained in isolated tanks whose water was continuously filtered and aerated at the animal holding facility of the Department of Anatomy & Neurobiology of the University of Puerto Rico, Medical Sciences Campus under a photoperiod of 12-h light/12-h dark. Experiments were done at the end of the dark photoperiod (the animals are nocturnal). Water temperature was maintained at 24–26 °C and the pH adjusted to 7.4 (safe range is 6.9–8.5). Animals were fed a marine high-protein (40%) pelleted chow once every other day. All procedures involving the use of animals were approved by the University of Puerto Rico Medical Sciences Campus Institutional Animal Care and Use Committee (IACUC) prior to the start of the experiments. Experimental manipulations and behavioral tracking The locomotion patterns was registered using a customized View Point System© that tracks the animal´s movements and quantifies the distance traveled and the duration spent at three ranges of velocity (very slow, slow, fast), previously defined in the software. Each prawn was placed alone in a specialized setup with water tank surrounded by infrared light boxes and cameras designed to track the animal in the water tank under very low levels of visible light (Fig.1). The water within the tank was constantly aerated and the temperature maintained at 77 degrees Fahrenheit. Following a habituation period of ten minutes, the animal was recorded for 30 minutes. This protocol was repeated daily, at the same time each day, under control conditions and then following injection of the contaminant of choice (or Prawn Ringer). Animals were cold-anesthetized for 2-3 min and then were manipulated to expose the ventral side up. While held in this position, the prawn then received a single injection of the contaminant of interest at a concentration equal to the limits established by EPA for drinking water (dissolved in prawn saline or Ringer) using a syringe needle (Hamilton, 30 gauge) inserted into the hemolymph sinus. We also performed control experiments where the prawn received a single injection of vehicle solution (prawn saline) in the same manner as the contaminant injections. The prawn saline solution had the following composition: 11mL NaCl 1M,

275uL KCl 1M, 170uL CaCl2, 125uL MgCl2.6H2O, 0.0032g NaHCO3, pH= 7.5. Only

175 | P a g e one animal in each pair received the injection, and each animal was only used once. The injections were performed by a trained lab member, and solutions were injected slowly (over a 10-s period). The animal was then placed in a 20-L glass tank (16 x 8 x 10 in ~ [40.6 x 20.3 x 25.4 cm]) for 20 min to allow recovery from the injection (recovery period). Finally, several videos of 30 minutes were performed on consecutive days to determine any persistency, delay or reversible effect in the overall activity of prawns. Travelled area quantification To gain information of the space preference of prawns inside the locomotion arena, and if it would affected by the contaminant exposure, we developed a technique for quantifying the area covered by the injected animals (Fig.3). Movement paths extracted from the View Point were modified by drawing a box in the external part simulating the dimensions of the experimental tank. That box was subdivided into 40 smaller squares of the same size (56px x 56px), to which they were assigned a specific number to separate the edges (1 to 25) and the center (26-40). Quantification of covered area in the experimental tank by injected prawns was carried out with ImageJ, a freely available java-based public-domain image processing and analysis program developed at the National Institutes of Health (NIH) (Girish and Vijayalakshmi 2004; Rasband 2008; Schneider, Rasband et al. 2012). With this program we established a scale for all the photos and we measured the color intensity in each box. The system allows changing the pixels of the picture to an extent area in cm2. Finally we sum the areas of the boxes belonging to the edges versus the areas of the boxes in the center. Data analysis All data was analyzed using GraphPad Prism 6, and SigmaPlot 12.3, two scientific graphing and statistical analysis platforms. Locomotion data (movement durations and travelled distances), and data from travelled pathways by each prawn was analyzed using One or Two Way ANOVA respectively to compare the means of repeated measures.

