gyrkppfngsifamide (gly-sifamide) modulates aggression in the freswater prawn

GYRKPPFNGSIFamide (Gly-SIFamide) ModulatesAggression in the Freshwater Prawn Macrobrachium

rosenbergii

NIETZELL VAZQUEZ-ACEVEDO1,2, NILSA M. RIVERA1,2,ALEJANDRA M. TORRES-GONZALEZ2, YARELY RULLAN-MATHEU2,

EDUARDO A. RUIZ-RODRIGUEZ3, AND MARIA A. SOSA1,2,*1Department of Anatomy and Neurobiology, School of Medicine, Medical Sciences Campus, University

of Puerto Rico, PO Box 365067, San Juan, Puerto Rico 00936-5067; 2Institute of Neurobiology,Medical Sciences Campus, University of Puerto Rico, 201 Blvd Del Valle, San Juan, Puerto Rico

00901; and 3Department of Social Sciences, Cayey Campus, University of Puerto Rico, Cayey, PuertoRico 00736

Abstract. The freshwater prawn Macrobrachium rosen-bergii is a tropical crustacean with characteristics similar tothose of lobsters and crayfish. Adult males develop throughthree morphological types—small (SC), yellow (YC), andblue claws (BC)—with each representing a level in thedominance hierarchy of a group, BC males being the mostdominant. We are interested in understanding the roleplayed by neuropeptides in the mechanisms underlying ag-gressive behavior and the establishment of dominance hi-erarchies in this type of prawn. SIFamides are a family ofarthropod peptides recently identified in the central nervoussystem of insects and crustaceans, where it has been linkedto olfaction, sexual behavior, and gut endocrine functions.One of the six SIFamide isoforms, GYRKPPFNGSIFamide(Gly-SIFamide), is highly conserved among decapod crus-taceans such as crabs and crayfish. We wanted to determinewhether Gly-SIFamide plays a role in modulating aggres-sion and dominant behavior in the prawn. To do this, weperformed behavioral experiments in which interactions be-tween BC/YC pairs were recorded and quantified before andafter injecting Gly-SIFamide directly into the circulatinghemolymph of the living animal. Behavioral data showed

that aggression among interacting BC/YC prawns was en-hanced by injection of Gly-SIFamide, suggesting that thisneuropeptide does have a modulatory role for this type ofbehavior in the prawn.

Introduction

Crustaceans such as lobsters and crayfish have been usedextensively as model systems for studying the neural basisof aggressive behavior. In these animals, status as dominantor subordinate during agonistic encounters is established onthe basis of body size and experience. The giant freshwaterprawn Macrobrachium rosenbergii (de Man, 1879) is a typeof clawed shrimp that lives in some tropical and subtropicalareas of the world and has characteristics similar to those oflobsters and crayfish concerning general body plan, internalanatomical organization, and some aspects of behavior(Huxley, 1973; Bliss, 1982). Sexually mature males developthrough three distinct morphological types that differ in theratio of claw to body length, growth rate, and claw color andmorphology. The morphotypes are designated blue claw(BC), yellow claw (YC), and small claws (SC), each rep-resenting a level in the dominance hierarchy of the popula-tion, with BCs being the most dominant and SCs the mostsubordinate (Ra’anan and Cohen, 1985).

Freshwater prawn adult males also show behavioral dif-ferences according to morphotype in terms of territorialityand behavior toward females (Ra’anan and Cohen, 1985).The BCs are territorial, sexually active, and tend to protect

Received 11 November 2008; accepted 28 July 2009.*To whom correspondence should be addressed: E-mail: [email protected]: Gly-SIFamide, GYRKPPFNGSIFamide; Val-SIFamide,

VYRKPPFNGSIFamide; Ala-SIFamide, AYRKPPFNGSIFamide; SC,male small claw; YC, male yellow claw; BC, male blue claw; DI, domi-nance index.

Reference: Biol. Bull. 217: 313–326. (December 2009)© 2009 Marine Biological Laboratory

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females; the YCs are nonterritorial, sexually incompetent,and tend to attack females; the nonterritorial SCs arestrongly attracted to females that are receptive to mating andoccasionally succeed in copulating with a female by sneak-ing in between it and a BC (Ra’anan and Sagi, 1985).

Even though the most obvious characteristic that distin-guishes the prawn morphotypes is claw size and coloration,this is not the only relevant cue in the establishment of thedominance hierarchy of a population. The relative propor-tions in an enclosed population of the three male morpho-types, SCs, YCs, and BCs, remain nearly constant at 5:4:1,respectively, under a wide range of environmental condi-tions (Brody et al., 1980; Cohen et al., 1981; Ra’anan et al.,1991). These ratios are maintained in a dynamic state, inwhich individual males are capable of undergoing a trans-formation from one morphotype to another, following anirreversible order: from SC to YC to BC. Such transforma-tions occur whenever large individuals either die or areselectively removed from the local habitat (Ra’anan andCohen, 1985). Thus, since the transition to a higher-ordermorphotype is influenced by the ratio of each morphotypealready present in the animal�s environment, it is possiblethat, in addition to the coloration of their claws, some formof chemical cues for status and size may be involved in theanimal’s assessment of its position within the hierarchy.Similar cues signaling sex, status, and size have been de-scribed in other crustaceans (e.g., Hazlett, 1985, 1990; Cald-well, 1987).

We are using this model system because the status of themales as dominant or subordinate is of a more fixed naturethan in other crustaceans, being established on the basis ofclaw type first and body size second (Barki et al., 1992).Also, the other differences in behavior, in terms of territo-riality and interactions with females, make this model sys-tem interesting for comparative behavioral studies.

