Mapping of serotonin-immunoreactive neurons of Anastrepha obJiqua Macquart larvae Isabel Cristina Boleli 1 Zilil Luz Paulino-Simoes 2 ABSTRACT. Serotonin-immunoreactive neurons were identified in the central nerv­ ous system (CNS) of Anaslrepha obliquo Macqualt, 1835 wandering stage larvae. The PAP immunocytochemical method was applied to the entire CNS (whole mounts). About 90 neurons were visualized in the CNS (20 in the brain and 70 in the ventral ganglion). Both somata and axons were strongly stained. These neurons showed a segmental arrangement and bilateral symmetry. All processes presented a basic projection pattern, in which the major fibres travel contra laterally. Comparison of these neurons with serotonergic neurons described in other insects suggests order-specific traits such as cerebral clusters and presence of only one 5-HT immunoreactive neuron in the 8th abdominal neuromere as well. KEY WORDS. Anaslrepha obliquo, flUit fly, serotonin, immunocytochemistry Larvae of Anastrepha obliqua Macquart, 1835 (Diptera, Tephritidae), as occuring in other tephritid species, live and feed on the pulp of many tropical fruits (MALA VASI et af. 1980), becoming them unviable to consumption and causing high economic losses for the fruit-growing industry. Developing inside the fruits, they are protected from many insectivides by the fruits' rind. So, studies about fruit flies controls have been carried out on adu lt population size (ALBRECHT & SHERMAN 1987; MARTINEZ & MORENO 1991; RAGA et af. 1993,1994). At the end of the larval stage, the mature larvae leave the fruits to look for pupation sites. This period is known as wandering stage (FRAENKEL & BHASKARAN 1973) and corresponds to unique developmental period in the larval life susceptible to control programs. Physiological and immunocytochemical studies have provided strong evi­ dence that 5-hydroxytryptamine (serotonin, 5-HT) is involved in the regulation of biological processes directly or indirectly related with wandering stage as, for example, visceral and skeletal muscle contractions (BANNER et of. 1987; VAN HAEFTEN et of. \993) and gut emptying (DAVEY & TREHERNE 1963), and also stimulation of the corpora allata activity (RACHlNSKY 1994) whose juvenile hormo­ ne synthesis has an important role in the maintenance of larval development of the insects (WIGGLESWORTH 1985; RrDDlFORD 1994). 1) Departamento de Morfologia e Fisiologia Animal, Faculdade de Cii'mcias Agrarias e Veterinarias, Universidade Estadual Paulista. Rodovia Paulo Donato Castellani Km 5, 14870-000 Jaboticabal, Sao Paulo, Brasil. 2) Departamento de Biologia, Faculdade de Filosofia, CiEmcias e Letras de Ribeirao Preto, Universidade de Sao Paulo. Rodovia Bandeirantes 3900, 14040-901 Ribeirao Preto, Sao Paulo, Brasil. Revta bras. Zool. 16 (4): 1099 -1107, 1999 1100 Boleli & Paulino-Simoes In view of the important role that 5-HT may play in the wandering behavior and maintenance of the juvenile stages it is undoubtedly of interest to determine the neuroanatomical organization of serotonergic system, which is expected to help in the understanding of the role of serotonergic neurons in this larval phase. Although cyclorraphous species ofDiptera have been submitted to mapping of serotonergic neurons (NASSEL & KLEMM 1983; NASSEL & CANTERA 1985 ; VALLES & WHITE 1988), until now, no information is available on the occurrence of5-HT in the nervous system of a true fruit fly (Tephritidae) . So, to provide a basis for future studies on the physiology of these neurons, the objective of the present study was to report the mapping of the 5-HT immunoreactive neurons in the CNS of A. obliqua wandering stage larvae. MATERIAL AND METHODS Experimental animals Third instar larvae of A. obliqua in the wandering stage were collected from infested fruits of Spondias lutea (caja-mirim) (Anacardiaceae). The larval stage and the species were identified according to FRAENKEL & BHASKARAN (1973) and TELES DA SILVA (1978), rescpectively. Imm u nocytochem istry Twenty larvae were used . The animals were immobilized and sacrificed by freezing in liquid nitrogen. The nervous system was dissected in phosphate buffer saline (0 .1 MpH 7.4; 0.9% NaCI) (PBS) and fixed for 2-3h with an ice-cold solution of 4% paraformaldehyde/8% saturated picric acid in phosphate buffer (0.1 M pH 7.4). After prolonged washing (72h) in PBS + 5% sucrose, the preparations were