Results

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According to the results obtained in the experiments of general locomotion, we found that all contaminants used (phthalates and heavy metals) cause specific effects on locomotor behavior of adult prawn species M. carcinus. Using concentrations allowed by EPA for phthalates in water was found that dibutyl phthalate, diethyl phthalate and benzilbutyl phthalate significantly affect patterns of locomotion, movement velocities and distances in the experimental tank. The effect of DBP on general activity was observed immediately the day of the injection and was reversible in the post-injection sessions (Fig.12). This plasticizer significantly increased slow and fast movement durations, as well as the slow and fast movement distances of prawns. Additionally, there were significant changes in the distances covered by the injected prawns, and animals ventured more into the center of the tank besides they covered a much greater area at the edges after exposure to the contaminant compared to controls (Fig.5). In the case of DEP, we found that there was a significant effect on the prawns after exposure. Prawns injected with 0.006ppm of DEP increased movement duration and distance travelled at slow and fast speeds. The effect on prawns was observed immediately after the injection and persisted in the following days. The pathways of prawns exposed to this phthalate also were affected and the animals seem to travel much more to the center of the tank the day of injection and in the post-injection sessions than in controls (Fig.4B). Analysis of the area showed that, after prawns received the contaminant, there was a significant increase in the covered area at the edges and also we identified a gradual increase in the space covered by prawns in the central region of the tank, but the results are not significant (Fig.6). Benzilbutyl phthalate, a high molecular weight phthalate, also affects general locomotor patterns of injected prawns. Plots created by prawns showed that the injection of BzBP increases activity in the center of the tank and was more pronounced in the recoveries (Fig.4C). The analysis of the total area covered by prawns showed that there are differences between edges and center in the controls and post-injections, but this is lost in the injection session since animals start venturing more to the center of the tank. However, exposure to BzBP apparently increases the area covered at the edges in the post-injection, but these data are not statistically significant (Fig.7). On the other part, the tracking system detected significant changes in the fast movement duration, as well as in the distances traveled by prawns at slow and fast motion (Fig.14).

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Heavy metals can also exert significant changes in the locomotor behavior of adult prawns. Injections of chromium (0.1ppm) affected the activity of prawns and a delay of one day was observed in the effect. During the post-injection 1, prawns increased their time traveling at very slow and fast speeds, as well as the distances traveled at slow and fast movements (Fig.15). Furthermore, there were changes in the locomotor patterns of those prawns after injection and on subsequent days (Fig.4D). Quantification of walked area indicates that Cr+3 had no significant effect, however a trend of the prawns toward to go over the central area and increase the covered area in the edges was observed (Fig.8). Our results are consistent with a study in rats exposed to Cr+3 that showed significantly reduction in the immobility time during a forced swimming test (Piotrowska, Siwek et al. 2013), suggesting that Cr+3 administration increase locomotor activity. Moreover, the use of cadmium in adult prawns caused a significant increase in activity patterns of locomotion in the center and edges of the tank (Fig.4E). Quantification of the total area covered by prawns after Cd+2 treatments showed a significantly increased in the total area covered at the edges of the tank in the post-injection session (Fig.9). This matched with the results of the movement and the distances of prawns in where a significant increase was observed in the slow and fast speeds in the post-injection sessions (Fig.16). Finally, the last heavy metal used (Mn+2, 0.05ppm) caused changes in the patterns of locomotion (Fig.4F), significantly increasing the duration and walked distance of prawns (Fig.17). Interestingly, immediately after Mn+2 injections, prawns significantly reduced the area covered at the edges presumably because they tend to explore more the center of the tank, but prawns preferred to remain at the edges of the tank instead to venture to the center in all sessions (Fig.10).

Discussion Phthalates may constitute up to 50 per cent of the total weight of PVC plastics, and their worldwide annual production is approximately 2.7 million metric tons (Bauer and Herrmann 1997), and the intense activity in the industrial and agriculture sectors inevitably increased the levels of heavy metals in natural waters. Plasticizers and heavy metals are not especially stable in products, so they can leach out and thus end up in the environment primarily affecting aquatic wildlife.