An extensive body of evidence links biogenic amines,such as serotonin and octopamine, with actions on thecrustacean nervous system that result in behaviors associ-ated with dominance or subordination. Some studies havefocused on understanding the role played by postural sys-tems controlling abdomen muscles in agonistic behavioralinteractions in lobsters and crayfish (Livingstone et al.,1980; Harris-Warrick, 1985; Kravitz, 1988; Ma et al., 1992;Sosa and Baro, 2002). Injections of biogenic amines inducespecific types of postures linked to social status, such asserotonin-flexed abdomen and octopamine-hyperextendedabdomen (Livingstone et al., 1980; Kravitz, 1988; Sosa andBaro, 2002). Moreover, injected serotonin induces aggres-sion in crayfish and lobsters (Livingstone et al., 1980;Kravitz, 1988; Huber et al., 1997a, b; Huber and Delago,1998; Tierney and Mangiamele, 2001; Tierney et al., 2004).We showed previously that injections of serotonin andoctopamine can modulate the levels of aggression in thefreshwater prawn (Sosa and Baro, 2002). Injections of se-

rotonin in YC males resulted in increased aggression, whileinjections of octopamine in BC males resulted in increasedsubmissiveness. This evidence suggests that biogenicamines play a role in modulating aggressive behavior inthese animals.

Escape responses (e.g., tailflips) and their underlyingneural basis have also been studied extensively in crusta-ceans (Wine and Krasne, 1972, 1982; Edwards et al., 1999;Krasne and Edwards, 2002; Herberholz et al., 2004; Espi-noza et al., 2006). Evidence also supports the role of sero-tonin in escape systems of crustaceans (Glanzman andKrasne, 1983; Yeh et al., 1996, 1997; Krasne et al., 1997;Teshiba et al., 2001; Edwards et al., 2002; Antonsen andEdwards, 2007). In the crayfish, it has been found thatmodulatory actions of serotonin on synaptic responseswithin a circuit involved in the escape response depend onthe initial social status of the animal (Yeh et al., 1996,1997). In subordinates, serotonin inhibits escape, but indominants it facilitates the lateral giant (LG) escape reflex.It has also been shown that burrowing, an activity notdirectly associated with fighting, is repressed in subordinatecrayfish while in the presence of a dominant animal, but isreestablished when the animal is alone for a period of timeafter the determination of its status as subordinate (Herber-holz et al., 2003). The simultaneous occurrence of theinhibition in burrowing with that of aggression in thesesubordinate animals suggests a common or similar under-lying mechanism. Thus, while the neural mechanisms in-volved in determining social status and aggressive behaviorinitially are not well known, there is ample evidence dem-onstrating the link between neuromodulatory influences andmaintenance or changes in said status.

Serotonin and octopamine play a primary role in modu-lating aggression and fighting behavior in crustaceans, in-cluding the freshwater prawn, but it is understood that theyare not the sole determinant elements. Existing modelsproposed to explain the roles of biogenic amines cannotfully explain all aspects of aggressive behavior and how itcan be adjusted to varying circumstances. In effect, nosingle chemical or transmitter system is responsible forgenerating or modulating these complex behaviors. Theinteraction and resulting balance of circulating transmittersis most probably responsible for controlling and regulatingsuch behaviors (Wood et al., 1995). Neuropeptides are verylikely candidates for acting as modulators of aggression. Athorough understanding of the roles neuropeptides are likelyto be playing in modulating agonistic behaviors in theprawn will help to expand the inventory of potential targetmolecules, binding sites, messenger systems, and genes forcontrolling or regulating various forms of interactive behav-iors. This knowledge will further our understanding of theneural basis of aggressive behavior.

Over the past decade, a number of studies have usedtechniques of mass spectrometry, immunohistochemistry, or

314 N. VAZQUEZ-ACEVEDO ET AL.

bioinformatics to identify peptides in the nervous system ofcrustaceans (Nilsson et al., 1998; Yasuda-Kamatani andYasuda, 2000, 2004; Skiebe et al., 2002, 2003; Li et al.,2002, 2003; Nichols, 2002; Huybrechts et al., 2003; Bulauet al., 2004; Yasuda et al., 2004; Messinger et al., 2005).These studies have identified a wide variety of novel neu-ropeptides whose distribution and physiological functions innervous tissues are not yet known (Christie et al., 2006).One such peptide is GYRKPPFNGSIFamide (Gly-SIF-amide), recently identified in the nervous system of variouscrustacean species (Sithigorngul et al., 2002; Huybrechts etal., 2003; Yasuda et al., 2004; Yasuda-Kamatani and Ya-suda, 2006; Messinger et al., 2005; Sullivan and Beltz,2005; Polanska et al., 2007; Gard et al., 2009; Ma et al.,2009a, b), including the freshwater prawn (Ma et al.,2008b). A related analog, VYRKPPFNGSIFamide (Val-SIFamide), has been described in the American lobsterHomarus americanus (Christie et al., 2006; Cape et al.,2008; Ma et al., 2008a; Dickinson et al., 2008), and morerecently in the European green crab Carcinus maenas (Maet al., 2009a). A third analog, AYRKPPFNGSIFamide(Ala-SIFamide), appears to be the native form in mostinsects (Janssen et al., 1996; Vanden Broeck, 2001; Auds-ley and Weaver, 2006; Christie, 2008a, b; Roller et al.,2008; Weaver and Audsley, 2008). Another three isoformshave been described in insects and crustaceans (Verleyen etal., 2009).

