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Effects of neuropeptides on feeding initiation in larvae of the silkworm, Bombyx mori
General and Comparative Endocrinology 172 (2011) 90–95
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Effects of neuropeptides on feeding initiation in larvae of the silkworm, Bombyx mori Shinji Nagata ⇑, Nobukatsu Morooka, Sumihiro Matsumoto, Takeshi Kawai, Hiromichi Nagasawa Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
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Article history: Available online 11 March 2011 Keywords: Bombyx mori Feeding behavior Myosuppressin Short neuropeptide F Tachykinin
a b s t r a c t 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. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Insects are the most prosperous members of the animal kingdom on earth. Among the widely spread insect species, a majority are obligate plant feeders. Although these phytophagous insects derive sustenance from their host dietary plants, they do not continuously feed. This indicates that some endogenous regulatory mechanisms function in the initiation and termination of meals. It also implies that regulation of insect feeding behavior occurs in response to both nutrient and nutritional storage status of the insect. An extensive number of physiological studies on feeding behavior have been performed on the desert locust, Schistocerca gregaria, and the migratory locust, Locusta migratoria [21], both of which are major agricultural pests. Physiological studies on insect feeding behavior have also displayed the pattern of feeding in various insect species, including locusts [20] and lepidopteran species [3,4]. Although feeding behaviors in most phytophagous insects occur at a regular frequency, several environmental factors such as natural enemies and poor weather conditions appear to disturb these regularly occurring feedings in wild insects. Recently, we also found regularly occurring feeding behavior in larvae of the domesticated silkworm, Bombyx mori [12]. Unlike other insect species, Bombyx larvae showed regularly occurring feeding behavior throughout their growth. It is also of interest that the feeding pattern in this species is independent of circadian rhythms [12], indicating that ⇑ Corresponding author. Fax: +81 3 5841 8022. E-mail address: [email protected] (S. Nagata). 0016-6480/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2011.03.004
this species can be a model organism for investigating the regulatory mechanisms underlying insect feeding behavior. Although a number of causal factors for insect feeding behavior have been characterized, little is known regarding the regulatory mechanisms at the molecular level. One of the reasons why molecular-based investigations of feeding behavior have lagged behind physiological investigations might be due to the difficulties in observing and evaluating the feeding behavioral activities in insects. For example, the most utilized bioassays to evaluate feeding activity include counting fecal pellets or measuring meal sizes, but no direct observations. Such assays can characterize the major principles to impact on feeding behavior. However, the regulatory mechanisms of regularly occurring feeding behavior in insects can be reset by each meal, similar to that of circadian rhythms, which are reset by light-stimulation. Therefore, observation of feeding behavior in insects is required for screening for the feeding-driving factors, since conventional evaluation methods such as measurement of fecal pellets and meal sizes are not enough to confirm the detailed behaviors. Because the causal factors of feeding behavior can influence the state of the hemolymph, it has been proposed that regulation of feeding behavior is mediated by nutrient status as well as several factors in the hemolymph [22]. This implies that endocrine regulation may play a key role in insect feeding behaviors similar to the regulatory mechanism of feeding in vertebrates. In addition, it has been demonstrated that such regulation of feeding behavior in vertebrates is strongly related to peptidyl factors [27]. To date, genomic information is available for several insect species. Those databases have allowed us to identify peptide factors
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comprehensively in silico. In addition, both genomic and transcriptomic data for the silkworm, B. mori, have provided information on the proteinous and peptidyl compounds [18]. Moreover, transcriptomic investigations building on the Bombyx genome and expression sequence tags (ESTs) have revealed that almost all of the peptidyl factors and their putative receptors (GPCRs; G-protein coupled receptors) have been identified [32]. To elucidate the molecular mechanisms underlying insect feeding behavior, we have screened a number of peptidyl factors for their ability to contribute to the feeding behaviors in larvae of the silkworm, B. mori. 2. Materials and methods
Table 1 List of examined peptides and their amino acid sequences.
a b
Injected peptides
Amino acid sequence
Tachykinin-1 (TK-1) Tachykinin-2 (TK-2) PDF Myosuppressin-1(BMS) Myosuppressin-2 (BMS-2) IMF amide sNPF-1 sNPF-2 sNPF-3 Proctolin
IPQGFLGMRa,b AANMHQFYGVRa,b NADLINSLLALPKDMNDAa DPSFIRFa SAIDRSMIRFa,b NYKNAPMNGIMFa,b APSMRLRFa,b TPVRLRFa SPSPPLRFa,b RYLPT
Amidated C-terminal. Found in silico.
2.1. Insects
Chemicals and reagents used in the present study were purchased from Nacalai-tesk (Osaka, Japan). The organic solvents and acetonitrile for RP–HPLC were purchased from Kanto Chemicals (Tokyo, Japan). Fmoc derivatives of amino acids were purchased from Watanabe Chemical Industries (Hiroshima, Japan).
were collected by centrifugation. The resulting crude synthetic peptides were subjected to Sep-Pak Vac C18. The column was washed with 0.1% TFA aqueous solution and then eluted with 60% acetonitrile/0.1% TFA. The eluate was subjected to an analytical reversed-phase HPLC (RP–HPLC) (JASCO SC-802, PU-880, UV875; JASCO; Tokyo, Japan) on a Senshu Pak Pegasil-300 ODS column (4.6 mm i.d. 250 mm; Senshu Kagaku; Tokyo, Japan) with a 25 min linear gradient of 10–60% acetonitrile containing 0.05% TFA at a flow rate of 1.0 ml/min. The elution was monitored by absorbance at 225 nm. The purified synthetic peptide was confirmed by measurement on a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer using Voyager-DE™ STR (Applied Biosystems, CA, USA) in the positive ion mode with a-cyano-4-hydroxycinnamic acid (CCA) as a matrix. Matrix solution was prepared by saturating CCA in 60% acetonitrile containing 0.1% TFA. Samples were applied after mixing with the matrix solution at a 1:1 ratio.
2.3. Bioassay for feeding behavior using Bombyx larvae
2.5. Statistical analyses
Only populations of larvae growing synchronously were used in assays. Before sample injection, larvae at day-2 of the last instar were starved for 16 h. The larvae were anesthetized by submerging in ice-cold water (4 °C) for 15 min. For maintenance of temperature changes by anesthetization, the anesthetized larvae were transferred into a paper towel to wipe off excess water and ice. Samples, which were dissolved in distilled water (100 ll), were immediately injected into the dorso-abdominal portion (at the fifth segment) of larvae after drying. Larvae were then transferred into a conditioned rearing room and were placed on new paper towels for facing an artificial diet block on a large sheet of wax paper in a plastic container. Behaviors of the larvae were observed until their first bites. The precisely conditioned sample injected larvae were confirmed by checking the moving initiation time at about 5 to 7 min after injection. Although larvae generally initiate nibbling behaviors at the beginning of each meal, these behaviors were not considered a bite. Observations were carried out using at most 10 larvae simultaneously. Because phosphate-buffered saline and Tris-buffered saline were used as a vehicle, the latencies after injection widely deviated compared with the use of distilled water (data not shown), consequently, we used distilled water as a vehicle for injection.