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In animals, a number of studies have reported that phthalate exposure was associated with altered neurobehaviors, including impaired spatial learning and reference memory (Tanaka 2005; Li, Zhuang et al. 2009), increased hyperactivity (Ishido, Masuo et al. 2004), and decreased grooming behavior (Hoshi and Ohtsuka 2009). However, the mechanisms of action of phthalate esters in the physiology of prawns are unclear. Studies on invertebrates demonstrated for example that in C. elegans phthalates cause toxic effects on locomotor behaviors including body bending, head thrashing, reversal frequency, and also affect thermosensory neurons (Tseng, Yang et al. 2013). According to studies using the giant freshwater prawn M. rosenbergii, it was found that phthalates esters damaged hemocytes, and BzBP or DEP treated hemocytes would primarily die via necrosis (Sung, Kao et al. 2003). Aditionally, four phthalates DEP, DHP, DPrP and DPP impacts the defense system of the prawn (Chen and Sung 2005). As hemocystes play a role in the immune system of invertebrates, a collapse would represent cell damage by water chemicals, which might cause specific abnormal behaviors on unprotected and vulnerable prawns. Other effects of DBP reported in crustaceans include decreased locomotor activity in Gammarus pulex at an exposure concentration of 500 ug/l (Thurén and Woin 1991). In our study we observed different effects of phthalates on the prawns. For example DEP and BzBP have a delayed effect after the day of injection. In contrast, the DBP acts quickly on the M. carcinus locomotor system, promoting an immediately activity and persisting for more than 24 hours after the injection. Comparisons of several phthalate esters (DMP, DEP, DBP, BBP, DHP and DEHP) in various aquatic organisms have shown differential acutely toxic effects for the concentration range tested (Call, Markee et al. 2001). This means that a same family of contaminants can have temporary effects on animal's physiology causing significant behavioral changes. A battery of behavioral markers in aquatic organisms have been revealed pollutant-induced stress syndrome (Viarengo, Lowe et al. 2007), suggesting that the altered locomotor activity (walking speed) of prawns may be due to the toxicant induced stress, which is an indication of neurotoxicity. Additionally, the resulted perturbation of locomotor behaviour (abnormal patterns) could be due by over-activation of enzymes that catalyze the process of locomotion (e.g. rapid breakdown of neurotransmitters at synaptic junctions), which in

179 | P a g e turn alters coordination between the nervous and muscular junctions (Rao, Begum et al. 2005). In all the assessments, water chemicals had a specific range of days that caused behavioral alterations, but the effects seem to return to normality. This is related to previous researches in which locomotor activity was affected after sub-lethal treatments with nickel (Bednarska, Gerhardt et al. 2010), and pesticides (Tooming, Merivee et al. 2014) in beetles, and after Cd+2 exposure in diptera larvae (Heinis, Timmermans et al. 1990), but the effects are of short-term duration. Water chemicals can be either accumulated or excreted. If accumulate, the location of accumulation varies between species (Hodson 2013), but is often in a detoxification organ, but also in the nervous system or at sites which are shed during molting. This suggested that pollutant exposure is more likely to produce a physiologic manifestation in the physiology of prawns, and the exposure time, contaminant concentration and its location at systemic level are those that can determine the range of effects on behavior and nervous system functions. The action of heavy metals in locomotion had been well documented using different scenarios. In vertebrates, locomotor activities of fish exposed to cadmium (0.1 and 0.25 mg 1−1 ), chromium (0.05, 2.4 and 24.0 mg 1−1), and zinc (0.1 and 5.0 mg 1−1) were respectively 1.3/3.8, 1.2/3.6/6.5, and 1.3/3.8 times as active as controls (Ellgaard, Tusa et al. 1978), and locomotor behaviors changed significantly in the larval zebrafish after the exposure to Cd+2 and Cr+3 (Jin, Liu et al. 2015). All contaminants tested in our assessments caused significant changes in the patterns of locomotion of prawns. Individuals exposed to Cr+3, Cd+2 and Mn+2 were more active than controls either the day of injection or posteriously. Neurobehavioral deficits, characterized by locomotor and emotional perturbations, and nigral glial activation are among the early signs of Mn+2 neurotoxicity caused by drinking water overexposure in mice (Krishna, Dodd et al. 2014). Studies on invertebrates showed that copper produced time- and concentration-dependent reductions in the ability of a freshwater oligochaete to evoke body reversal and helical swimming and significant reductions in giant nerve fiber conduction velocities (O’Gara, Bohannon et al. 2004). This suggests that if the contaminant is causing damage to the nervous system of an organism, cells can be initially activated to repair damage and nutrient levels and normalize