While members of the SIFamide family have been iden-tified in a number of arthropods and appear to be widelydistributed in the central nervous system, very little isknown about the physiological roles they play in any spe-cies. The fact that the sequence of SIFamide is extremelyconserved among species suggests an important function forthis neuropeptide (Verleyen et al., 2009). In Drosophila,Ala-SIFamide is restricted to only four neurons in the brainand appears to be involved in modulating sexual behavior(Terhzaz et al., 2007). In other insects and crayfish, theexpression patterns of the SIFamide peptides in specificareas of the brain support involvement in the integration ofvisual, tactile, and olfactory information (Verleyen et al.,2004, 2009; Yasuda et al., 2004; Yasuda-Kamatani andYasuda, 2006; Polanska et al., 2007). In the Americanlobster, immunohistochemical and physiological data aresuggestive of modulatory actions of Val-SIFamide in thestomatogastric nervous system (Christie et al., 2006; Rehmet al., 2008). The recent identification of Gly-SIFamide inthe crab midgut implies a possible gut endocrine/paracrinerole for members of the SIFamide family (Christie et al.,2007). However, widespread expression patterns in otherareas of the nervous systems of various arthropods clearlysuggest that much remains to be learned about the functionsof these neuropeptides.

Dominance and aggression are involved in the sexualbehavior of many species, including the prawn. These ele-

ments of interactive behavior are also influenced directly byinformation the animal receives from its immediate externalenvironment by means of visual, tactile, and olfactory cues.Since, as mentioned above, SIFamide has been suggested tobe involved precisely in the integration of visual, tactile, andolfactory information (Verleyan et al., 2009), as well as inregulating sexual behavior (Terhzaz et al., 2007), we pos-tulated that maybe this amide also functions in modulatingdominance and aggressive behavior in the prawn.

The purpose of this study was thus to determine the rolethat Gly-SIFamide plays in modulating the prawn�s aggres-sive behavior during agonistic encounters, as a means tostart elucidating how neuropeptides function in the mecha-nisms underlying plasticity of interactive behaviors. Wefocused on the question of whether exogenous Gly-SIF-amide increases or decreases the level of aggressivenessdisplayed by interacting prawns. To address this question,we performed behavioral observation experiments in whichthe interactions between BC/YC pairs were recorded andquantified before and after Gly-SIFamide was injected di-rectly into the circulating hemolymph of the living animal.

Materials and Methods

Experimental animals

The freshwater prawn Macrobrachium rosenbergii is acrustacean model system characterized by adults with threeclaw morphotypes that differ in color, relative size, andspination, each morphotype correlating with a fixed domi-nance status and other defining features of the animal’smodes of interaction with others, including territoriality andcourtship behavior (Ra’anan and Cohen, 1985; Ra’anan andSagi, 1985; Barki et al., 1992). The fixed nature of thedominance hierarchies established by the prawn makes thismodel interesting for behavioral studies.

Male prawns measuring 8–15 cm in length from eyestalkto telson were obtained from private aquaculture farms inLajas and Canovanas, Puerto Rico. They were maintained incommunity tanks whose water was continuously filtered andaerated at the Animal Care Facility of the Institute ofNeurobiology of the University of Puerto Rico MedicalSciences Campus under a photoperiod of 12-h light/12-hdark. Experiments were done at the end of the dark photo-period (the animals are nocturnal). Water temperature wasmaintained at 26–28 °C and the pH adjusted to 7.4 (saferange is 6.9–8.5). Animals were fed a high-protein (�40%)pelleted Purina chow once every other day. All proceduresinvolving the use of animals were approved by the Univer-sity of Puerto Rico Medical Sciences Campus InstitutionalAnimal Care and Use Committee (IACUC) prior to the startof the experiments.

315SIFAMIDE AND CRUSTACEAN AGGRESSION

Behavioral observations

Since the dominance hierarchy of this species of prawn ispredetermined on the basis of the animal�s morphotype(Ra’anan and Cohen, 1985; Ra’anan and Sagi, 1985; Barkiet al., 1992), specific pairs of BC and YC prawns did notneed to be placed together before experiments to establishdominance, as is required with other crustacean models. Allanimals used for behavioral observations had been previ-ously kept in a communal tank containing male prawns ofall morphotypes, as well as females, in the proportions theyare normally found in their natural habitats (50% SC, 40%YCs, 10% BCs; Brody et al., 1980; Cohen et al., 1981;Ra’anan et al., 1991). Interactions were observed in an 80-lglass tank (24 � 12 � 16 in [�60.9 � 30.5 � 40.6 cm]).All behavioral observations were recorded on videotapeusing a camcorder (Panasonic Palmcorder PVA228) or dig-ital camera (SONY Handycam PC350) placed in front of thetank at the same time as parameters of interest were notedon paper. Observations (and their corresponding numericalvalues) during each session were registered at 30-s intervalswhen analyzing the video recordings. A grid of squares withsides of 4 cm was marked at the bottom of the tanks usingwhite tape against a black background. All other sides of thetanks, except the front, were also covered with a blackbackground to increase contrast. To minimize the possibil-ity of the animals reacting to the presence or movements ofthe observer, a black curtain was placed in front of theobservation tank with a small opening for the lens of thecamera and another for the eyes of the observer. Waterfilters, heaters, and aeration tubes were temporarily re-moved during observation sessions to minimize sources ofdistraction for the animals. Before reuse, each test aquar-ium was thoroughly rinsed and filled with freshly preparedwater.

Quantitative assessment of aggressive behavior

We measured parameters that serve as indicators of dom-inance/subordination in instances in which a BC and a YCare placed in the same tank. These parameters were thesame as those used for prawns by Barki et al. (1992) andsimilar to those used for crayfish by Huber and Kravitz(1995), with some additions and modifications based on ourown observations (Table 1). The values in the Level columndesignate the weight of the parameter as an indicator ofdominance/subordination, 6 indicating the most highlydominant behavior and 0 indicating complete or total sub-ordination (based on Barki et al., 1992). Thus, the weightvalues ascribed to each parameter at each instance of ob-servation serve as a measure of the level of aggression.These values were used to calculate a dominance index (DI)for each individual tested in the paired interactions, calcu-lated as the sum of the weights of all the instances recordedduring the observation period, divided by the total number

of instances. All DI values are shown as mean � S.E.M. Todetermine whether specific parameters could be differen-tially affected by the injections in comparison to the others,the mean level of aggression reached for each parameterwas also calculated for each animal during the observationperiods.