submitted to immunocytochemistry. The immunoreaction was carried out on whole mounts by the indirect peroxidase-anti peroxidase (PAP) technique (BOURNE 1984). To reduce nonspecific background staining, the specimens were incubated for one hour in O. IM Tris-HCl, pH 7.4, containing 10% normal guinea-pig serum (Dako­ patts). Rabbit-antiserotonin antiserum (kindly donated by Dr. Jean Lauder; Univer­ sity of North Carolina, Chapell Hill) was diluted I :500 in PBS + 0.25% TritonX-100 (Sigma) (PBS-T) and specimens were incubated for 72h at 4°C. After washing in PBS-T, the specimens were incubated with unlabeled guinea-pig anti rabbit antise­ rum (Dakopatts) and rabbit PAP-complex (Dakopatts) diluted 1 :50 and 1: 100 in PBS-T for 12h at 4°C. The peroxidase reaction was visualized in 0.025% 3,3' diaminobenzidine tetrahydrochloride (DAB, Sigma) with 0.01% H20 2 in PBS. After staining, some preparations were dehydrated in alcohol, cleared in xylene, and mounted in Entellan, and others were embedded in paraplast for serial sectioning (10 fJ.m). The absence of endogenous peroxidase was confirmed by incubating the entire nervous system in the absence of the primary antiserum. The specificity of the immunoreaction was tested with antiserum preincubated with antiserum (STETNBUSCH et al. 1983). RESULTS Serotonergic neurons were immunocytochemically identified in the central nervous system of A. obliqua third instar larvae. Revta bras. Zool. 16 (4): 1099 -1107,1999 Mapping of serotonin-immunoreactive neurons of Anastrepha ... 1101 Staining of whole mounts revealed immunoreactive cell bodies, axons and fine dendrites . A total of 90 neurons were located (20 in the brain and 70 in the ventral ganglion), whose cell body and axons were strongly stained. These neurons are uniform in size and show a bilateral symmetrical arran­ gement. The larval brain The proto-, deuto- and tritocerebrum are not clearly distinguishable in the larval's central nervous system. Thus, the proto cerebral cell bodies were considered to be the most dorsal somata in the cerebral hemisphere and the tritocerebral cell bodies were considered to be the most ventral. The cerebral 5-HT immunoreactive neurons were grouped into four distinc­ tive clusters, as follows: cluster 1, is formed by one neuron localized in the dorso-anterior cortex (protocerebral region). The axons of this cluster project ventro-posteriorly within the ipsilateral brain hemisphere toward the intercerebral is commissure (Figs 1 a,b, 2a); cluster 2, consists of four neurons situated in the dorso-medial cortex (protocerebrum), after cluster I. Their axons travel ventrally to the contralateral hemisphere forming an immunoreactive tract and arborizing in the dorsal contralateral neuropil lamina (Figs la,b, 2a); cluster 3, is formed by two neurons in the dorso-posterior cortex (protocerebrum). The axons run anteroven­ trally to the ipsilateral neuropil and then laterally to the intercerebral is commissure, where they also form another tract (Figs la, 2a); cluster 4, is formed by three neurons lying in the ventro-posterior cortex (tritocerebrum). Their axons travel ventro-an­ teriorly to the contralateral side forming a tract (Figs I b, 2a) . The axons of clusters 2 and 3 form a large-field of fine immunoreactive arborization in the dorsal contralateral midbrain neuropile, while the axons belon­ ging to cluster 4 arborize in the ventral neuropile. Ventral ganglion The ventral ganglion is formed by the fusion of the suboesophageal, thoracic and abdominal neuromeres (Figs Ic, 2b). About 26 neurons were identified in the suboesophageal neuromeres. These neurons are distributed unevenly among the three neuromeres that form this gan­ glion, e.g., mandibular, maxillary and labial neuromeres. The number and arrange­ ment of the 5-HT immunoreactive neurons in the mandibular neuromere differ from those observed in the maxillary and labial neuromere . The mandibular neuromere presents a cluster formed by three somata located laterally in each hemiganglion, and whose axons ascend contralaterally into the tritocerebrum. The maxillary and labial neuromeres have five somata per hemiganglion, which were grouped into two clusters: cluster I, consisting of three neurons located in the lateral region and corresponding to the maxillary neuromere cluster; and cl uster 2, formed by two neurons situated ventrally in the hemiganglion. The axonal