180 | P a g e neurotransmitters. However, this may cause the death of neuronal groups, which can translate into an effect at the level of behavior. In other cases, copper affects decapod crustacea with significant differences in the overall orientation ability of crayfish to locate an odor source and avoidance when previously exposed to copper (Sherba, Dunham et al. 2000; Lahman, Trent et al. 2015), and MeHg exposure caused behavioral impairment in lobsters as evidenced by a significant decrease in distance travelled, time spent walking, turn angle and body rotation (Rocha, Adedara et al. 2015). Macrobrachium lanchesteri (Palaemonidae) individuals exposed to acid mine drainage decreased locomotor activity levels (Mohti, Shuhaimi-Othman et al. 2012), indicating that the metals in the drainage played a role as a stress factors to the neuro- motor system. These results explain that exposure to pollutants can have a huge range of consequences in vertebrates and invertebrates, causing an impairment on orientation, location, escape and avoidance behaviors that may or may not be beneficial to aquatic organisms. In the natural environment reduced locomotor performance in prawns could be advantageous in decreasing their detectability by the instream and river adjacent predators. In our study, chemicals increase locomotive patterns, and contaminated prawns had abnormal pathways, spent more time in the open field and exhibited fast speed suggesting that they may be more visible to aquatic predators. This suggests that contaminants can be a cause for the decline of this species in urban rivers of the island, making it more vulnerable in their habitat by the continued degradation of freshwater bodies. Different behavioural states (e.g. locomotion) would be associated with different patterns of modulator release and modulatory effect (Blitz, Christie et al. 1999; Kravitz 2000). At the morphological level, toxicants may damage nerve cell bodies, axons, and myelin sheaths, but at the biochemical and neurochemical levels, they can alter synaptic transmission and neuromodulators, which may be associated with behavioral changes. Altered locomotor behavior may be a result of a modulation or toxicant damage to the nervous system. In arthropods, serotonin, dopamine, acetylcholine, and GABA are involved in locomotion, aggression, and feeding behavior. Many water pollutants act by raising levels of those molecules in the nervous system, thus creating a noticeable effect on the behavior of aquatic animals. For example, organic chemicals such as polychlorinated biphenyls (PCBs) alter brain levels of dopamine and

181 | P a g e norepinephrine affecting locomotor activity in fish (Fingerman and Russell 1980). Exposure to lead caused increased serotonin and an increase in feeding time and miscues in fathead minnows, and also produced elevated brain 5-HT and decreased GABA in catfish (Katti and Sathyanesan 1986; Weber, Russo et al. 1991). Locomotion in crabs was significantly increased after anti-depressant fluoxetine exposure, with animals spending more time moving, walking longer distances than controls (Mesquita, Guilhermino et al. 2011). Fluoxetine binds to serotonin transporters inhibiting the reuptake from the synaptic cleft and potentiating its effects. The neurophysiology and locomotor behavior of prawns can be influenced by water chemicals that potentiate the levels of serotonin helping to maintain the ChE activity and increasing neuromuscular cholinergic transmission. Altered ChE activity has been shown to cause behavioral impairments in locomotion, swimming capability, prey capture, and avoidance behavior (Jensen, Garsdal et al. 1997; Vieira, Gravato et al. 2009; Almeida, Oliveira et al. 2010). In crayfish, recordings of the pairs of large serotonergic neurons in the last thoracic (T5) and first abdominal (A1) ganglia revealed that these active neurosecretory cells modulate the abdominal postural and thoracic locomotor systems in both crayfish and lobsters (Beltz and Kravitz 1983; Beltz and Kravitz 1987). The over-activation or suppression of central monoaminergic pathways whose function is to set the level of excitability for the locomotor escape responses in crayfish, can be associated with the possible effects of water chemicals on neuromodulators in M. carcinus. Many of those neurotransmitters mentioned above have been identified in M. carcinus, so those toxicant-dependent mechanisms that alter chemical substances at the neural level, could be present in our model of study, and may contribute to the behavioural locomotor changes. Endocrine disruptors are known to cause hyperactivity in rats and decrease expression of dopamine receptor D1 by DBP and DEHP (Ishido, Morita et al. 2005), whereas increase dopamine receptor D2 by DBP. This mechanism may also be correlated with the observed hyperactivity in the prawns various days after the injection. Phthalates may act probably regulating the levels of gene expression, catecholamines, and protein-coupled receptor and dopaminergic neurotransduction systems. Also, locomotor aberrant behavior coincide with significantly elevated body concentrations of heavy metals and lowered protein and glycogen levels in isopods

