Immunocytochemical localization of serotonin in the central and peripheral chemosensory system of mosquitoes

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Abstract

Female mosquitoes depend on blood to complete their reproductive cycle and rely mainly on chemosensory systems to obtain blood meals. An immunocytochemical analysis reveals a number of serotonin-immunoreactive neurons that innervate the chemosensory systems, suggesting a potential role of serotonin in modulating chemosensory processes. In the primary olfactory system, we identify a single ipsilateral centrifugal neuron with arborizations in higher brain centers; the varicosities of this neuron display volumetric changes in response to both blood feeding and during a circadian rhythm. Six to eight pairs of serotonin-immunoreactive neurons are identified in the primary gustatory neuropil, including the subesophageal ganglion and tritocerebrum. The peripheral chemosensory organs, i.e. the antenna, the maxillary palp and the labium, are described as having extensive serotonergic neurohemal plexi. In addition, we describe the presence of serotonin-immunoreactive fibers in the mechanosensory Johnston's organ. Taking these results together, we discuss the potential role of serotonin as a neuromodulator in the chemosensory system of disease vector mosquitoes.

Introduction

Many animals fine tune and modulate their sensory systems in order to adapt to complex environmental and behavioral conditions. Various neurochemicals present in the nervous system orchestrate these crucial changes. Biogenic amines, metabolic derivatives of amino acids, play important roles as neuromodulators and neurohormones, whereby they modify the sensitivity of both peripheral and central nervous targets (Dolzer et al., 2001, Monastirioti, 1999, Nässel, 1988, Orchard, 2006). Serotonin, or 5-hydroxytryptamine, is a biogenic amine found in all vertebrate and invertebrate nervous systems (Nässel, 1988). From the role of serotonin in learning and memory, control of circadian rhythm, motor activity and neural development, it is well established that serotonin acts as a neurotransmitter as well as a neuromodulator in the central nervous system (CNS) of insects (Menzel and Muller, 1996, Mercer et al., 1996, Nässel, 1988, Yuan et al., 2005). Apart from its action in the CNS, serotonin has been demonstrated to be present in the insect hemolymph (Lange et al., 1989) and shown to act peripherally on salivary glands (Ali and Orchard, 1996, Ali et al., 1993), malphigian tubules (Te Brugge et al., 2002) and the heart (Koladich et al., 2002), indicating that it may act as a neurohormone.

Immunocytochemical localization of serotonin in the primary olfactory neuropil of insects, the antennal lobe (AL), has been studied in cockroaches, moths, locusts, beetles, honeybees, flies and many other insects (Dacks et al., 2006, Hill et al., 2002, Ignell, 2001, Nässel, 1988, Rehder et al., 1987, Salecker and Distler, 1990, Sun et al., 1993). These studies have generally revealed the presence of a serotonin-immunoreactive (SI) centrifugal neuron with branches in the AL and in defined neuropil regions of higher brain centers. From anatomical and physiological studies, it has been postulated that SI neurons may function as feedback neurons by providing efferent centrifugal input from higher centers of the brain and modulating olfactory information processing in the AL (Kloppenburg and Hildebrand, 1995, Mercer et al., 1996).

Serotonin immunoreactivity has not only been found throughout the central nervous system, but also in the peripheral nervous system. Immunocytochemical studies of serotonin in Periplaneta americana, Rhodnius prolixus, Leptinotarsa decemlineata and Calliphora erythrocephala have revealed the presence in the peripheral nervous system of a unique system of serotonergic processes that form extensive neurohemal areas (Davis, 1987, van Haeften and Schooneveld, 1993, Lange et al., 1988, Nässel and Elekes, 1984). The diffuse system of serotonergic axons is carried by several nerves in the head and targets the peripheral appendages and muscles. The main nerves involved in this serotonergic system are associated with the mouthparts, which suggest that serotonin plays a crucial role in modulating the feeding behavior of these insects.

In the present study, we provide a detailed map of serotonin immunoreactivity in the central chemosensory system of mosquitoes that, in principle, compares to previous studies in insects. However, we provide the first morphological evidence of a unique serotonergic investment of the peripheral chemosensory system. Furthermore, in the AL we show volumetric changes in immunoreactive varicosities, the putative serotonin release sites, in response to both blood feeding and during a circadian cycle emphasizing that serotonin may play an important role in modulating functional characteristics of the chemosensory systems.

Section snippets

Mosquitoes

The Rockefeller strain of Aedes aegypti and a colony of Anopheles gambiae originating from Suakoko, Liberia were used for the experiments. Mosquitoes were reared at 27 °C, 70–80% relative humidity and at a photoperiod cycle of 12 h light/12 h dark. Adults were kept in plastic cages and were fed ad libitum with 6% sugar solution. We used 4- to 10-day post-emergence, sugar-fed (non-blood-fed) females as well as male mosquitoes for the experiments.

Serotonin immunoreactivity in primary and higher olfactory centers

A single serotonin-immunoreactive (SI) putative centrifugal neuron was found in each AL. The intensely stained, 15–20 μm diameter soma was located in the anterolateral region of the AL (Fig. 1A,B). The primary neurite extended through the AL without arborizing and entered the ipsilateral protocerebrum through the base of the inner antennocerebral tract (IACT) (Fig. 1B). The primary neurite bifurcated posterolaterally to the central complex, and sent a recurrent axon branch back into the AL,

Discussion

We have localized serotonergic pathways in the chemosensory systems of two disease vector mosquitoes with the help of immunocytochemical techniques. The salient features of the serotonergic system in the chemosensory system of mosquitoes are the presence of a single pair of SI neurons and 6–8 pairs of SI neurons in the central olfactory and gustatory neuropils, respectively. Along with their investments of the central nervous system, SI fibers were found in Johnston's organ, the antenna, the

Acknowledgments

We thank Teun Dekker and Sharon Rose Hill for comments on the manuscript. Schematic diagram of mosquito brain in Figs. 2 and 6A is courtesy of Teun Dekker. We greatly appreciate help with statistics from Sharon Rose Hill. We are grateful to Maryam Ghadimi, Majid Ghaninia and Mari-Louise Skoog for valuable technical help. This study was supported by the Swedish Research Council, Formas and the Swedish Linnaeus Initiative ICE.

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