projections of this second cluster were not clearly observed. In the thoracic neuromeres, 14 neurons were stained. They are arranged as follows: three pairs of serotonergic somata in the prothoracic neuromere, and two Revta bras. Zoo I. 16 (4): 1099 -11 07,1999 1102 Boleli & Paulino-Simoes Fig. 1. Serotonin immunoreactive neurons of wandering third instar larvae of Anastrepha obliqua. Photomicrographs of whole mount preparations of cerebral ganglion in dorsal (a) and frontal (b) view, and suboesophageal ganglia in a ventral view (c). Arrows in the Figure 1b indicate the commissural tracts. Scale bar: in (a) and (b) = 180 flm , in (c) = 190 flm, (1-4) cerebral clusters. pairs in the meso- and metathoracic neuromeres. The somata are located laterally in each hemineuromere and the axons run contralaterally and then appear to project in an ascending and descendeing fashion. Thirty neurons arranged in pairs were mapped in the neuromeres correspon­ ding to the abdominal ganglia. All the abdominal neuromeres present two somata per hemineuromere, except the last, which contains only one. The axonal projections travel contralaterally and appear to ascend into the anterior ganglia and to descend into the posterior ganglia. Revta bras. Zoo I. 16 (4): 1099 -1107,1999 Mapping of serotonin-immunoreactive neurons of Anastrepha ... 1103 o v a ---- --- -------- b Sl S2 S3 T1 T2 T3 A1 A2 A3 A4 AS A6 A7 AS Fig. 2. Camara lucida drawing indicating the neuronal clusters of the brain (a) and of the ventral ganglion (b) of Anastrepha obliqua. Scale bar: in (a) and (b) = 200l1m, (1-4) cerebral clusters, (D) dorsal, (V) ventral, (S1-3) suboesophageal clusters, (T1-3) thoracic clusters, (A1-8) abdominal clusters. DISCUSSION This paper contains a basic description of serotonin-like immunoreactive neurons in the eNS of A. obliqua wandering stage larvae. Altogether 90 neurons and their processes were identified in the brain and ventral ganglion, where they showed a segmental and bilateral symmetrical arran­ gement. This organizational pattern of serotonergic neurons reflects the segmenta­ tion and bilateral symmetry plan of the nervous system of Arthropods, which are believed to have descended from segmented, annelid-like ancestors (BRUSCA & BRUSCA 1990). The segmental neuroanatomical disposition of the 5-HT immunoreactive neurons in the ventral ganglion unab1ed us to distinguish the neuromeres ofthe basic Revta bras. Zool. 16 (4): 1099 -1107,1999 1104 Boleli & Paulino-Simoes functional units or tagmas of insects: three suboesophageal neuromeres, three thoracic neuromeres and eight abdominal neuromeres. The presence of bilateral symmetric pairs of neurons in all of the neuromeres suggests a serial homology (LONGLEY & LONGLEY 1986), which has long been recognized as a result of repetition of development instructions to form several similar segments along the antero-posterior axis (BATE 1976). The immunoreactivity pattern also showed some segment-specific variations in the number of cells, whose function is not clear. An interesting aspect of these neurons is the contralateral projections of their axons. The functional role of these axonal projections toward the opposite hem i­ ganglion and to the somata appears to be the interaction of the right and left nervous system (HOMBERG & HILDEBRAND 1989), supposedly important to coordinate the movements of lateral body undulations during the wandering behavior, since the muscles are organized as segmental bands and muscle contractions appear to be regulated by 5-HT (BANNER et at. 1987; VAN HAEFTEN et at. 1993). However, the ausence of serotonin immunoreactivity in the peripheral nerves makes it difficult to consider the serotonergic neurons mapped in the ventral nervous system of A. obliqua wandering stage larvae as motoneurons. On the physiological view, another important aspect about the serotonergic system of A. obliqua is the strong immunoreactivity showed by the neurons, axons and areas of the neuropi le, whose presence in the wandering stage indicates that large amounts of 5-HT are sinthetized and secreted. In th is same developmental phase, it was observed a high synthesis of juvenile hormone in vitro by corpora allata (data not showed). So, considering that, according to RACHINSKY (1994), serotonin is a strong stimulant of the corpora allata activity, it's probably that the 5-HT really has a stimulant role on the