Previous studies carried out in our laboratory have shownthat when a pair of BC and YC prawns are placed together,

Table 1

Parameters used to calculate a dominance index (DI) for eachindividual tested in the pair interactions

Parameter Level

Movement toward or away from each otherRush toward other prawn while facing it 6Walk toward other prawn while facing it 5Turn toward other prawn but not walking to it 4Turn away from facing other prawn, without walking

away from it 3Walk away from other prawn while facing it 2Walk away from other prawn without facing it 1Rush to move away from other prawn without facing it 0

Abdomen postureFully flexed 6Partially flexed 4, 5Extended (horizontally) 3Partially hyperextended 1, 2Fully hyperextended 0

Position of body and walking legsLegs fully flexed/body touching bottom 0, 1Legs partially flexed/body partially lifted 2, 3, 4Legs fully extended/body fully lifted 5, 6

Movement and position of clawsCrossed in front of eyes 0Touching bottom while extended 1Touching bottom while flexed 2At body level 3Above body level 4Extended at or above body level 5Quickly thrust forward 6Used for lifting the other prawn 6

Movement of chelaClosed/not touching other prawn 0Closed/touching other prawn 1Open/not touching other prawn 2Open/touching other prawn 3Scissoring in front of other prawn 4Open, grabbing other prawn 5Nipping or tearing 6

The values in the Level column indicate the weight or level of the parameteras an indicator of dominance/subordination, 6 indicating the most highlydominant behavior, 0 indicating complete or total subordination. These pa-rameters are based on those used by Barki and colleagues (1992) with thefollowing modifications: (1) a more detailed specification of levels or weightsby describing in words what each whole number in the scale represents; (2)subdividing the parameter of claw movement into the two parameters“move-ment and position of claws” and “movement of chelas”; and (3) changing thescale from one that included negative numbers (�3 through �3) to one withonly positive numbers (0 through �6).


30 min of recording is sufficient to obtain a reliable quan-titative estimate of the behaviors of interest, and that furtherextending these observation periods does not significantlyalter the resulting values of the DI (Sosa and Baro, 2002).Therefore, animals were observed and videotaped for 30min before the injection (control or pre-injection behavior).A session following the same sequence was conducted afterone of the animals was injected with Gly-SIFamide, or withprawn saline (vehicle solution) in the case of control injec-tions (post-injection behavior). Tapes were watched in arandom order, and data were registered by three trainedobservers who were blind to the injection status of theanimals being observed.

Injections

The approach we used to inject the agents of interest wassimilar to that used by Glanzman and Krasne (1983), withsome modifications. An animal whose interaction with an-other had been observed and recorded was cold-anesthe-tized for 5–7 min and was then held, dorsal side up, on theedge of a deep preparation dish containing aerated tankwater in such a manner that the animal was flexed at thethoracoabdominal junction and its cephalothorax was sub-merged in the water so it could breath. While held in thisposition, the prawn then received a single injection of Gly-SIFamide (GenScript, Piscataway, NJ) at a concentration of1 � 10�3 mol l�1 (dissolved in prawn saline or Ringer)using a syringe needle (Hamilton, 30 gauge) inserted intothe dorsal abdominal artery, just before it entered the heart.We also performed control experiments where the prawnreceived a single injection of vehicle solution (prawn saline)in the same manner as the Gly-SIFamide injections. Theprawn saline solution had the following composition (inmmol l�1): 220 NaCl, 5.5 KCl, 13.5 CaCl2, 2.5 MgCl2, 2.0NaHCO3, pH � 7.4. Only one animal in each pair receivedthe injection, and each animal was only used once. Injec-tions were carried out both in dominant BC and submissiveYC animals. The injections were performed by a trainedindividual, and solutions were injected slowly (over a 10-speriod). The animal was then placed in a 20-l glass tank(16 � 8 � 10 in [�40.6 � 20.3 � 25.4 cm]) for 45 min toallow recovery from the injection (recovery period). Theanimal was not placed in contact with the same animal ithad been tested with during the control sessions until itsmobility level was reestablished to a level comparable tothat observed before the injection (see below). After the45-min recovery period, the animal was placed in the be-havioral observation 80-l glass tank (24 � 12 � 16 in[�60.9 � 30.5 � 40.6 cm]) in contact with the other prawn,and their interactions were recorded and compared withthose of the pre-injection observation session. After testing,prawns were placed in isolated tanks and observed for 4

days to make sure they would not molt. Data from animalsthat molted during this period were discarded.

Number and duration of attacks

Besides measuring the overall DI and the DI level forindividual parameters, we also counted the number of at-tacks and the duration of fights for each BC/YC pair, beforeand after injections. The beginning of an attack was regis-tered when one of the animals thrust forward its chelatowards the other animal. The attack was considered to beover once one of the animals retreated, escaped, or stoppedpursuing the other. Retreating was defined as walking awayfrom an attacking animal until being positioned out of reachof the latter. Escaping was defined as the triggering ofreflexive flexion of abdominal muscles so that the animalquickly pulls itself away from the attacking party.

Locomotor activity

To assess possible changes in locomotion as a result ofinjections of vehicle (prawn saline) or Gly-SIFamide, wecounted the number of lines demarcating a square pattern atthe bottom of the tank that each prawn crossed during eachconsecutive 30-s interval for each 30-min recording period,before and after injections.