All examined assay data were statistically analyzed by the method of analysis of variant (one-way ANOVA). The reproducibility of all examined bioassays was confirmed at least two times with different populations of larvae.
Silkworm eggs from the hybrid B. mori strain (Kinshu Showa) were purchased from UEDA SANSHU Ltd. (Ueda, Japan). Larvae were reared in plastic containers at 26 ± 1 °C with 70 ± 10% relative humidity under long-day lighting conditions (16L:8D), using SILKMATE 2S artificial diet purchased from NIPPON NOSAN Co. Ltd. (Yokohama, Japan). Larvae were provided with fresh diet on a daily basis. Only larvae whose growth was synchronized were utilized for staged experiments. 2.2. Chemicals and reagents
3. Results 3.1. Effects of water To identify the endogenous factors modulating insect feeding behavior, we previously established a bioassay using larvae of the silkworm, B. mori [13]. The established bioassay was setup as described in Fig. 1A. Feeding activity was evaluated by measuring the latency to the first bite after the sample injection into 16 h starved larvae. During the initial phases of setting up the bioassay, we noticed that injection volume influenced the latency to the first bite. We tested the effect of injection volume (ranging from 5 to 100 ll) of distilled water into starved Bombyx larvae. The prolonged latency to the first bite correlated with increased injection volumes (Fig. 1B). Among the different volumes tested, the resulting latency to the first bite after injection of distilled water deviated less at a volume of 100 ll; we, therefore, injected samples of interest dissolved in 100 ll of distilled water.
2.4. Preparation of synthetic peptides 3.2. Effects of synthetic neuropeptides on the latency to the first bite All peptides listed in Table 1 were chemically synthesized based on the Fmoc method using an automated peptide synthesizer (APEX-SC, apex396, Advanced ChemTech, Louisville, KT, USA) according to the manufacturer’s instruction. After deprotection and cleavage from resins, the synthetic peptides in diethylether
Although it has been proposed that several peptidyl factors modulate feeding behavior, little has been demonstrated so far. To elucidate the mechanism underlying insect feeding behavior and to determine the identity of peptidyl factors contributing to
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A
**
B
Latency to the first bite (min)
30
20
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** 10
proctolin
sNPF-2
myosupressin-1
PDF
IMF amide
tachykinin-1
vehicle
0
Injected peptides
Fig. 1. Schematic diagram of the delayed feeding assay. (A) Assay schedule for delayed feeding activity. Bombyx larvae were starved for 16 h and then injected with samples after anesthetization. The latency was compared with vehicle (distilled water) injected larvae as a control. The biological activity was evaluated by measuring the latency to the first bite. (B) Effects of distilled water on the latency to the first bite. 100, 50, and 10 ll of distilled water were injected. Zero microliter of distilled water indicates sham-operated data. An asterisk represents significant difference (⁄p < 0.05, one-way ANOVA). Data represent mean ± SD (n = 5).
feeding regulation, we assayed various chemically synthesized biologically active peptidyl factors. Since Roller and his colleagues comprehensively identified peptidyl factors from genomic information of the silkworm, B. mori, in silico [18], we selected several biologically active peptides from a list of peptides: IMF amide, pigment dispersing factor (PDF), tachykinin-1 (TK-1), Bombyx myosuppressin-1 (Bommyosuppressin-1: BMS-1), and short neuropeptide F-2 (sNPF-2) (Table 1). TK and sNPF were tested to assess their possible roles in modulating feeding in B. mori larvae as those peptides have been reported to be feeding-related peptides in the fruit fly, Drosophila melanogaster [2,9,29]. PDF was tested for the possibility of feeding modulation based on its ability to modulate locomotor activity in a number of insects including D. melanogaster [6] and L. migratoria [16]. We also tested proctolin, an endogenic cardiac stimulator that is widely conserved among arthropod species, even though its presence has not been confirmed in the Bombyx genome [18]. Since these peptides share the similar C-terminal motif, RF-amide and F-amide, we also tried IMFamide, which is one of the peptides identified in silico possessing a similar C-terminal F-amide [18]. The chemically synthesized peptides were purified by reversed-phase HPLC prior to use in the bioassay. First, two doses (0.2 and 1.0 lg) of synthetic peptides were injected into day-2 last instar larvae (Fig. 2). Once the injected samples were wholly dispersed in the body, their hemolymph concentration would be expected to be in the range of 50 nM to 150 lM. Among the examined peptides, BMS-1 prolonged the latency to the first bite, whereas the other tested peptides did not. Interestingly, TK-1 and sNPF-2 shortened the latency to the first bite. In addition, a high dose of PDF showed a tendency toward prolonged latency to the first bite, as shown in Fig. 2. Observation of the PDF-injected larvae indicated that the delayed latency might
Fig. 2. Effects of chemically synthesized biologically active peptides on the latency to the first bite. Gray and black bars indicate latencies to the first bites following injection of 0.2 and 1.0 lg of peptide, respectively. The dotted line and the white bar represent the time of the latency to the first bite after injection of 100 ll of distilled water. Asterisks ( and ) indicate significant differences (p < 0.05 and p < 0.01, respectively) compared with vehicle-injected larvae by one-way ANOVA. Data represent mean ± SD (n = 5).
be due to exaggerated active locomotor. As shown in Fig. 2, although BMS-1 and sNPF share the same C-terminal sequence, RF-amide, the resulting biological activities in this bioassay had opposite effects. In contrast, IMF amide, which is characterized by the F-amide instead of the RF-amide, did not show any difference in latency compared with the vehicle injection. Because several analogous peptides to BMS-1 and sNPF-2 are potentially encoded by the respective cDNAs, we further analyzed the effect of those peptides (BMS-2, sNPF-1 and sNPF-3). 3.3. Effects of myosuppressin on the latency to the first bite To confirm the effects of BMS-1 on the delayed latency to the feeding initiation, we injected different amounts of this peptide (Fig. 3). Latency was prolonged in a dose-dependent manner by BMS-1 injections. The effects of prolonged latency were observed with as little as 10 ng (approximately 5 pmol) injection. The latency to the first bite did not reach the plateau level even with 5 lg injection. Another analog of BMS-1, BMS-2 [31] was also assayed. In contrast to BMS-1, BMS-2 had a less severe effect on latency (Fig. 3). In addition, because decreased locomotor activity was not observed in BMS-injected larvae, the prolonged latency following BMS-1 injection might not be due to the suppression of locomotor activity, which would be expected to affect the larvae’s ability to approach the artificial diet block. 3.4. Effects of tachykinin on the latency to the first bite As observed in Fig. 2, the latency to the first bite was shortened following TK-1 injection. To confirm the effects of TK-1, we injected different amounts of TK-1 ranging from 10 ng to 5 lg; approximately 10 pmol to 5 nmol (Fig. 4). The result showed that injection of TK-1 shortened the latency in a dose-dependent manner (Fig. 4). Since it has been proposed that five TKs are encoded in a single cDNA, we injected another type of tachykinin, TK-2
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**
Latency to the first bite (min)
** 30
*
*
*
20
10
0
Latency to the first bite (min)
30 40
20
*
0 0
10
100
1000
5000
0
myosuppressin (ng)
10
100
1000
5000
sNPF (ng)
Fig. 3. Dose-dependent effect of myosuppressin (BMS-1 and BMS-2) on the latency to the first bite. The injected doses of BMS-1 (closed circles) and BMS-2 (closed triangles) were 0.01, 0.1, 1.0, and 5.0 lg/ larva. Asterisks ( and ) indicate significant differences (p < 0.05 and p < 0.01, respectively) compared with vehicleinjected larvae by one-way ANOVA. Data represent mean ± S. D. (n = 5). A similar data tendency was observed in three trials, confirming the reproducibility.
