Abstract
The male accessory glands (AG) and gonoducts of moths develop during metamorphosis and are essential for successful fertilization of females. We found that these reproductive organs are innervated by a sex-specific cluster of peptidergic neurons in the posterior 9th neuromere of the terminal abdominal ganglion (TAG). This cluster of ~20 neurons differentiate during metamorphosis to innervate the accessory glands and sperm ducts. Using immunohistochemistry and in situ hybridization (ISH) we showed that these neurons express four neuropeptide precursors encoding calcitonin-like diuretic hormone (CT-DH), allatotropin (AT) and AT-like peptides (ATLI-III), allatostatin C (AST-C), and myoinhibitory peptides (MIPs). We used contraction bioassay in vitro to determine roles of these neuropeptides in the gonoduct and accessory gland activity. Spontaneous contractions of the seminal vesicle and AG were stimulated in a dose depended manner by CT-DH and AT, whereas AST-C and MIP elicited dose dependent inhibition. Using quantitative RT-PCR we confirmed expression of receptors for these neuropeptides in organs innervated by the male specific cluster of neurons. Our results suggest a role of these neuropeptides in regulation of seminal fluid movements during copulation.
Highlights
Insects are the most widespread and common group of terrestrial animals due to their effective reproductive strategies
Both males and females of 5th instar larvae show calcitonin-like diuretic hormone (CT-DH) expression in three groups of prominent midline neurons in the terminal abdominal ganglion (TAG) which is composed of fused abdominal neuromeres 7–9 (AN7-9); a pair of large neurons in the AN7 (∼35 μm in diameter), four large neurons in the AN8 (∼30 μm) and 4–6 smaller neurons in the abdominal neuromere 9 (AN9) (∼25 μm) (Fig. 1a,b)
In this paper we used immunohistochemistry, in situ hybridization (ISH) and electrophysiology to determine differentiation and possible function of sex-specific cluster of neurons (MAN9) that innervate the male accessory glands and gonoducts in B. mori. This male-specific neuronal cluster has been first identified in M. sexta by cobalt backfilling and named “imaginal midline neurons of the abdominal segment 9” (A9 IMN)[4,5]. These authors described the appearance of peptide production in A9 IMN in pharate pupae that persisted to the adult stage using antibodies to small cardioactive peptide B (SCP-B) and FMRFamide[5,6]
Summary
Insects are the most widespread and common group of terrestrial animals due to their effective reproductive strategies. Movements of seminal fluids within the reproductive organs are facilitated by the visceral muscles that form outer layer of gonoducts and associated glands. This musculature is innervated by neurons from the terminal abdominal ganglion (TAG) which have been first described in the tobacco moth Manduca sexta[4]. These different neuropeptides are coexpressed in a male-specific cluster of neurons that differentiates during metamorphosis and innervates the male reproductive organs. We detected expression of receptors for these neuropeptides in the male gonoducts and accessory glands These data indicate a complex neuropeptide-receptor signaling that controls activity of the male reproductive organs during expression was detected by ISH (a) or immunostaining (b) in midline neurons PM7 and medial neurosecretory. Possible roles of these neuropeptides on spontaneous muscle activity of the accessory glands, seminal vesicles and ejaculatory duct were examined in vitro using electrophysiology
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