Statistical analysis

We used Sigma Stat ver. 3.0 (Systat Software, Inc.,Chicago, IL) for all the statistical analysis of the behavioraldata. Parametric one-way analysis of variance (ANOVA)was used to compare parameters of the DI between thedifferent morphotypes, under control conditions and afterinjections. When significant differences were identified, apost hoc multiple comparison test (Tukey’s test) was used todetermine whether the observed significant differences werebetween the DIs of BCs and YCs either before or afterinjection, and when comparing pre- and post-injection DIsfor BCs or pre- and post- injection DIs for YCs. We used thesame statistical analysis to compare differences betweenhow many times each animal performed each of the param-eters used to calculate the DI. In all cases, results wereconsidered significant when P 0.05.

Results

The effect of Gly-SIFamide on aggressive behavior wasassessed in the prawn by injecting this neuropeptide into thecirculating haemolymph through the dorsal abdominal ar-tery. To determine the potential effects of the injections onthe interactive behavior between morphotypes, we paired aBC individual and a YC individual under various experi-mental conditions, described below. The animal�s morpho-type was determined on the basis of the coloration andrelative size of its claws. The ratio of claw-to-body length of


BCs is normally above 1.5, and that of YCs is normallyabove 0.5 and below 1.5. Values for animals used in thevehicle and Gly-SIFamide injections experiments are shownin Tables 2 and 3, respectively. A dominance index (DI)was calculated for each individual tested in all the pairedinteractions (the higher the DI value, the higher the domi-nance level).

Typical behavior of paired BC and YC prawns undernormal conditions

Under normal (control) conditions, when a BC and a YCare placed together in an 80-l glass tank (24 � 12 � 16 in[�60.9 � 30.5 � 40.6 cm]), the BC establishes itself as thedominant animal and tends to attack the YC. This behaviorpattern continues until an escape response is elicited in theYC or the animals are separated. In control experimentsprior to assessing the effect of injecting submissive animalswith Gly-SIFamide (e.g., pre-injection behavior: Fig. 1), themean DI for BCs was 2.10 � 0.10 (n � 5), while that ofYCs was 1.37 � 0.09 (n � 5). A similar pattern wasobserved in the pre-injection behavior for other experimentscarried out to assess the effect of injecting Gly-SIFamide indominant animals (Fig. 2), as well as in experiments inwhich only prawn Ringer was injected to either morphotype(Figs. 3 and 4).

Gly-SIFamide injection in the YC prawn

When the YC was injected with Gly-SIFamide (1 � 10�3

mol l�1), the typical behavior of paired BC and YC prawnsusually observed under normal conditions appeared to bereversed (Fig. 1). YCs injected with Gly-SIFamide behavedvery much like BCs do under control conditions (mean DIof 2.16 � 0.08; n � 5). Interestingly, this increase inaggression in the normally submissive prawn was accom-panied by a statistically significant decrease (one-wayANOVA, Tukey’s test, P � 0.041) in the levels of aggres-sion of the normally dominant BC (mean DI of 1.62 � 0.17,n � 5) when compared to control conditions (Fig. 1), eventhough the BC animal did not receive an injection.

Gly-SIFamide injection in the BC prawn

We also injected Gly-SIFamide into the circulating he-molymph of BC in other BC/YC pairs. Injections of Gly-SIFamide (1 � 10-3 mol l�1) increased even more the BC�snormal aggression toward the YC (Fig. 2). The BC injectedwith Gly-SIFamide very quickly approached the YC andstarted pushing and grabbing him. The YC was alwayscornered and repeatedly attempted to push the BC away

Table 2

Mean and standard deviation of body and claw measurements of prawnsused in vehicle injection experiments

Parameter

Blue injected Yellow injected

BC(n � 5)

YC(n � 5)

BC(n � 5)

YC(n � 5)

Body length (cm) 14.7 � 1.3 13.3 � 0.7 13.9 � 0.2 13.3 � 0.9Claw length (cm) 28.3 � 0.5 15.8 � 0.0 26.6 � 0.3 14.9 � 0.2

Right 27.9 � 2.7 15.7 � 1.5 26.8 � 2.0 15.0 � 2.7Left 28.7 � 4.1 15.8 � 1.6 26.4 � 1.8 14.7 � 2.5

Ratio (Claw:Body) 1.9 � 0.1 1.2 � 0.1 1.9 � 0.1 1.1 � 0.1

Table 3

Mean and standard deviation of body and claw measurements of prawnsused in SIFamide injection experiments

Parameter

Blue injected Yellow injected

BC(n � 5)

YC(n � 5)

BC(n � 5)

YC(n � 5)

Body length (cm) 14.8 � 0.9 12.7 � 1.5 14.1 � 0.2 13.5 � 1.7Claw length (cm) 29.3 � 0.8 14.3 � 0.0 27.5 � 0.4 16.4 � 0.3

Right 29.9 � 1.7 14.3 � 2.0 27.8 � 1.3 16.6 � 3.5Left 28.8 � 2.1 14.2 � 2.0 27.2 � 1.1 16.2 � 3.7

Ratio (Claw:Body) 2.0 � 0.1 1.1 � 0.1 2.0 � 0.1 1.2 � 0.1

Figure 1. Gly-SIFamide injection into YCs. Effect on mean domi-nance index (DI) before (pre) and after (post) injection of the YCs with 1 �10�3 mol l�1 Gly-SIFamide (n � 5). Before the injection, BCs establishedthemselves as the dominant animals and YCs as the subordinates (one-wayANOVA, Tukey’s test, P � 0.001). When the YCs were injected withGly-SIFamide, they behaved very much as do BCs under control condi-tions. The behavior of BCs confronted with Gly-SIFamide-injected YCswas also modified, showing a significant decrease in the levels of aggres-sion compared to control conditions. The asterisk (*) over the YC post-injection group represents a significant difference from the YCs in controlconditions (before the injection). The cross (†) over the BC post-injectiongroup represents a significant difference from the BCs in control condi-tions. BC: blue clawed prawns; YC: yellow clawed prawns; filled barindicates the mean DI of the injected (YC) prawns; *P � 0.001; **P �

0.025; †P � 0.050.


with its claws. The BC kept up this incessant attack until anescape response was elicited in the YC. Though the YCtried to get away, the BC kept fighting and would notretreat. The mean DI for BCs in these experiments was2.57 � 0.06 (n � 5), while that of YCs was 1.44 � 0.06(n � 5). Thus, levels of aggression were further enhanced inthe BC by injection of Gly-SIFamide.