30
Latency to the first bite (min)
**
10
Fig. 5. Dose-dependent effect of short neuropeptide Fs (sNPF-1, sNPF-2, and sNPF3) on the latency to the first bite. The injected doses of sNPF-1 (closed triangles), sNPF-2 (closed circles), and sNPF-3 (open circles) were 0.01, 0.1, 1.0, and 5.0 lg/ larva. Asterisks ( and ) indicate significant differences (p < 0.05 and p < 0.01, respectively) compared with vehicle-injected larvae by one-way ANOVA. Data represent mean ± SD (n = 5). A similar data tendency was observed in three trials, confirming the reproducibility.
at all doses injected, whereas sNPF-2 showed a shortened latency to the first bite after peptide injection in a dose-dependent manner. The effect of sNPF-2 on the shortened latency to the first bite was observed with injections of more than 100 ng.
20
* **
10
*
4. Discussion
**
In the present study, we screened several biologically active peptides in terms of their effect on latency to the first bite in starved Bombyx larvae. The activities of the tested peptides were assessed by prolonged or shortened latency. Of the examined peptides, BMS had a prolonged effect, while TK-1 and sNPF-2 reduced latency.
0 0
10
100
1000
5000
tachykinin (ng) Fig. 4. Dose-dependent effect of tachykinin (TK-1 and TK-2) on the latency to the first bite. The injected doses of TK-1 (closed circles) and TK-2 (closed triangles) were 0.01, 0.1, 1.0, and 5.0 lg/ larva. Asterisks ( and ) indicate significant differences (p < 0.05 and p < 0.01, respectively) compared with vehicle-injected larvae by oneway ANOVA. Data represent mean ± SD (n = 5). A similar data tendency was observed in three trials, confirming the reproducibility.
[18,19,14]. The difference in the amino acid sequences between TK-1 and TK-2 exists in the C-terminal portion, which is relatively conserved and contributes to the biological activities of TKs. Both TK-1 and TK-2 shortened the latency to the first bite similarly in a dose-dependent manner. The effects of TK-1 and -2 on the shortened latencies to the first bites were observed with more than 100 ng and 1 lg injection, respectively. 3.5. Effects of short neuropeptide F on the latency to the first bite As observed in Fig. 2, the latency to the first bite was also shortened by sNPF-2 injection. To confirm this, we next assayed different amounts of sNPF-2 ranging from 10 ng to 5 lg; approximately 10 pmol to 5 nmol (Fig. 5). Similar to TK peptides, three different sNPFs are encoded by a single cDNA [32]. We consequently injected sNPF-1, -2 and -3 and measured their effects on latency to the first bite. sNPF-1 and sNPF-3 had minor effects on latency
4.1. Effect of Bommyosuppressins BMS delayed the initiation of feeding, which might be attributed to: decreased myotropic activity by locomotor depression and weakened gut contraction activity. Several explainable facts on the BMS effects have been reported so far. Myosuppressin was originally isolated from head extracts of the cockroach, Leucophaea maderae as an inhibitor of spontaneous visceral muscle contraction [7]. It has been demonstrated that myosuppressin exhibits several biological activities, including inhibition of muscle and gut contractions [1,7,17], antifeeding activity [28], and inhibition of neuropeptide secretion [30]. There are several peptides analogous to myosuppressin encoded by a single cDNA including BMS-2 [30]. As a result, BMS-2 showed weaker prolonging activity. They also might be involved in feeding behavior similar to BMS, most likely by modulating gut contraction. Since the expression sites of myosuppressin are the central and stomatogastoric nervous systems and the endocrine cells in the midgut of the fruit fly, D. melanogaster [10] and other species [11,25], BMS could also potentially function as a brain-gut regulatory neuropeptide modulating feeding behavior. It has been reported that ecdysteroid secretion from the prothoracic glands is regulated by BMS [30], suggesting that BMS is involved in the general secretory mechanisms of neuroendocrine cells involved in producing other hormones.
