Specific distribution of expression and enzymatic activity of cholesterol biosynthetic enzyme DHCR24 orthologs in the phytophagous insect.

Insects must intake sterol compounds because of their inability to synthesize cholesterol de novo. In phytophagous insects, enzymatic conversion of phytosterols to cholesterol involving 24-dehydrocholesterol reductase (DHCR24) exerts to acquire cholesterol. Here, we reported the presence of two DHCR24 homologs in the silkworm Bombyx mori, BmDHCR24-1 and -2, with several transcript variants. Consistent with the data of spatial expression analyses by RT-PCR, predominant enzymatic activity of DHCR24 was observed in B. mori larval midgut whereas weak activity was observed in the other tissues examined. In addition, BmDHCR24-1 expression in HEK293 cells showed an enzymatic activity, but BmDHCR24-2 did not, although both BmDHCR24s were localized in the endoplasmic reticulum, where the mammalian DHCR24s are located to exert their enzymatic activities. The present data indicated that BmDHCR24-1 but not BmDHCR24-2 contributes to conversion of phytosterols to cholesterol mainly in the midgut of the phytophagous lepidopteran larvae.

Enteroendocrine peptides regulate feeding behavior via controlling intestinal contraction of the silkworm Bombyx mori.

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.

Antagonistic Effect of Short Neuropeptide F on Allatotropin-Inhibited Feeding Motivation of the Silkworm Larva, Bombyx mori.

Here, we demonstrated an antagonistic effect of short neuropeptide F (sNPF) in modulating feeding motivation in the silkworm Bombyx mori; sNPF reduced the feeding-delaying effects caused by administration of an inhibitory peptide, allatotropin (AT). In situ hybridization and MALDI-TOF MS analysis revealed the presence of three subtypes of sNPFs (sNPF-1, -2, and -3) in the midgut enteroendocrine cells. Ca2+-imaging analyses revealed that three subtypes of sNPF receptors (sNPFRs) (BNGR-A7, -A10, and -A11) showed different affinities with the three subtypes of sNPFs. In addition, sNPF activated its signaling via ERK phosphorylation in the midgut, while mixture of sNPF and AT reduced the phosphorylation level, agreeing with the results of behavioral assay. Together, our current findings suggest that intestinal sNPF positively modulates the feeding motivation by reducing the inhibitory effects by AT within the midgut.

Effects of starvation on brain short neuropeptide F-1, -2, and -3 levels and short neuropeptide F receptor expression levels of the silkworm, Bombyx mori.

In our previous report, we demonstrated the possibility that various regulatory neuropeptides influence feeding behavior in the silkworm, Bombyx mori. Among these feeding-related neuropeptides, short neuropeptide F (sNPF) exhibited feeding-accelerating activity when injected into B. mori larvae. Like other insect sNPFs, the deduced amino acid sequence of the cDNA encoding the sNPF precursor appears to produce multiple sNPF and sNPF-related peptides in B. mori. The presence of three sNPFs, sNPF-1, sNPF-2, and sNPF-3, in the brain of B. mori larvae was confirmed by direct MALDI-TOF mass spectrometric profiling. In addition, all three sNPFs are present in other larval ganglia. The presence of sNPF mRNA in the central nervous system (CNS) was also confirmed by Reverse transcription-polymerase chain reaction. Semi-quantitative analyses of sNPFs in the larval brain using matrix-assisted laser desorption ionization time-of-flight mass spectrometry further revealed that brain sNPF levels decrease in response to starvation, and that they recover with the resumption of feeding. These data suggest that sNPFs were depleted by the starvation process. Furthermore, food deprivation decreased the transcriptional levels of the sNPF receptor (BNGR-A10) in the brain and CNS, suggesting that the sNPF system is dependent on the feeding state of the insect and that the sNPF system may be linked to locomotor activity associated with foraging behavior. Since the injection of sNPFs accelerated the onset of feeding in B. mori larvae, we concluded that sNPFs are strongly related to feeding behavior. In addition, semi-quantitative MS analyses revealed that allatostatin, which is present in the larval brain, is also reduced in response to starvation, whereas the peptide level of Bommyosuppressin was not affected by different feeding states.

Identification of cDNAs encoding allatotropin and allatotropin-like peptides from the silkworm, Bombyx mori.

The cDNAs encoding allatotropin (AT) and allatotropin-like peptides (ATLPs) were isolated from the silkworm, Bombyx mori. Similar to those of the tobacco hornworm, Manduca sexta, four peptides (AT, ATLP1, ATLP2, and ATLP3) are present in three different variants generated by alternative splicing. RT-PCR analyses showed that these splice variants are expressed in the central nervous system with differing expression patterns in each ganglion. Immunohistochemistry using an anti-AT antibody confirmed that AT-expressing cells were located in these central nervous ganglia as well as in two large anterior cells of the frontal ganglia. Injection of synthetic AT and ATLP-1 into B. mori larvae increased the latency to feed, indicating that AT and ATLP might function in the regulation of feeding behavior in B. mori.

Effects of neuropeptides on feeding initiation in larvae of the silkworm, Bombyx mori.

In insects, especially phytophagous insects, feeding behavior occurs at a regular frequency. Although a number of physiological studies have revealed various causal factors leading to feeding behavior in insects, little has been demonstrated regarding the regulatory mechanisms underlying insect feeding behavior. To confirm the presence of an endocrinological regulatory mechanism in feeding behavior, we tested the effects of several biologically active peptides on silkworm, Bombyx mori larvae feeding behaviors. To evaluate the effects of the biologically active peptides, we measured the period of latency to the first bite following sample injection into starved Bombyx larvae. Of the chemically synthesized peptides tested, myosuppressin exhibited a prolonged latency, indicating that myosuppressin is a possible inhibitory peptide in Bombyx larvae. In contrast, injections of tachykinin and short neuropeptide F, which are members of the structurally related RF-amide peptide family, had a shorter latency period, indicating that these two peptides are possible stimulatory peptides. In addition, the present study suggests that this bioassay will be advantageous for screening for peptides that regulate insect feeding behavior.

Characterization of feeding-delaying factors from the silkworm Bombyx mori.

In several phytophagous insects, feeding behavior occurs regularly. Recently, we demonstrated that feeding behavior in larvae of the silkworm Bombyx mori had a regular frequency. To address the control of the feeding cycle in B. mori, we aimed to characterize factors influencing feeding initiation and termination. Injection of extracts of the midgut, foregut, and fat body into starved Bombyx larvae delayed the initiation of feeding. This result indicates the presence in these tissues of factors capable of decreasing the likelihood of feeding initiation.