Vehicle injection in the BC and YC prawns

To rule out the possibility that the observed changes inbehavior could be a result of the injection process, experimentswere done in which the BC or the YC was injected only withvehicle solution (prawn saline). There were no significantchanges in the level of aggression in these animals, as shownby comparing the DI before and after the injection of thevehicle into the YC (Fig. 3: P � 0.325 when comparing pre-and post-injection DIs for BCs; P � 0.215 when comparingpre- and post-injection DIs for YCs) or into the BC (Fig. 4: P� 0.621 when comparing pre- and post-injection DIs for BCs;P � 1.000 when comparing pre- and post-injection DIs forYCs). Thus, we can conclude that the injection process is notresponsible for the changes in DI observed following injectionof Gly-SIFamide into the BCs or YCs.

Effects of Gly-SIFamide on specific parameters ofaggressive behavior

To determine which specific behavioral components ofagonistic behavior increase or decrease after the Gly-SIF-amide injection, we group our data in terms of how manytimes each animal performed each of the parameters used tocalculate the DI before and after the injection of the neu-ropeptide. For data collected during pre- and post-injectionsessions, a one-way ANOVA analysis found significantoverall differences among the parameters for 4 of the 5measured behavioral categories (n � 5; P � 0.05). Theseincluded position of body and walking legs, movement andposition of claws, movement of chela, and instances of

Figure 2. Gly-SIFamide injection into BCs. Effect on the meandominance index (DI) before (pre) and after (post) injection of the BCswith 1 � 10�3 mol l�1 Gly-SIFamide (n � 5). When the BCs wereinjected, they further increased their aggression toward YCs when com-pared to control conditions (asterisk [*] over the BC post-injection group,P � 0.001). BC: blue clawed prawns; YC: yellow clawed prawns; filled barindicates the mean DI of the injected (BC) prawns; *P � 0.001.

Figure 3. Vehicle injection into YCs. Effect on the mean dominanceindex (DI) before (pre) and after (post) injection of the YC with prawnsaline (n � 5). Injection of vehicle solution alone did not change the DI forYCs. BC: blue clawed prawns; YC: yellow clawed prawns; filled barindicates the mean DI of the injected (YC) prawns; #P � 0.002; ##P �

0.040.

Figure 4. Vehicle injection into BCs. Effect on the mean dominanceindex (DI) before (pre) and after (post) injection of the BCs with prawn saline(n � 5). Injection of vehicle solution alone did not change the DI for BCs. BC:blue clawed prawns; YC: yellow clawed prawns; filled bar indicates the meanDI of the injected (BC) prawns; *P � 0.001; #P � 0.002.


movement toward or away from each other (Table 4). Todetermine the origin of the significant differences amonggroups, Tukey’s tests were performed to compare BC andYC post-injection data to their corresponding control con-dition data. In general, these tests revealed that injection ofGly-SIFamide produced an increase in the number of timesthe animal performed a more dominant behavior, resultingin a decrease in the more submissive behaviors. We alsofound that the greater effects of the Gly-SIFamide injectionwere on the movement and position of claws, as well as onthe movement of chela. Interestingly, we did not find anysignificant differences in the abdomen posture after theinjection of the neuropeptide compared to control condi-tions.

The only significant differences seen in the experimentswhere the BCs were injected with Gly-SIFamide were in 2of the 5 measured behavioral categories, movement andposition of claws, and movement of chela (Table 2). In theseexperiments, the injected BCs and the non-injected YCssignificantly increased the number of times they positionedtheir claws at body level. However, the difference in theinjected BCs is higher than in the non-injected YCs. Wealso found that the above increase in BCs was accompaniedby a significant decrease in the number of times they posi-tioned claws touching the bottom of the tank while flexed.

The major significant differences were seen in the exper-iments where the YCs were injected with Gly-SIFamide(Table 4). In these experiments, injected YCs showed an

Table 4

Significant differences in agonistic behaviors among BCs and YCs receiving Gly-SIFamide injections

Behavioral Parameters

BC Injected YC injected

BC* YC BC YC*

Position of Body and Walking LegsLegs fully flexed/body touching bottom [0] �(38) �36Legs fully flexed/body touching bottom [1] �29Legs partially flexed/body partially lifted [2] �12Legs partially flexed/body partially lifted [3]Legs partially flexed/body partially lifted [4]Legs fully extended/body fully lifted [5]Legs fully extended/body fully lifted [6]

Movement and Position of ClawsCrossed in front of eyes [0]Touching bottom while extended [1] �39Touching bottom while flexed [2] �35 �(22)At body level [3] �34 �12 �(24) �34Above body level [4]Extended at or above body level [5]Quickly thrust forward/lifting other prawn [6]

Movement of ChelaClosed/not touching the other prawn [0] �60 �37 �65Closed/touching the other prawn [1] �11 �17Open/not touching the other prawn [2] �58 �68Open/touching the other prawn [3]Scissoring in front of the other prawn [4]Open, grabbing or lifting the other prawn [5]Nipping or Tearing [6]