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4.2. Effects of tachykinin-related peptides In the present study, we found two peptides, TK and sNPF, which when injected shortened the feeding latency period. Both peptides have been established as feeding-related factors in the fruit fly, D. melanogaster [2,8,9,29]. In contrast to tachykinin-related peptides, there have been no reports to date indicating that sNPF functions in the feeding behavior of other species. Insect TK was originally identified from L. migratoria [19]. By searching in silico, a cDNA encoding TK was identified from B. mori [18]. The TK cDNA encodes five different TK peptides including TK1 and TK-2, which were used for assay in the present study. TK mRNA is widely expressed in the CNS, indicating that TK also functions as a pleiotropic factor. These TK peptides share the C-terminal sequence, GXRamide. In the present study, we utilized two TKs whose C-terminal sequences were GMRamide and GVRamide. In the present experiment, we could not find any differences in the feeding stimulating activity between them (data not shown). Because of the conservation of the C-terminal amino acid, all five tachykinin-like peptides may exhibit shortening activity in Bombyx larvae. 4.3. Effect of short neuropeptide F sNPF was first identified from the Colorado potato beetle, Leptinotarsa decemlineata as a homolog of a mammalian feeding regulatory peptide, neuropeptide Y (NPY) [23]. To date, sNPF is well known as a neuropeptide conserved among arthropods [15]. In Drosophila, sNPF regulates its calorimetric control in combination with insulin-like peptides [8,9]. In B. mori, sNPF was recently identified as a factor regulating JH biosynthesis by inducing the secretion of alltotropin from the corpora cardiaca [32]. The possibility exists that, similar to allatotropin–sNPF regulation, sNPF may regulate the secretion of an unidentified neuropeptide(s) or hormones that function in feeding behavior. The present data showed that three sNPFs exhibited somewhat different strengths of activity (Fig. 5). The difference in the activity of sNPF might be due to differences in receptors as well as the difference of peptide sequences. In fact, transcriptome analysis of GPCRs showed the presence of two receptors for sNPFs in B. mori [32]. Therefore, for a more detailed understanding of sNPF regulation, further studies including expression and distribution analyses of the sNPF receptors and their binding activities are strongly required. 4.4. Effects of other examined peptides Of the injected peptides, we also tested the effect of proctolin, which was originally identified from the cockroach, Periplaneta americana [24]. It has been thought that there is no proctolin in lepidopteran species. Although proctolin is absent in the Bombyx genomic sequence [18], previous reports indicate that proctolin is detectable by immunostaining in the gypsy moth, Lymantria dispar [5], and the tobacco hormworm, Manduca sexta [33]. Consequently, we tested this peptide. In the present study, we could not find any significant effect of proctolin (Fig. 1) on latency to the first bite. Therefore, we concluded that the lack of an effect by proctolin is due to two possibilities: the respective receptor is not expressed in B. mori or that activity was below the threshold for detection in this assay. To clarify whether proctolin can influence biological events in B. mori other investigations such as a gut contraction assay are required. The effects of PDF on the latency to the first bite resulted in data with an expanded deviation (Fig. 2). Our observations revealed more excited behaviors in terms of foraging-like behaviors such as nibbling and walking. Injection of PDF might disturb the
pacemaker neuron related locomotor activity which is regulating circadian rhythms in many species including B. mori [6,16,26]. 4.5. The possibilities for screening biologically active peptides using this assay In the present study, we demonstrated the effects of known biologically active peptides on feeding behavior by observing changes in the amount of time silkworm larvae spent in a latency period larvae prior to first bites following peptide injection. Despite similar C-terminal motifs among the peptides tested, the assay detected three different peptide effects on the latency period: prolonged, shortened and no effect. Although the functional details of the effective peptides remain to be elucidated, we suggest that our assay would serve as a good screening step for judging whether the testing sample constitutes a feeding-modulating peptide or not. However, because a large amount of peptide sample is required for a single bioassay, large numbers of animals might be needed for identification from crude extracts. In summary, we identified several possible feeding modulating factors from known biologically active peptides. However, we have to identify the true biologically active factors regulating feeding behavior in insects. Our previous report showed that several biologically active factors are present in the crude extract [13], we will now work toward identifying strong candidate feeding-modulating factors in the near future. Acknowledgments This work was supported in part by Grants-in-Aid for Scientific Research (#18780083 and 22780099) from the Ministry of Education, Science, Sports, and Culture of Japan, and the NAITO Foundation. The authors thank Dr. J. Joe Hull (USDA-ARS,Maricopa, AZ) for critical reading and assistance in manuscript preparation. References [1] R. Aguilar, J.L. Maestro, L. Vilaplana, C. Chiva, D. Andreu, X. Bellés, Identification of leucomyosuppressin in the German cockroach Blattella germanica, as an inhibitor of food intake, Regul. Peptide 119 (2004) 105–112. [2] B. Al-Anzi, E. Armand, P. Nagamei, M. Olszewski, V. Sapin, C. Waters, K. Zinn, R.J. Wyman, S. Benzer, The leucokinin pathway and its neurons regulate meal size in Drosophila, Curr. Biol. 20 (2010) 969–978. [3] E.A. Bernays, M.S. Singer, A rhythm underlying feeding behaviour in a highly polyphagous caterpillar, Ecol. Entomol. 23 (1998) 295–302. [4] E.A. Bernays, H.A. Woods, Foraging in nature by larvae of Manduca sexta – influenced by an endogenous oscillation, J. Insect Physiol. 46 (2000) 825–836. [5] N.T. Davis, S.G. Velleman, T.G. Kingan, H. Keshishian, Identification and distribution of a proctolin-like neuropeptide in the nervous system of the gypsy moth, Lymantria dispar, and in other Lepidoptera, J. Comp. Neurol. 283 (1989) 71–85. [6] C. Helfrich-Förster, M. Täuber, J.H. Park, M. Mühlig-Versen, S. Schneuwly, A. Hofbauer, Ectopic expression of the neuropeptide pigment-dispersion factor alters behavioral rhythms in Drosophila melanogaster, J. Neurosci. 20 (2000) 3339–3353. [7] G.M. Holman, B.J. Cook, R.J. Nachman, Isolation, primary structure and synthesis of leucomyosuppressin, an insect neuropeptide that inhibits spontaneous contractions of the cockroach hindgut, Comp. Biochem. Physiol. C 85 (1986) 329–333. [8] K.S. Lee, K.H. You, J.K. Choo, Y.M. Han, K. Yu, Drosophila short neuropeptide F regulates food intake and body size, J. Biol. Chem. 279 (2004) 50781–50789. [9] K.S. Lee, O.Y. Kwon, J.H. Lee, K. Kwon, K.J. Min, S.A. Jung, A.K. Kim, K.H. You, M. Tatar, K. Yu, Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling, Nat. Cell Biol. 10 (2008) 468–475. [10] J. McCormick, R. Nichols, Spatial and temporal expression identify dromyosuppressin as a brain-gut peptide in Drosophila melanogaster, J. Comp. Neurol. 338 (1993) 278–288. [11] S.M. Meola, M.S. Wright, G.M. Holman, J.M. Thompson, Immunocytochemical localization of leucomyosuppressin-like peptides in the CNS of the cockroach, Leucophaea maderae, Neurochem. Res. 16 (1991) 543–549. [12] S. Nagata, H. Nagasawa, Effects of diet-deprivation and physical stimulation on the feeding behaviour of the larvae of the silkworm, Bombyx mori, J. Insect Physiol. 52 (2006) 807–815.
