J_ID: CNE Customer A_ID: CNE23548 Cadmus Art: CNE23548 Ed. Ref. No.: 13-0333.R2 Date: 21-March-14 Stage: Page: 1 RESEARCH ARTICLE Localization of Tyrosine Hydroxylase-Like Immunoreactivity in the Nervous Systems of Biomphalaria glabrata and Biomphalaria alexandrina, Intermediate Hosts for Schistosomiasis Deborah Vallejo,1 Mohamed R. Habib,2,3 Nadia Delgado,1 Lee O. Vaasjo,1 Roger P. Croll,2 and Mark W. Miller1* 1Institute of Neurobiology and Department of Anatomy & Neurobiology, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico 00901 2Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada 3Medical Malacology Laboratory, Theodor Bilharz Research Institute, Giza, Egypt ABSTRACT (LPeD1) in the pedal ganglion described previously in Planorbid snails of the genus Biomphalaria are major several pulmonates. The majority of TH-like immunore- intermediate hosts for the digenetic trematode parasite active neurons were detected in the peripheral nervous Schistosoma mansoni. Evidence suggests that levels of system (PNS), especially in lip and foot regions of the the neurotransmitter dopamine (DA) are reduced during anterior integument. Independent observations support- the course of S. mansoni multiplication and transforma- ing the dopaminergic phenotype of THli neurons tion within the snail. This investigation used immunohis- included 1) block of LPeD1 synaptic signaling by the tochemical methods to localize tyrosine hydroxylase D2/3 antagonist sulpiride, and 2) the similar localiza- (TH), the rate-limiting enzyme in the biosynthesis of cat- tion of aqueous aldehyde (FaGlu)-induced fluorescence. echolamines, in the nervous system of Biomphalaria. The distribution of THli neurons indicates that, as in The two species examined, Biomphalaria glabrata and other gastropods, dopamine functions as a sensory Biomphalaria alexandrina, are the major intermediate neurotransmitter and in the regulation of feeding and hosts for S. mansoni in sub-Saharan Africa, where more reproductive behaviors in Biomphalaria. It is hypothe- than 90% of global cases of human intestinal schistoso- sized that infection could stimulate transmitter release miasis occur. TH-like immunoreactive (THli) neurons from dopaminergic sensory neurons and that dopami- were distributed throughout the central nervous system nergic signaling could contribute to modifications of (CNS) and labeled fibers were present in all commis- both host and parasite behavior. J. Comp. Neurol. sures, connectives, and nerves. Some asymmetries 000:000–000, 2014. were observed, including a large distinctive neuron VC 2014 Wiley Periodicals, Inc. INDEXING TERMS: Schistosoma mansoni; dopamine; pulmonate mollusk; pond snail; trematode The parasitic disease schistosomiasis (snail fever) is Grant sponsor: National Institutes of Health; Grant numbers: RCMI estimated to impact 77 developing countries, where its RR-03051, G12-MD007600, NIGMS MBRS: GM-087200; Grant sponsor: socioeconomic toll is second only to malaria (Hotez, National Science Foundation; Grant numbers: DBI-0115825, DBI-0932955, HRD-1137725; Grant sponsor: Partnership and 2008; King, 2010; World Health Organization Media Ownership Initiative (ParOwn 0911), Ministry of High Education and Centre, 2012). The trematode species Schistosoma Scientific Research of Egypt. mansoni that causes the most widespread form of *CORRESPONDENCE TO: Dr. Mark W. Miller, Institute of Neurobiology, University of Puerto Rico, 201 Blvd del Valle, San Juan, Puerto Rico intestinal schistosomiasis utilizes the planorbid snail 00901. E-mail: [email protected] Biomphalaria as its major intermediate host (Rollinson Received November 3, 2013; Revised January 20, 2014; and Chappell, 2002; Bayne, 2009; Toledo and Fried, Accepted January 22, 2014. DOI 10.1002/cne.23548 Published online Month 00, 2014 in Wiley Online Library VC 2014 Wiley Periodicals, Inc. (wileyonlinelibrary.com) The Journal of Comparative Neurology | Research in Systems Neuroscience 00:00–00 (2014) 1 ID: jwweb3b2server Time: 22:25 I Path: //xinchnasjn/01Journals/Wiley/3b2/CNE#/VOL00000/140017/APPFile/JW-CNE#140017 J_ID: CNE Customer A_ID: CNE23548 Cadmus Art: CNE23548 Ed. Ref. No.: 13-0333.R2 Date: 21-March-14 Stage: Page: 2 D. Vallejo et al. 2010). Biomphalaria glabrata and Biomphalaria alexan- can regulate entire neural circuits in the gastropod CNS drina are the principal intermediate hosts for S. man- (Wieland and Gelperin, 1983; Trimble and Barker, 1984; soni in sub-Saharan Africa, where 90% of the global Kyriakides and McCrohan, 1989; Kabotyanski et al., cases occur. Within the gastropod host, schistosome 2000). Such modulation has been intensively studied in larvae undergo multiplication and transformation into the central networks that control