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(Freddy Sorensen 2009). It is known the capacity of the hepatopancreas to detoxify heavy metals in crustaceans, but it may become saturated resulting in a rising blood concentration of metals that might interfere with biochemical reactions in which the metals are not normally involved. In addition, heavy metals are known to directly affect a number of voltage-gated ion channels (Arhem 1980; Hinkle, Kinsella et al. 1987; Atchison 2003; Marchetti and Gavazzo 2012). Among the channels affected by heavy metals are the voltage-gated calcium, sodium and potassium channels that underlie the production of action potentials. The alteration of such channels and synaptic transmission could explain behavioral impairment in general locomotion induced by contaminants in the present study. However, there is little evidence in crustaceans that phthalate or heavy metal exposure causes lethal toxicity, and the existing data are insufficient to evaluate the adverse effects of those water pollutants in the brain and the Central Nervous System.

Conclusions Locomotor behavior is an important trait of the integrity of organisms because it includes vital activities such as feeding, foraging, mating and, defense in their attempt to use environmental resources. In this perspective, laboratory studies in behavioral neuro-ecology are an aid in understanding how anthropogenic chemicals affect animal’s neural structure and functions and individual behaviors. From the present study it can be seen that sub-lethal effects of two types of urban river compounds, phthalates and heavy metals, alter the locomotor behavior of Puerto Rican native prawns. Exposure to sub-lethal concentrations of toxicants can produce biochemical changes resulting in disturbance of their general physiology and impairment in their normal behavior. Different behavioural analyses were obtained from video tracks, and pollutants affected all of them in different ways. They caused an increase of the mean walking speed, but also enhanced different traits related to activity and space use. Contaminated prawns (in most cases), spent less time moving at very slow speed, but walked and travelled shorter and larger distances more often. The video-tracking analysis showed that the effects were even more complex as prawns moved not only more, but also differently. Control individuals typically moved more along the edges of the tank, whereas prawns exposed to specific chemicals stayed more in open areas.

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In addition, our results showed that there was a rapid locomotor response of prawns to sub-lethal concentrations of phthalates from urban rivers. Other compounds such as cadmium and manganese cause a delayed effect in the activity of freshwater prawns. These results imply that chemicals strongly affect locomotor repertory of prawns but in different ways depending on their incorporation and metabolization, thus suggesting that they have different modes of action. Although administration of pollutants into prawns did not cause their death, immediate and persistent effects that resulted in aberrant movement patterns could be a cause for them to be more vulnerable in their natural environment. Our study confirms that urban anthropogenic chemicals exert effects at sub-lethal doses, but environmental concentrations may be higher of those used in the present work due to the elevated contamination near Puerto Rican watersheds. The observed behaviors thus far confirm our hypothesis of the toxicity of phthalates and heavy metals within the locomotor assessments. This study represents the first attempt to quantify behavioral responses of this species to urban toxicants using an automated method. Through accurate tracking analyses in lab conditions, specific behavioural effects have been highlighted, providing an initial understanding of the mode of action of various substances in the nervous system. The new possibilities offered by video-tracking software now make it possible to go even further in characterizing more behavioral endpoints from video recording. Our study helps with quantitative data to add new knowledge of varied locomotive patterns such as distance moved, space preference, speed, and general activity in a decapod crustacean. Future research is needed to produce new behavioral markers to improve our understanding of the toxicity of chemicals in our model, and also neurobiological approaches to determine the mechanisms that underlying the resulted behaviors. Unmask these mechanisms in an invertebrate organism with a relatively simple nervous system, could help us to be able to find clues to extrapolate the results in order to elucidate similar mechanisms in higher evolutionary organisms such as vertebrate mammals. With this research additional emphasis has been put on the importance of assessing physiological responses suitable for evaluating animal's individual performance and possible effects at population level, linking observed changes in biomarkers of activity to putative contaminant effects.

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Appendix

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Figure 1: Specialized setup of water tank for recording and assessment of general locomotion activity.

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Figure 2: A schematic representation of the experimental design. After acclimatization, prawns were allowed to walk in the experimental arena during six days. Afterwards prawns were exposed to the contaminant of interest, and the effects of the pollutant were observed until reversibility. Behavior assessments were performed using video recordings of a specialized tracking system.