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[24] A.N. Starratt, B.E. Brown, Structure of the pentapeptide proctolin, a proposed neurotransmitter in insects, Life Sci. 17 (8) (1975) 1253–1256. [25] L.S. Swales, P.D. Evans, Distribution of SchistoFLRFamide-like immunoreactivity in the adult ventral nervous system of the locust, Schistocerca gregaria, Cell Tissue Res. 281 (1995) 339–348. [26] P.H. Taghert, O.T. Shafer, Mechanisms of clock output in the Drosophila circadian pacemaker system, J. Biol. Rhythms 21 (2006) 445–457. [27] F. Takenoya, H. Kageyama, K. Shiba, Y. Date, M. Nakazato, S. Shioda, Neuropeptide W: a key player in the homeostatic regulation of feeding and energy metabolism?, Ann N.Y. Acad. Sci. 1200 (2010) 162–169. [28] L. Vilaplana, N. Pascual, N. Perera, D. Leira, X. Bellés, Antifeeding properties of myosuppressin in a generalist phytophagous leafworm, Spodoptera littoralis (Boisduval), Regul. Pept. 148 (2008) 68–75. [29] A.M.E. Winther, D.R. Nässel, Intestinal peptides as circulating hormones: release of tachykinin-related peptide from the locust and cockroach midgut, J. Exp. Biol. 204 (2001) 1269–1280. [30] N. Yamanaka, Y.J. Hua, A. Mizoguchi, K. Watanabe, R. Niwa, Y. Tanaka, H. Kataoka, Identification of a novel prothoracicostatic hormone and its receptor in the silkworm Bombyx mori, J. Biol. Chem. 280 (2005) 14684–14690. [31] N. Yamanaka, D. Zitnan, Y.J. Kim, M.E. Adams, Y.J. Hua, Y. Suzuki, M. Suzuki, A. Suzuki, H. Satake, A. Mizoguchi, K. Asaoka, Y. Tanaka, H. Kataoka, Regulation of insect steroid hormone biosynthesis by innervating peptidergic neurons, Proc. Natl. Acad. Sci. USA 103 (2006) 8622–8627. [32] N. Yamanaka, S. Yamamoto, D. Zitnan, K. Watanabe, T. Kawada, H. Satake, Y. Kaneko, K. Hiruma, Y. Tanaka, T. Shinoda, H. Kataoka, Neuropeptide receptor transcriptome reveals unidentified neroendocrine pathways, Plos One 3 (2008) e3048. [33] D. Zitnan, T.G. Kingan, S.J. Kramer, N.E. Beckage, Accumulation of neuropeptides in the cerebral neurosecretory system of Manduca sexta larvae parasitized by the braconid wasp Cotesia congregata, J. Comp. Neurol. 356 (1995) 83–100.
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General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen
Effects of neuropeptides on feeding initiation in larvae of the silkworm, Bombyx mori Shinji Nagata ⇑, Nobukatsu Morooka, Sumihiro Matsumoto, Takeshi Kawai, Hiromichi Nagasawa Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
a r t i c l e
i n f o
Article history: Available online 11 March 2011 Keywords: Bombyx mori Feeding behavior Myosuppressin Short neuropeptide F Tachykinin
a b s t r a c t 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. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Insects are the most prosperous members of the animal kingdom on earth. Among the widely spread insect species, a majority are obligate plant feeders. Although these phytophagous insects derive sustenance from their host dietary plants, they do not continuously feed. This indicates that some endogenous regulatory mechanisms function in the initiation and termination of meals. It also implies that regulation of insect feeding behavior occurs in response to both nutrient and nutritional storage status of the insect. An extensive number of physiological studies on feeding behavior have been performed on the desert locust, Schistocerca gregaria, and the migratory locust, Locusta migratoria [21], both of which are major agricultural pests. Physiological studies on insect feeding behavior have also displayed the pattern of feeding in various insect species, including locusts [20] and lepidopteran species [3,4]. Although feeding behaviors in most phytophagous insects occur at a regular frequency, several environmental factors such as natural enemies and poor weather conditions appear to disturb these regularly occurring feedings in wild insects. Recently, we also found regularly occurring feeding behavior in larvae of the domesticated silkworm, Bombyx mori [12]. Unlike other insect species, Bombyx larvae showed regularly occurring feeding behavior throughout their growth. It is also of interest that the feeding pattern in this species is independent of circadian rhythms [12], indicating that ⇑ Corresponding author. Fax: +81 3 5841 8022. E-mail address: [email protected] (S. Nagata). 0016-6480/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2011.03.004
this species can be a model organism for investigating the regulatory mechanisms underlying insect feeding behavior. Although a number of causal factors for insect feeding behavior have been characterized, little is known regarding the regulatory mechanisms at the molecular level. One of the reasons why molecular-based investigations of feeding behavior have lagged behind physiological investigations might be due to the difficulties in observing and evaluating the feeding behavioral activities in insects. For example, the most utilized bioassays to evaluate feeding activity include counting fecal pellets or measuring meal sizes, but no direct observations. Such assays can characterize the major principles to impact on feeding behavior. However, the regulatory mechanisms of regularly occurring feeding behavior in insects can be reset by each meal, similar to that of circadian rhythms, which are reset by light-stimulation. Therefore, observation of feeding behavior in insects is required for screening for the feeding-driving factors, since conventional evaluation methods such as measurement of fecal pellets and meal sizes are not enough to confirm the detailed behaviors. Because the causal factors of feeding behavior can influence the state of the hemolymph, it has been proposed that regulation of feeding behavior is mediated by nutrient status as well as several factors in the hemolymph [22]. This implies that endocrine regulation may play a key role in insect feeding behaviors similar to the regulatory mechanism of feeding in vertebrates. In addition, it has been demonstrated that such regulation of feeding behavior in vertebrates is strongly related to peptidyl factors [27]. To date, genomic information is available for several insect species. Those databases have allowed us to identify peptide factors
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comprehensively in silico. In addition, both genomic and transcriptomic data for the silkworm, B. mori, have provided information on the proteinous and peptidyl compounds [18]. Moreover, transcriptomic investigations building on the Bombyx genome and expression sequence tags (ESTs) have revealed that almost all of the peptidyl factors and their putative receptors (GPCRs; G-protein coupled receptors) have been identified [32]. To elucidate the molecular mechanisms underlying insect feeding behavior, we have screened a number of peptidyl factors for their ability to contribute to the feeding behaviors in larvae of the silkworm, B. mori. 2. Materials and methods
Table 1 List of examined peptides and their amino acid sequences.
a b
Injected peptides
Amino acid sequence
Tachykinin-1 (TK-1) Tachykinin-2 (TK-2) PDF Myosuppressin-1(BMS) Myosuppressin-2 (BMS-2) IMF amide sNPF-1 sNPF-2 sNPF-3 Proctolin
IPQGFLGMRa,b AANMHQFYGVRa,b NADLINSLLALPKDMNDAa DPSFIRFa SAIDRSMIRFa,b NYKNAPMNGIMFa,b APSMRLRFa,b TPVRLRFa SPSPPLRFa,b RYLPT
Amidated C-terminal. Found in silico.
2.1. Insects
Chemicals and reagents used in the present study were purchased from Nacalai-tesk (Osaka, Japan). The organic solvents and acetonitrile for RP–HPLC were purchased from Kanto Chemicals (Tokyo, Japan). Fmoc derivatives of amino acids were purchased from Watanabe Chemical Industries (Hiroshima, Japan).
were collected by centrifugation. The resulting crude synthetic peptides were subjected to Sep-Pak Vac C18. The column was washed with 0.1% TFA aqueous solution and then eluted with 60% acetonitrile/0.1% TFA. The eluate was subjected to an analytical reversed-phase HPLC (RP–HPLC) (JASCO SC-802, PU-880, UV875; JASCO; Tokyo, Japan) on a Senshu Pak Pegasil-300 ODS column (4.6 mm i.d. 250 mm; Senshu Kagaku; Tokyo, Japan) with a 25 min linear gradient of 10–60% acetonitrile containing 0.05% TFA at a flow rate of 1.0 ml/min. The elution was monitored by absorbance at 225 nm. The purified synthetic peptide was confirmed by measurement on a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer using Voyager-DE™ STR (Applied Biosystems, CA, USA) in the positive ion mode with a-cyano-4-hydroxycinnamic acid (CCA) as a matrix. Matrix solution was prepared by saturating CCA in 60% acetonitrile containing 0.1% TFA. Samples were applied after mixing with the matrix solution at a 1:1 ratio.