feeding, where spe- cercariae that are capable of infecting humans. cific dopaminergic neurons exert broad and coordinated Survival and propagation of trematode larvae within influence over the central pattern generator (CPG) net- the snail both depend on complex bidirectional signal- works that control consummatory actions (Rosen et al., ing between host and parasite (de Jong-Brink et al., 1991; Teyke et al., 1993; Quinlan et al., 1997; Kabo- 2001; Yoshino et al., 2001). Specific neurotransmitters tyanski et al., 1998; Narusuye and Nagahama, 2002). that are shared by both schistosomes and gastropods, The ability of these interneurons to implement qualita- such as the biogenic amine dopamine (DA; 3,4-dihy- tive and quantitative specification of feeding motor pro- droxyphenethylamine), are leading candidates for host– grams is attributable to their capacity to reconfigure parasite communication, as both species possess the multifunctional CPG networks (Kupfermann and Weiss, requisite synthetic enzymes, receptors, and uptake 2001; Murphy, 2001; Cropper et al., 2004). It has been mechanisms necessary to achieve such signaling (Ham- proposed that such features of motor system control dan and Ribeiro, 1998; Taman and Ribeiro, 2009; can provide opportunities for parasites to alter host Larsen et al., 2011). behavior (see de Jong-Brink et al., 1999; Katz and Dopamine (DA) is a major neurotransmitter in the gas- Edwards, 1999; Adamo, 2002, 2005). tropod central nervous system (CNS) where it can pro- In this study, immunohistochemical methods were duce both excitatory and inhibitory synaptic actions used to localize tyrosine hydroxylase (TH), the rate- (Sweeney, 1963; Carpenter et al., 1971; Osborne and Cot- limiting enzyme in catecholamine biosynthesis (see trell, 1971; Ascher, 1972; Berry and Cottrell, 1973; McCa- Osborne et al., 1975, 1976; Osborne, 1977), in the man et al., 1973). The presence of DA in B. glabrata was CNS and cephalopedal sensory organs of B. glabrata originally examined using spectrofluorometric measure- and B. alexandrina. Emphasis was placed on identifica- ments and its localization within the nervous system was tion of catecholaminergic neurons that could serve as demonstrated with histochemical fluorescence micros- 1) sources of host-derived DA in the developmental and copy (Chiang et al., 1974). Significant reductions in DA reproductive program of S. mansoni, and 2) potential content were measured in the Biomphalaria CNS during targets for parasite-induced modification of snail behav- the course of S. mansoni infection (Manger et al., 1996). ior. Preliminary reports of these observations were pre- Recently, a DA transporter (SmDAT) was shown to be sented in abstract form (Delgado et al., 2010; Vallejo expressed at high levels in the parasitic stages of S. man- et al., 2011). soni and it was proposed that the larval trematode could reduce its metabolic costs by scavenging DA from its host (Larsen et al., 2011). To date, however, potential neural MATERIALS AND METHODS sources of DA in Biomphalaria and the stimuli that could Specimens, dissections, and nomenclature promote its release and availability to parasites have not Experiments were conducted on B. glabrata that were been identified. reared in the laboratory in Puerto Rico and B. alexan- Histological findings indicate that DA also partici- drina that were collected from water courses in Giza pates in sensory signaling by the peripheral nervous governorate, Egypt. The latter snails were held for 6 system (PNS) of gastropods (Osborne and Cottrell, weeks in the Medical Malacology Laboratory, Theodor 1971; Croll, 2001; Faller et al., 2008). While the modal- Bilharz Research Institute, Egypt, and examined on a ity of peripheral dopaminergic neurons remains uncer- weekly basis for natural infections before being shipped tain, they are generally associated with cephalic to Nova Scotia. All snails in the laboratory colonies sensory organs (CSOs) that mediate contact chemore- were housed in glass or plastic aquaria at room temper- ception and mechanoreception (Salimova et al., 1987; ature (21–25C) and fed carrots or lettuce ad libitum. Croll et al., 2003; Wyeth and Croll, 2011). Peripheral Tanks contained distilled water with Instant Ocean dopaminergic neurons project to the CNS where their (Kingman AZ) added (1 g per gallon) to approximate synaptic actions can influence the expression of behav- pond water and crushed oyster shells or blackboard ior (Nargeot et al., 1999; Martınez-Rubio et al., 2009; chalk as calcium supplements. Snails dissected at 6–10 Wyeth and Croll, 2011; Bedecarrats et al., 2013). mm shell diameter were considered sexually mature, as In addition to mediating rapid synaptic signaling, evidenced by their