Abstract
Our previous study demonstrated that predominant feeding inhibitory effects were found in the crude extracts of foregut and midgut of the silkworm Bombyx mori larvae. To address the entero-intestinal control crucial for the regulation of insect feeding behavior, the present study identified and functionally characterized feeding inhibitory peptides from the midgut of B. mori larvae. Purification and structural analyses revealed that the predominant inhibitory factors in the crude extracts were allatotropin (AT) and GSRYamide after its C-terminal sequence. In situ hybridization revealed that AT and GSRYamide were expressed in enteroendocrine cells in the posterior and anterior midgut, respectively. Receptor screening using Ca2+-imaging technique showed that the B. mori neuropeptide G protein-coupled receptor (BNGR)-A19 and -A22 acted as GSRYamide receptors and BNGR-A5 acted as an additional AT receptor. Expression analyses of these receptors and the results of the peristaltic motion assay indicated that these peptides participated in the regulation of intestinal contraction. Exposure of pharynx and ileum to AT and GSRYamide inhibited spontaneous contraction in ad libitum-fed larvae, while exposure of pharynx to GSRYamide did not inhibit contraction in non-fed larvae, indicating that the feeding state changed their sensitivity to inhibitory peptides. These different responses corresponded to different expression levels of their receptors in the pharynx. In addition, injection of AT and GSRYamide decreased esophageal contraction frequencies in the melamine-treated transparent larvae. These findings strongly suggest that these peptides exert feeding inhibitory effects by modulating intestinal contraction in response to their feeding state transition, eventually causing feeding termination.
Highlights
Most phytophagous insects live on or around their preferred host plants [1]
B. mori tachykinin-related peptides (TRPs) and short neuropeptide F -2 have feeding acceleratory effects that can shorten the latency to the initiation of feeding [16]
To identify the feeding inhibitory factors in B. mori larvae, we purified these factors into two fractions at the sixth purification step of reversed-phase high-performance liquid chromatography (RP-HPLC) according to their biological activities, Enteroendocrine control of feeding behavior in B. mori which significantly prolonged the latency to the first bite after sample injection compared with those by vehicle injection (A1 and B3 in Fig 1A and 1B)
Summary
Most phytophagous insects live on or around their preferred host plants [1]. Observations of these insects have demonstrated the presence of a regularly occurring pattern switching from a feeding mode to a quiescent mode, indicating unidentified endogenous regulatory mechanisms in both upregulation and downregulation of feeding motivation. The regulatory mechanisms in feeding motivation have been characterized by a number of physiological investigations, illuminating the importance of hemolymph factors corresponding to the nutritional status [2], eventually inducing in modifications in locomotor activities and neuronal signaling to maintain homeostasis [3, 4] These biological homeostatic processes, including sequential feeding behavior and fluctuation in motivation, are known to be regulated by a number of neuropeptides in the central nervous system (CNS) and the peripheral organs [5, 6]. B. mori TRPs and short neuropeptide F (sNPF) -2 have feeding acceleratory effects that can shorten the latency to the initiation of feeding [16] These reports demonstrate that endogenous endocrine factors regulate feeding motivation, little is known about their mode of action or how feeding state transition occurs
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