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Figure 3: Developed methodology for quantifying prawn's travelled area in the locomotion tank. Modified photos of the patterns of locomotion were subjected to the ImageJ platform to obtain measures of covered area and place preference.

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Figure 3: Locomotor patterns of prawns using the View Point Customized Tracking System. Plots show M. carcinus behavior before and after exposure to DBP(n=5), DEP(n=8), BzBP(n=5), Cr+3(n=5), Cd+2(n=5), Mn+2(n=6), and Ringer solution (n=5). Examples of prawn tracking (black lines) during 30min video recording revealed that general locomotion pathways appear to be affected by specific water contaminants by increasing the overall level of activity and altering the locomotor patterns.

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Figure 4: Quantification of total area (mean ± SEM) of total area covered by prawns in the locomotion assessment using ImageJ. Physiological Ringer do not increased total area covered by prawns (n=5) either at the edges (p=0.187) or the center (p=0.980) of the tank after saline administration. The behavior of the shrimp in the tank appears to be similar to the natural environment. ANOVA Two Way (p<0.05).

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Figure 5: Quantification of total area (mean ± SEM) of total area covered by prawns in the locomotion assessment using ImageJ. DBP injected animals (n=5) increased total area covered at the edges (p=0.002), and ventured more towards the center of the tank (p=0.021). ANOVA Two Way (p<0.05).

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Figure 6: Quantification of total area (mean ± SEM) of total area covered by prawns in the locomotion assessment using ImageJ. DEP injected animals (n=8) increased total area covered at the edges of the tank in the post-injection session (p=0.015). The prawns decided to remain at the edges instead to venture to the center in all sessions. ANOVA Two Way (p<0.05).

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Figure 7: Quantification of total area (mean ± SEM) of total area covered by prawns in the locomotion assessment using ImageJ. Injected prawns (n=5) preferred to remain at the edges of the tank instead to venture to the center in control (p=0.038) and post-injection (p=0.005) sessions, but this was abolished within the injection session by the contaminant because the animals start venturing more into the center of the experimental arena (p=0.086). BzBP exposure apparently causes an increase in the area covered by prawns at the edges, but the data do not reach significant values (p=0.055). ANOVA Two Way (p<0.05).

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Figure 8: Quantification of total area (mean ± SEM) of total area covered by prawns in the locomotion assessment using ImageJ. Animals exposed to 0.1ppm of chromium (n=5) tend to increase total area covered at the edges of the tank in the post-injection session, but results are not significantly different (p=0.103). As a natural behavior, prawns preferred to remain at the edges of the tank instead to venture to the center in control (p=0.002) and post-injection (p<0.001) sessions, but this was abolished with the contaminant injection (p=0.063). ANOVA Two Way (p<0.05).

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Figure 9: Quantification of total area (mean ± SEM) of total area covered by prawns in the locomotion assessment using ImageJ. Animals exposed to 0.005ppm of cadmium (n=5) significantly increased total area covered at the edges of the tank in the post-injection session (p=0.015). After Cd+2 injections, prawns tend to increase their visits to the center of the tank, but preferred to remain at the edges in all sessions. ANOVA Two Way (p<0.05).

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Figure 10: Quantification of total area (mean ± SEM) of total area covered by prawns in the locomotion assessment using ImageJ. Animals exposed to 0.05ppm of manganese (n=6) significantly increased total area covered at the edges of the tank in the post-injection session (p<0.001). After Mn+2 injections, prawns significantly reduced the area covered at the edges (p=0.008), presumably because they tend to explore more the center of the tank. As expected, prawns preferred to remain at the edges of the tank instead to venture to the center in all sessions. ANOVA Two Way (p<0.05).

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Figure 11: Measures of general locomotion using the View Point Customized Tracking System. Effects of 0.1cc dose of saline on mobility (mean ± SEM) of prawns. Saline solution does not increase mobility in the open field test for all days of testing. ANOVA revealed no significant effect in the locomotion of prawns (p>0.05; n=5).

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Figure 12: Measures of general locomotion using the View Point Customized Tracking System. Effects of 0.006ppm dose of DBP on mobility (mean ± SEM) of prawns. ANOVA revealed a significant effect in the mobility of prawns in the open field test (p<0.0001; n=5), when compared with the effect of saline. Injections of low concentrations of DBP significantly affect movement velocities and distances travelled by prawns in the experimental arena. Effects in the locomotor behavior of prawns were observed immediately after the contaminant exposure.