Movement Toward or Away From Each OtherRush to move away from other prawn w/o facing it [0] �1Walk away from other prawn w/o facing it [1] �(7)Walk away from other prawn while facing it [2]Turn away from other prawn, w/o walking away from it [3] �4Turn towards other prawn, w/o walking away from it [4] �(5)Walk toward the other prawn while facing it [5] �6Rush toward the other prawn while facing it [6]

The asterisk represents the animal injected with Gly-SIFamide in each pair interactions. Data values shown indicate where statistically significantdifferences were observed. These numbers are the mean percentage of the difference between how many times each animal performed each parameterbefore and after injection of the neuropeptide (the higher the number, the higher the magnitude of the effect). White spaces indicate parameters wherenonsignificant differences were found. The values in brackets indicate the weight, or level, of the parameter as an indicator of dominance/subordination,6 indicating the most highly dominant behavior, 0 indicating complete or total subordination. Numbers in brackets indicates a tendency. Plus and minussymbols represent a significant increase or decrease in the performed behavior after the injection compared to the control (before the injection), respectively.


increase in how many times the body was partially liftedrelative to the bottom, the claws were at body levels and thechelae were open and touching the other prawn. We alsofound a significant reduction in the number of times injectedYCs positioned the body touching the bottom of the tank,their claws touching the bottom while extended, and theirchelae closed without touching the other prawn. Interest-ingly, the non-injected BCs exhibited more submissive be-haviors after the injection of Gly-SIFamide into the YCscompared to control conditions (Table 4). These animalstended to maintain their bodies touching the bottom andtheir claws touching the bottom while flexed. Also, non-injected BCs showed a significant increase in instances ofmovement toward or away from each other such as turning

away from the other prawn without walking away from it,and walking toward the other prawn while facing it. Thus,Gly-SIFamide acted to increase or decrease the performanceof several important agonistic behaviors.

Effects of injections on number and duration of attacks

The effects of vehicle (prawn saline) and Gly-SIFamideinjection were also examined in terms of number and du-ration of attacks between prawns. Data analysis showed nostatistically significant effects when we compared the num-ber of attacks and their duration before and after the injec-tion of vehicle solution or Gly-SIFamide (Fig. 5). Never-theless, nonsignificant trends toward an increase in the

Figure 5. Effects of vehicle and SIFamide injections on number and duration of attacks between prawns.No statistically significant effects were observed on the number of attacks and fight duration for any of theinjection experiments. Effect on the mean number of attacks (A) and attack duration (B) before (pre) and after(post) injection of the BC (n � 5) and YC (n � 5) with prawn saline, respectively. Effect on the mean numberof attacks (C) and attack duration (D) before (pre) and after (post) injection of the BC (n � 5) and YC (n � 5)with Gly-SIFamide, respectively. BC: blue clawed prawns; YC: yellow clawed prawns; Pre: pre-injectionbehavior; Post: post-injection behavior; filled bar indicates the injected prawns in each set of experiments.


number of attacks and fighting duration were observedwhen BCs were injected with Gly-SIFamide (Fig. 5C, D;n � 5, P � 0.216 and P � 0.271, respectively). A similarnonsignificant trend toward an increase in fighting durationwas also observed when BCs and YCs were injected withvehicle solution (Fig. 5B; n � 5, P � 0.264 and P � 0.053,respectively).

Effects of injections on locomotor activity

To examine the effects of vehicle (prawn saline) andGly-SIFamide injection on locomotor activity, we assessedgeneral locomotion by counting the number of lines crossed

by each prawn during a 30-s period before and after theinjection. Our data revealed that neither vehicle nor SIF-amide injections had any statistically significant effect onlocomotion of the animals (Fig. 6). However, in the exper-iments where we injected vehicle solution, we observed anonsignificant trend toward a decrease in the number oflines crossed after injecting the BCs (Fig. 6A; n � 5, P �0.195) and YCs (Fig. 6B; n � 4, P � 0.306). A similarnonsignificant trend toward a decrease in locomotion wasalso observed when YCs were injected with Gly-SIFamide(Fig. 6D; n � 5, P � 0.156). Interestingly, in the latterexperiments, the non-injected BCs also showed a nonsig-

Figure 6. Effects of vehicle and SIFamide injections on the prawn�s locomotion. No statistically significanteffects were observed on locomotor activity for any of the injection experiments. (A) and (B) Effect on the meannumber of lines crossed in 30-s intervals during a 30-min recording period, before (pre) and after (post) injectionof the BCs (n � 5) and YCs (n � 4) with prawn saline, respectively. (C) and (D) Effect on the mean numberof lines crossed in 30-s intervals during a 30-min recording period, pre- and post-injection of the BCs (n � 5)and YCs (n � 5) with Gly-SIFamide, respectively. BC: blue clawed prawns; YC: yellow clawed prawns; Pre:pre-injection behavior; Post: post-injection behavior; filled bar indicates the mean number of lines crossed in a30-s interval by the injected (BC or YC) prawns.


nificant trend toward a decrease in locomotor activity (Fig.6D; n � 5, P � 0.056).

Discussion

The different patterns of behavior associated with each ofthe three male morphotypes and their status within a fixeddominance hierarchy are the advantages of using the fresh-water prawn as a model system to study aggressive behav-ior. In several studies on aggressive interactions amongconspecifics in crustaceans, the parameters used to deter-mine levels of aggression or degree of dominance haveinvolved observing who initiates fighting behavior, whoretreats first, how many times contact is established usingclaws, and the posture of the abdomen (Livingstone et al.,1980; Kravitz, 1988; Huber and Delago, 1998; Tierney andMangiamele, 2001; Herberholz et al., 2003, 2004). Fightingbehavior may be further characterized by breaking down theanimal’s behavioral sequences into individual actions,movements, or both that can then be assigned numericalvalues according to how important or relevant they are asindicators of aggression or submission. This approach hasbeen used previously in determining the effect of size andmorphotype on dominance hierarchies of Macrobrachiumrosenbergii (Barki et al., 1992).