corpora allata of the wandering stage. This point of view is reinforced by the presence of strongly stained processes in the neuropile, from which the corpora allata nerve emerge. By means of detection of BUdR incorporation, TRUMAN & BATE (1988) have demonstrated that a large number of new neurons are produced during larval life by embryonic neurob lasts persisting into the larval stage. According to LAUDER (1993), neuronal development is regulated by serotonin. So it is possible that the immunoreactivity to serotonin found in areas of neuropile of A. obliqua wandering stage larvae is involved in the control of neurogenesis. Comparison among different insect species A comparison of the arrangement of serotonergic neurons in the larval nervous system of Diptera, e.g., A. obliqua (present data), Calliphora erythroce­ phala Meigen, 1830 (Call iphoridae) and Sarcophaga bullata Parker, 1976 (Sarco­ phagidae) (NASSEL & CANTERA 1985) and Drosophila melanogaster Meigen, 1830 (Drosophi lidae) (VALLES & WHITE 1988), shows some similarities in the location and axonal projection pattern. Concerning the cerebral neurons, it was observed that these similarities among the four species mentioned are also related to the clusters . However, with respect to the number of neurons per cluster, there is correspondence only between A. obliqua and D. melanogaster, a fact reflecting the higher phyloge­ netical proximity between Tephritidae and Drosophilidae. Revta bras. Zoo I. 16 (4): 1099 -11 07, 1999 Mapping of serotonin-immunoreactive neurons of Anastrepha ... 1105 In the suboesophageal ganglia, A. obliqua, Calliphora and Sarcophaga all present three neuron pairs in the mandibular neuromere and five in the maxillary neuromere; in Drosophila only two mandibular and three maxillary neurons are identified. A total offive neurons were stained in the labial neuromere of A. obliqua. But, this number does not correspond to that obseved in the other three dipteran species, i.e., of three neurons. These differences may be related to some species­ specific role whose involvement in insect development is still unknown. No difference was observed among these species in the total number of thoracic and abdominal somata. All of them presented three 5-HT immunoreactive neurons in the prothoracic neuromere, two in the mesothoracic, metathoracic and I st_ i h abdominal neuromeres, and one in the last (8 th) abdominal neuromere. The presence of bilateral symmetrical neurons, also denominated twin neurons, in the ventral ganglion has been demonstrated in cockroach (BISHOP & O'SHEA 1983), grasshopper (TAGHERT & GOODMAN 1984), dragonfly (LONGLEY & LONGLEY 1986), beetle (BREIDBACH 1991 ; VAN HAEFTEN & SCHOONEVELD 1992), honeybee (BOLEL! ef al. 1995), fly (NASSEL & CANTERA 1985; VALLES & WHITE 1988). This similarity of cellular and process staining among a wide range of insects belonging to distinct orders, as cited by LONGLEY & LONGLEY (1986), indicates that this organizational pattern of serotonergic neurons was maintained phylogenetically. However, particularly interesting is the fact that no other insects present a single neuron pair in the 8th abdominal neuromere, except Diptera. Further studies are needed to determine whether the absence of this neuron pair in the last neuromere corresponds to a loss of activity related to some different role of the abdominal ganglia. Another interesting fact about the serotonergic system of A. obliqua wande­ ring stage larvae is its correspondence in the location of cells with the groups of cerebral paraldehyde fuchsin (PF) positive neurosecretory cells studied by BOLELl et af. (1994). PF-neurosecretory cells have been described as A-type cells and their significance has often been debated. The presente paper, however, indicates that in A. obliqua wandering stage larvae these cells contain biogenic amine as occurring in locust (VIElLLEMARlNGE et af. 1982). ACKNOWLEDGEMENTS. We thank J. Lauder (University of North Carolina, Chapell Hill) for the kind donation of rabbit antiserotonin serum. We are also grateful to Brazilian Gover­ nment (CNPq) tor financial support tor rCB. REFERENCES ALBRECHT, C.P. & M. SHERMAN. 1987. Lethal and sublethal effects ofovermectin B 1 on three fruit fly species (Diptera: Tephritidae). Jour. Econ. Entomol. 80: 344-347. BANNER, S.E.; R.H OSBORNE & K.J CATELL. 1987. 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