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Figure 13: Measures of general locomotion using the View Point Customized Tracking System. Effects of 0.006ppm dose of DEP on mobility (mean ± SEM) of prawns. ANOVA revealed a significant effect in the mobility of prawns in the open field test (p<0.00;, n=8), when compared with the effect of saline. Movement velocities and distances travelled by prawns in the experimental arena were affected by the toxicant chemical. Effects in the locomotor behavior of prawns were observed immediately after the contaminant exposure and persisted until the recovery sessions.

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Figure 14: Measures of general locomotion using the View Point Customized Tracking System. Effects of 0.006ppm dose of BzBP on mobility (mean ± SEM) of prawns. ANOVA revealed a significant effect in the mobility of prawns in the open field test (p<0.0001; n=5), when compared the treatment with the controls. EPA allowed drinking water standard for BzBP significantly affect the duration and distances travelled by prawns in the experimental arena at fast speed. Locomotor behavior of prawns was affected in the post-injection 1.

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Figure 15: Measures of general locomotion using the View Point Customized Tracking System. Effects of 0.1ppm dose of Cr+3 on mobility (mean ± SEM) of prawns. ANOVA revealed a significant effect in the mobility of prawns in the open field test (p<0.0001; n=5), when compared the treatment with the controls. Locomotor behavior of prawns was affected in the post-injection session.

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Figure 16: Measures of general locomotion using the View Point Customized Tracking System. Effects of 0.006ppm dose of Cd+2 on mobility (mean ± SEM) of prawns. ANOVA revealed a significant effect in the mobility of prawns in the open field test (p<0.0001; n=5), when compared the treatment with the controls. Cd+2 significantly affect slow and fast movement velocities, as well as the distances travelled by prawns in the experimental arena. Locomotor behavior of prawns was affected in the post-injection session.

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Figure 17: Measures of general locomotion using the View Point Customized Tracking System. Effects of 0.006ppm dose of Mn+2 on mobility (mean ± SEM) of prawns. ANOVA revealed a significant effect in the mobility of prawns in the open field test (p<0.0001; n=6), when compared the treatment with the controls. Mn+2 significantly affect fast movement velocity, as well as the short and large distances travelled by prawns in the experimental arena. Locomotor behavior of prawns was affected in the post-injection session.

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Table 1: Overview of results obtained in the experiments of locomotion. All contaminants used provoke a significant effect on the general activity of prawns. The phthalates had their effects immediately or several days after its administration. Conversely, all heavy metals had a delayed effect on locomotor behavior of adult prawns. Saline injections caused no significant effect, and in all cases the effect was reversible.