2.3. Bioassay for feeding behavior using Bombyx larvae
2.5. Statistical analyses
Only populations of larvae growing synchronously were used in assays. Before sample injection, larvae at day-2 of the last instar were starved for 16 h. The larvae were anesthetized by submerging in ice-cold water (4 °C) for 15 min. For maintenance of temperature changes by anesthetization, the anesthetized larvae were transferred into a paper towel to wipe off excess water and ice. Samples, which were dissolved in distilled water (100 ll), were immediately injected into the dorso-abdominal portion (at the fifth segment) of larvae after drying. Larvae were then transferred into a conditioned rearing room and were placed on new paper towels for facing an artificial diet block on a large sheet of wax paper in a plastic container. Behaviors of the larvae were observed until their first bites. The precisely conditioned sample injected larvae were confirmed by checking the moving initiation time at about 5 to 7 min after injection. Although larvae generally initiate nibbling behaviors at the beginning of each meal, these behaviors were not considered a bite. Observations were carried out using at most 10 larvae simultaneously. Because phosphate-buffered saline and Tris-buffered saline were used as a vehicle, the latencies after injection widely deviated compared with the use of distilled water (data not shown), consequently, we used distilled water as a vehicle for injection.
All examined assay data were statistically analyzed by the method of analysis of variant (one-way ANOVA). The reproducibility of all examined bioassays was confirmed at least two times with different populations of larvae.
Silkworm eggs from the hybrid B. mori strain (Kinshu Showa) were purchased from UEDA SANSHU Ltd. (Ueda, Japan). Larvae were reared in plastic containers at 26 ± 1 °C with 70 ± 10% relative humidity under long-day lighting conditions (16L:8D), using SILKMATE 2S artificial diet purchased from NIPPON NOSAN Co. Ltd. (Yokohama, Japan). Larvae were provided with fresh diet on a daily basis. Only larvae whose growth was synchronized were utilized for staged experiments. 2.2. Chemicals and reagents
3. Results 3.1. Effects of water To identify the endogenous factors modulating insect feeding behavior, we previously established a bioassay using larvae of the silkworm, B. mori [13]. The established bioassay was setup as described in Fig. 1A. Feeding activity was evaluated by measuring the latency to the first bite after the sample injection into 16 h starved larvae. During the initial phases of setting up the bioassay, we noticed that injection volume influenced the latency to the first bite. We tested the effect of injection volume (ranging from 5 to 100 ll) of distilled water into starved Bombyx larvae. The prolonged latency to the first bite correlated with increased injection volumes (Fig. 1B). Among the different volumes tested, the resulting latency to the first bite after injection of distilled water deviated less at a volume of 100 ll; we, therefore, injected samples of interest dissolved in 100 ll of distilled water.
2.4. Preparation of synthetic peptides 3.2. Effects of synthetic neuropeptides on the latency to the first bite All peptides listed in Table 1 were chemically synthesized based on the Fmoc method using an automated peptide synthesizer (APEX-SC, apex396, Advanced ChemTech, Louisville, KT, USA) according to the manufacturer’s instruction. After deprotection and cleavage from resins, the synthetic peptides in diethylether
Although it has been proposed that several peptidyl factors modulate feeding behavior, little has been demonstrated so far. To elucidate the mechanism underlying insect feeding behavior and to determine the identity of peptidyl factors contributing to
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A
**
B
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30
20
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** 10
proctolin
sNPF-2
myosupressin-1
IMF amide
tachykinin-1
vehicle
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Fig. 1. Schematic diagram of the delayed feeding assay. (A) Assay schedule for delayed feeding activity. Bombyx larvae were starved for 16 h and then injected with samples after anesthetization. The latency was compared with vehicle (distilled water) injected larvae as a control. The biological activity was evaluated by measuring the latency to the first bite. (B) Effects of distilled water on the latency to the first bite. 100, 50, and 10 ll of distilled water were injected. Zero microliter of distilled water indicates sham-operated data. An asterisk represents significant difference (⁄p < 0.05, one-way ANOVA). Data represent mean ± SD (n = 5).
feeding regulation, we assayed various chemically synthesized biologically active peptidyl factors. Since Roller and his colleagues comprehensively identified peptidyl factors from genomic information of the silkworm, B. mori, in silico [18], we selected several biologically active peptides from a list of peptides: IMF amide, pigment dispersing factor (PDF), tachykinin-1 (TK-1), Bombyx myosuppressin-1 (Bommyosuppressin-1: BMS-1), and short neuropeptide F-2 (sNPF-2) (Table 1). TK and sNPF were tested to assess their possible roles in modulating feeding in B. mori larvae as those peptides have been reported to be feeding-related peptides in the fruit fly, Drosophila melanogaster [2,9,29]. PDF was tested for the possibility of feeding modulation based on its ability to modulate locomotor activity in a number of insects including D. melanogaster [6] and L. migratoria [16]. We also tested proctolin, an endogenic cardiac stimulator that is widely conserved among arthropod species, even though its presence has not been confirmed in the Bombyx genome [18]. Since these peptides share the similar C-terminal motif, RF-amide and F-amide, we also tried IMFamide, which is one of the peptides identified in silico possessing a similar C-terminal F-amide [18]. The chemically synthesized peptides were purified by reversed-phase HPLC prior to use in the bioassay. First, two doses (0.2 and 1.0 lg) of synthetic peptides were injected into day-2 last instar larvae (Fig. 2). Once the injected samples were wholly dispersed in the body, their hemolymph concentration would be expected to be in the range of 50 nM to 150 lM. Among the examined peptides, BMS-1 prolonged the latency to the first bite, whereas the other tested peptides did not. Interestingly, TK-1 and sNPF-2 shortened the latency to the first bite. In addition, a high dose of PDF showed a tendency toward prolonged latency to the first bite, as shown in Fig. 2. Observation of the PDF-injected larvae indicated that the delayed latency might
Fig. 2. Effects of chemically synthesized biologically active peptides on the latency to the first bite. Gray and black bars indicate latencies to the first bites following injection of 0.2 and 1.0 lg of peptide, respectively. The dotted line and the white bar represent the time of the latency to the first bite after injection of 100 ll of distilled water. Asterisks ( and ) indicate significant differences (p < 0.05 and p < 0.01, respectively) compared with vehicle-injected larvae by one-way ANOVA. Data represent mean ± SD (n = 5).