Although injecting substances such as biogenic amines orneuropeptides in the hemolymph of a living animal willmost likely induce general systemic effects, these types ofexperiments can nevertheless be an essential initial step inelucidating the mechanisms that underlie a specific behav-ior, by narrowing down the range of agents that should bestudied in more detail at the circuit or cellular level. Weused this quantitative approach to measure the level ofaggression/submission in interactions between BC/YC pairsbefore and after injecting Gly-SIFamide directly into thecirculating hemolymph of the living animal.

In this study, we observed that YCs receiving a Gly-SIFamide injection showed a level of aggression that isnormally characteristic of the BCs. These animals becameso aggressive that they actively attacked BCs, a behaviorthat rarely occurred under normal circumstances. We alsoobserved that Gly-SIFamide injections into the circulatinghemolymph of BCs increased their aggressiveness towardYCs even more. Together, these behavioral data suggestthat levels of aggression among interacting BC/YC prawnscan be enhanced by injection of the neuropeptide SIFamideinto the circulating hemolymph.

It is interesting that when only the YC received a Gly-SIFamide injection, the behavior of the BC was also mod-ified. Under these circumstances, a significant decrease wasobserved in the levels of aggression of the normally domi-nant BC. This change in the behavior of BC could beexplained as an effect of observation of the behavior of itsrival or by influence from water-borne agents produced by

the injected YC. In crayfish, injection of serotonin in asubordinate animal results in a reduction in the likelihood ofthe animal deciding to retreat during a fight (Huber et al.,1997a, b). Although this change can indeed be considered asless subordinate behavior, a complete reversal in status ofthe fighting animals, like that seen here in the prawn, has notbeen recorded. The results observed in our experimentsfollowing injection of Gly-SIFamide in a subordinate YCsuggest a full reversal in status of both the injected andnon-injected animals of a fighting BC/YC pair.

It has been reported that injection of serotonin in juvenilelobsters at concentrations required for observing an effecton aggressive behavior also inhibits locomotion, makinginterpretation of results more difficult (Peeke et al., 2000).This is not the case in the freshwater prawn, where injectionof Gly-SIFamide at a concentration affecting aggression(1 � 10�3 mol l�1) does not affect the animal’s mobilityany more than does placing the animal on ice to perform asaline injection, a transient effect from which the animalrecovers in a short time. Our results demonstrated that therewere no statistically significant effects on locomotion afterthe injection of vehicle solution or Gly-SIFamide. However,a nonsignificant trend toward a decrease in locomotor ac-tivity was observed in YCs after the injection of eithervehicle or Gly-SIFamide. YCs used in this study had ashorter body length than BCs (Tables 3 and 4) and may havehad a smaller diameter in the dorsal abdominal artery, thesite of injection. This may have resulted in a higher suscep-tibility to a reduction in general level of activity followinginjections. Nevertheless, these changes were not significantand thus did not mask the observed increase in aggressivebehavior after injection of Gly-SIFamide. Interestingly,when YCs were injected with Gly-SIFamide, a nonsignifi-cant reduction was also observed in the non-injected animal.Under these conditions, this reduction in the locomotoractivity of BCs could be explained as an effect of observa-tion of the behavior of its rival or by influence from water-borne agents produced by the injected YC. In the literature,we found only two functional studies on the physiologicalroles played by SIFamides, one in a crustacean and the otherin an insect. In the American lobster Homarus americanus,exogenous application of Val-SIFamide to the isolated sto-matogastric ganglion produced a profound modulation ofthe output of the pyloric neural circuit (Christie et al., 2006).In the fruit fly Drosophila melanogaster, results from stud-ies using ablation of the SIFamide neurons and RNA inter-ference have implicated Ala-SIFamide in the control ofsexual behavior (Terhzaz et al., 2007). Also, several immu-nohistochemical and in situ hybridization studies suggestadditional neuromodulatory roles for the SIFamides withinthe CNS, including modulation of the olfactory system(Yasuda et al., 2004; Yasuda-Kamatani and Yasuda, 2006).Recently, Christie et al. (2007) found that Gly-SIFamide islocated in and released by midgut epithelial endocrine cells


in several Cancer crabs, suggesting a possible gut paracrine/endocrine role for the SIFamide family. In the present study,we performed behavioral experiments in which the interac-tions between prawn pairs were recorded and quantifiedbefore and after injecting Gly-SIFamide directly into thecirculating hemolymph of the living animal. Our data showthat levels of aggression among interacting BC/YC prawnscan be enhanced by injection of the Gly-SIFamide, suggest-ing a function in the modulation of aggression in the prawn.Our study thus constitutes the first report demonstrating arole for Gly-SIFamide in modulating aggressive behaviorand, to our knowledge, the first description of the effects ofinjecting a neuropeptide in the circulatory system of a livinganimal. It remains to be determined through future experi-ments whether this neuropeptide may also play a part inother forms of the prawn’s interactive behaviors (e.g., mat-ing) or in regulating the mechanisms by which the animalprogresses from one morphotype to the next. Further exper-iments are also needed to characterize the function of Gly-SIFamide at the circuit and molecular levels. A better un-derstanding of the mechanisms involved in mediatingand/or modulating interactive behaviors in simple modelsystems such as crustaceans will ultimately help enable theidentification of novel sources for treatments of a widevariety of mental health disorders, through identification ofnew potential targets for drugs or gene therapies.

Acknowledgments

This investigation was supported by NIH/MBRS SCORES06GM008224, NIH/MRISP MH48190, NIH-NCRR/RCMIG12RR03051, and NIH/MBRS RISE R25-GM061838.

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gyrkppfngsifamide (gly-sifamide) modulates aggression in the freswater prawn

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