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General Discussion

The study of population dynamics, the behavior of the species and the factors that influence the natural environment are critical to understanding the link with the nervous system. However, these processes have not been fully explored and are much more complex when we refer to riparian areas in tropical ecosystems. Puerto Rico is a tropical island with tourist development that has about 4 million inhabitants and is known for its wide variety of environments, which makes this an excellent place for various types of environmental neuroscience studies. The freshwater habitats on the island have a variety of organisms including fish and decapod crustaceans, many of which provide recreational and research ends. However, it has been suggested that populations of aquatic organisms in watersheds are vulnerable to varying degrees by huge human activities which includes the introduction of exotic species, building bridges and dams, canals, and water pollution. Many of these environmental problems could have consequences in terms of animal’s physiology, behavior and nervous system function, but this paradigm is poorly elucidated. For this reason, our work was designed to contribute to the overall knowledge and management of communities of organisms in the rivers of Puerto Rico using an ecological approach and noting the possible links to neural level in a macro- invertebrate model. We attained this goal with three main objectives. In the first part (chapter 1) we determined quantitatively the effects of the degree of urbanization on the relative abundance of prawn species M. carcinus on several representative rivers of the island. The second component (Chapter 2) describes how urban river contaminants can affect the neural basis of aggression in adult prawns, and finally, in the third part (Chapter 3), we identify changes in locomotor activity of adult prawns caused by the exposure to chemicals of anthropogenic origin. Our results demonstrate and strengthen the evidence of the effects of high urban incidence near rivers on sensitive species such as shrimps. However, we also examine and quantify the effects of other physical, chemical and geographical factors on the abundance of prawns to have a more comprehensive idea of the scenario in the non-urban and urban aquatic environments. The data obtained after 15 months of sampling indicated that prawn populations of the species M. carcinus were significantly lower in urban rivers compared to the reference non-urban river. In the sampling effort 231 | P a g e we captured a total of 172 individuals in the non-urban river compared with 0, 0, and 4 prawns in comparable areas of the three other urban streams. In addition high temperatures, high concentrations of sediment, salts and metal ions in urban rivers were recorded. All parameters measured in each river suggest that is a set of anthropogenic factors that are negatively acting on the population densities of native shrimps in urban rivers of Puerto Rico. The knowledge presented here on the distribution and abundance of prawns and their relationship with their environment is critical to the management and to detect trends over time. Available results from the monthly census showed that prawn populations and ecosystem status can be applied to decisions about damming, and alteration/regulation of normal watershed flow. This study provides guidance for the protection of unique resources in rivers and reduces their impacts on urban hotspots. The results of these techniques can be apply to decisions concerning surface water management in Puerto Rico, where macro- invertebrate community assessment can be used as a planning tool for managing water uses, for ambient monitoring, and for evaluating the effectiveness of pollution control measures. Additionally, for having knowledge of the impact of urban pollution on the behavior of aquatic fauna, we exposed different groups of adult prawns to several chemicals of interest to measure changes in aggressiveness. In these experiments we determined that specific pollutants had the ability to alter the aggressive behavior of submissive prawns after receiving a small dose of the contaminant (EPA Drinking Water Standards Allowed [ALDWS]). Dibuthyl phthalate (DBP), Chromium (Cr+3), Cadmium (Cd+2), and Manganese (Mn+2) increased the aggressive behavior of subordinates in a pair, and in some cases decreased the dominance status of alphas. Similarly, all the contaminants tested (three phthalates and three heavy metals) caused a significant alteration in prawn's locomotor patterns. Rapid and erratic movements and a significant increase of covered area in the experimental tank were the most pronounced effects created by the administration of urban chemicals. The effects observed in all trials were reversible, though not in all cases in the same time range after chemical injection. The arrival of pollutants into the circulatory system of prawns seems to be the threshold for the initiation of immediate or late responses in the aggressive and locomotive behaviors. In this case we believe that physiological

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Figure 18: Proposed model to explain the effects of urbanization and water pollution on rivers. Toxic chemicals can pass directly to the circulatory hemolymph and can reach the Nervous System of macro-crustaceans. This can cause possible physiological and neural disruptions that are reflected in behavior and in turn alter population dynamics within those ecosystems.

outcomes depend on several factors such as chemical concentration, and rapid accumulation of toxic metabolites within the nervous system. Also each particular effect may be associated to changes in substances, molecules or neuromodulators that are activated by the presence of toxic urban chemicals, but these assumptions need further research for a more accurate explanation.

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The nervous system is often regarded as a central processing unit that uses environmental cues and its internal state to plan future actions, and then generates behavioral commands. Recent results suggest that behavior can best be understood within the context of the biomechanics of the body, the structure of an organism’s environment, and the continuous feedback between the nervous system, the body and the environment (Chiel and Beer 1997). Based in our work, what happens around an ecosystem can directly affect existing organisms (Fig.18). In freshwater ecosystems massive constructions and emerging contaminants (everyday wastes), are probably the main factors for the decline of species of macro-crustaceans such as M. carcinus. Furthermore, the physiological effects of pollutants may be linked to the low population rates found in urban rivers because the increase in aggressive behavior and aberrant movement patterns could place them in a vulnerable spot within the food web (e.g. predators). The set of human-induced factors were negatively correlated with the abundance of prawns in the rivers of urbanized areas, suggesting an alarming issue with the survival of this species. However, the high sensitivity of this species to environmental pressures forced by humans could put it as bioindicator in highly susceptible waterways. Because living systems depend on their environment, we argue that we need to understand how nervous system activity-dependent adaptation mechanisms integrate ecological pressures in order to explain individual variations in performance, and ultimately to explain the adverse consequences on population assemblages.

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