be due to exaggerated active locomotor. As shown in Fig. 2, although BMS-1 and sNPF share the same C-terminal sequence, RF-amide, the resulting biological activities in this bioassay had opposite effects. In contrast, IMF amide, which is characterized by the F-amide instead of the RF-amide, did not show any difference in latency compared with the vehicle injection. Because several analogous peptides to BMS-1 and sNPF-2 are potentially encoded by the respective cDNAs, we further analyzed the effect of those peptides (BMS-2, sNPF-1 and sNPF-3). 3.3. Effects of myosuppressin on the latency to the first bite To confirm the effects of BMS-1 on the delayed latency to the feeding initiation, we injected different amounts of this peptide (Fig. 3). Latency was prolonged in a dose-dependent manner by BMS-1 injections. The effects of prolonged latency were observed with as little as 10 ng (approximately 5 pmol) injection. The latency to the first bite did not reach the plateau level even with 5 lg injection. Another analog of BMS-1, BMS-2 [31] was also assayed. In contrast to BMS-1, BMS-2 had a less severe effect on latency (Fig. 3). In addition, because decreased locomotor activity was not observed in BMS-injected larvae, the prolonged latency following BMS-1 injection might not be due to the suppression of locomotor activity, which would be expected to affect the larvae’s ability to approach the artificial diet block. 3.4. Effects of tachykinin on the latency to the first bite As observed in Fig. 2, the latency to the first bite was shortened following TK-1 injection. To confirm the effects of TK-1, we injected different amounts of TK-1 ranging from 10 ng to 5 lg; approximately 10 pmol to 5 nmol (Fig. 4). The result showed that injection of TK-1 shortened the latency in a dose-dependent manner (Fig. 4). Since it has been proposed that five TKs are encoded in a single cDNA, we injected another type of tachykinin, TK-2
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**
Latency to the first bite (min)
** 30
*
*
*
20
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0
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30 40
20
*
0 0
10
100
1000
5000
0
myosuppressin (ng)
10
100
1000
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sNPF (ng)
Fig. 3. Dose-dependent effect of myosuppressin (BMS-1 and BMS-2) on the latency to the first bite. The injected doses of BMS-1 (closed circles) and BMS-2 (closed triangles) were 0.01, 0.1, 1.0, and 5.0 lg/ larva. Asterisks ( and ) indicate significant differences (p < 0.05 and p < 0.01, respectively) compared with vehicleinjected larvae by one-way ANOVA. Data represent mean ± S. D. (n = 5). A similar data tendency was observed in three trials, confirming the reproducibility.
30
Latency to the first bite (min)
**
10
Fig. 5. Dose-dependent effect of short neuropeptide Fs (sNPF-1, sNPF-2, and sNPF3) on the latency to the first bite. The injected doses of sNPF-1 (closed triangles), sNPF-2 (closed circles), and sNPF-3 (open circles) were 0.01, 0.1, 1.0, and 5.0 lg/ larva. Asterisks ( and ) indicate significant differences (p < 0.05 and p < 0.01, respectively) compared with vehicle-injected larvae by one-way ANOVA. Data represent mean ± SD (n = 5). A similar data tendency was observed in three trials, confirming the reproducibility.
at all doses injected, whereas sNPF-2 showed a shortened latency to the first bite after peptide injection in a dose-dependent manner. The effect of sNPF-2 on the shortened latency to the first bite was observed with injections of more than 100 ng.
20
* **
10
*
4. Discussion
**
In the present study, we screened several biologically active peptides in terms of their effect on latency to the first bite in starved Bombyx larvae. The activities of the tested peptides were assessed by prolonged or shortened latency. Of the examined peptides, BMS had a prolonged effect, while TK-1 and sNPF-2 reduced latency.
0 0
10
100
1000
5000
tachykinin (ng) Fig. 4. Dose-dependent effect of tachykinin (TK-1 and TK-2) on the latency to the first bite. The injected doses of TK-1 (closed circles) and TK-2 (closed triangles) were 0.01, 0.1, 1.0, and 5.0 lg/ larva. Asterisks ( and ) indicate significant differences (p < 0.05 and p < 0.01, respectively) compared with vehicle-injected larvae by oneway ANOVA. Data represent mean ± SD (n = 5). A similar data tendency was observed in three trials, confirming the reproducibility.
[18,19,14]. The difference in the amino acid sequences between TK-1 and TK-2 exists in the C-terminal portion, which is relatively conserved and contributes to the biological activities of TKs. Both TK-1 and TK-2 shortened the latency to the first bite similarly in a dose-dependent manner. The effects of TK-1 and -2 on the shortened latencies to the first bites were observed with more than 100 ng and 1 lg injection, respectively. 3.5. Effects of short neuropeptide F on the latency to the first bite As observed in Fig. 2, the latency to the first bite was also shortened by sNPF-2 injection. To confirm this, we next assayed different amounts of sNPF-2 ranging from 10 ng to 5 lg; approximately 10 pmol to 5 nmol (Fig. 5). Similar to TK peptides, three different sNPFs are encoded by a single cDNA [32]. We consequently injected sNPF-1, -2 and -3 and measured their effects on latency to the first bite. sNPF-1 and sNPF-3 had minor effects on latency
4.1. Effect of Bommyosuppressins BMS delayed the initiation of feeding, which might be attributed to: decreased myotropic activity by locomotor depression and weakened gut contraction activity. Several explainable facts on the BMS effects have been reported so far. Myosuppressin was originally isolated from head extracts of the cockroach, Leucophaea maderae as an inhibitor of spontaneous visceral muscle contraction [7]. It has been demonstrated that myosuppressin exhibits several biological activities, including inhibition of muscle and gut contractions [1,7,17], antifeeding activity [28], and inhibition of neuropeptide secretion [30]. There are several peptides analogous to myosuppressin encoded by a single cDNA including BMS-2 [30]. As a result, BMS-2 showed weaker prolonging activity. They also might be involved in feeding behavior similar to BMS, most likely by modulating gut contraction. Since the expression sites of myosuppressin are the central and stomatogastoric nervous systems and the endocrine cells in the midgut of the fruit fly, D. melanogaster [10] and other species [11,25], BMS could also potentially function as a brain-gut regulatory neuropeptide modulating feeding behavior. It has been reported that ecdysteroid secretion from the prothoracic glands is regulated by BMS [30], suggesting that BMS is involved in the general secretory mechanisms of neuroendocrine cells involved in producing other hormones.
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4.2. Effects of tachykinin-related peptides In the present study, we found two peptides, TK and sNPF, which when injected shortened the feeding latency period. Both peptides have been established as feeding-related factors in the fruit fly, D. melanogaster [2,8,9,29]. In contrast to tachykinin-related peptides, there have been no reports to date indicating that sNPF functions in the feeding behavior of other species. Insect TK was originally identified from L. migratoria [19]. By searching in silico, a cDNA encoding TK was identified from B. mori [18]. The TK cDNA encodes five different TK peptides including TK1 and TK-2, which were used for assay in the present study. TK mRNA is widely expressed in the CNS, indicating that TK also functions as a pleiotropic factor. These TK peptides share the C-terminal sequence, GXRamide. In the present study, we utilized two TKs whose C-terminal sequences were GMRamide and GVRamide. In the present experiment, we could not find any differences in the feeding stimulating activity between them (data not shown). Because of the conservation of the C-terminal amino acid, all five tachykinin-like peptides may exhibit shortening activity in Bombyx larvae. 4.3. Effect of short neuropeptide F sNPF was first identified from the Colorado potato beetle, Leptinotarsa decemlineata as a homolog of a mammalian feeding regulatory peptide, neuropeptide Y (NPY) [23]. To date, sNPF is well known as a neuropeptide conserved among arthropods [15]. In Drosophila, sNPF regulates its calorimetric control in combination with insulin-like peptides [8,9]. In B. mori, sNPF was recently identified as a factor regulating JH biosynthesis by inducing the secretion of alltotropin from the corpora cardiaca [32]. The possibility exists that, similar to allatotropin–sNPF regulation, sNPF may regulate the secretion of an unidentified neuropeptide(s) or hormones that function in feeding behavior. The present data showed that three sNPFs exhibited somewhat different strengths of activity (Fig. 5). The difference in the activity of sNPF might be due to differences in receptors as well as the difference of peptide sequences. In fact, transcriptome analysis of GPCRs showed the presence of two receptors for sNPFs in B. mori [32]. Therefore, for a more detailed understanding of sNPF regulation, further studies including expression and distribution analyses of the sNPF receptors and their binding activities are strongly required. 4.4. Effects of other examined peptides Of the injected peptides, we also tested the effect of proctolin, which was originally identified from the cockroach, Periplaneta americana [24]. It has been thought that there is no proctolin in lepidopteran species. Although proctolin is absent in the Bombyx genomic sequence [18], previous reports indicate that proctolin is detectable by immunostaining in the gypsy moth, Lymantria dispar [5], and the tobacco hormworm, Manduca sexta [33]. Consequently, we tested this peptide. In the present study, we could not find any significant effect of proctolin (Fig. 1) on latency to the first bite. Therefore, we concluded that the lack of an effect by proctolin is due to two possibilities: the respective receptor is not expressed in B. mori or that activity was below the threshold for detection in this assay. To clarify whether proctolin can influence biological events in B. mori other investigations such as a gut contraction assay are required. The effects of PDF on the latency to the first bite resulted in data with an expanded deviation (Fig. 2). Our observations revealed more excited behaviors in terms of foraging-like behaviors such as nibbling and walking. Injection of PDF might disturb the
pacemaker neuron related locomotor activity which is regulating circadian rhythms in many species including B. mori [6,16,26]. 4.5. The possibilities for screening biologically active peptides using this assay In the present study, we demonstrated the effects of known biologically active peptides on feeding behavior by observing changes in the amount of time silkworm larvae spent in a latency period larvae prior to first bites following peptide injection. Despite similar C-terminal motifs among the peptides tested, the assay detected three different peptide effects on the latency period: prolonged, shortened and no effect. Although the functional details of the effective peptides remain to be elucidated, we suggest that our assay would serve as a good screening step for judging whether the testing sample constitutes a feeding-modulating peptide or not. However, because a large amount of peptide sample is required for a single bioassay, large numbers of animals might be needed for identification from crude extracts. In summary, we identified several possible feeding modulating factors from known biologically active peptides. However, we have to identify the true biologically active factors regulating feeding behavior in insects. Our previous report showed that several biologically active factors are present in the crude extract [13], we will now work toward identifying strong candidate feeding-modulating factors in the near future. Acknowledgments This work was supported in part by Grants-in-Aid for Scientific Research (#18780083 and 22780099) from the Ministry of Education, Science, Sports, and Culture of Japan, and the NAITO Foundation. The authors thank Dr. J. Joe Hull (USDA-ARS,Maricopa, AZ) for critical reading and assistance in manuscript preparation. References [1] R. Aguilar, J.L. Maestro, L. Vilaplana, C. Chiva, D. Andreu, X. Bellés, Identification of leucomyosuppressin in the German cockroach Blattella germanica, as an inhibitor of food intake, Regul. Peptide 119 (2004) 105–112. [2] B. Al-Anzi, E. Armand, P. Nagamei, M. Olszewski, V. Sapin, C. Waters, K. Zinn, R.J. Wyman, S. Benzer, The leucokinin pathway and its neurons regulate meal size in Drosophila, Curr. Biol. 20 (2010) 969–978. [3] E.A. Bernays, M.S. Singer, A rhythm underlying feeding behaviour in a highly polyphagous caterpillar, Ecol. Entomol. 23 (1998) 295–302. [4] E.A. Bernays, H.A. Woods, Foraging in nature by larvae of Manduca sexta – influenced by an endogenous oscillation, J. Insect Physiol. 46 (2000) 825–836. [5] N.T. Davis, S.G. Velleman, T.G. Kingan, H. Keshishian, Identification and distribution of a proctolin-like neuropeptide in the nervous system of the gypsy moth, Lymantria dispar, and in other Lepidoptera, J. Comp. Neurol. 283 (1989) 71–85. [6] C. Helfrich-Förster, M. Täuber, J.H. Park, M. Mühlig-Versen, S. Schneuwly, A. Hofbauer, Ectopic expression of the neuropeptide pigment-dispersion factor alters behavioral rhythms in Drosophila melanogaster, J. Neurosci. 20 (2000) 3339–3353. [7] G.M. Holman, B.J. Cook, R.J. Nachman, Isolation, primary structure and synthesis of leucomyosuppressin, an insect neuropeptide that inhibits spontaneous contractions of the cockroach hindgut, Comp. Biochem. Physiol. C 85 (1986) 329–333. [8] K.S. Lee, K.H. You, J.K. Choo, Y.M. Han, K. Yu, Drosophila short neuropeptide F regulates food intake and body size, J. Biol. Chem. 279 (2004) 50781–50789. [9] K.S. Lee, O.Y. Kwon, J.H. Lee, K. Kwon, K.J. Min, S.A. Jung, A.K. Kim, K.H. You, M. Tatar, K. Yu, Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling, Nat. Cell Biol. 10 (2008) 468–475. [10] J. McCormick, R. Nichols, Spatial and temporal expression identify dromyosuppressin as a brain-gut peptide in Drosophila melanogaster, J. Comp. Neurol. 338 (1993) 278–288. [11] S.M. Meola, M.S. Wright, G.M. Holman, J.M. Thompson, Immunocytochemical localization of leucomyosuppressin-like peptides in the CNS of the cockroach, Leucophaea maderae, Neurochem. Res. 16 (1991) 543–549. [12] S. Nagata, H. Nagasawa, Effects of diet-deprivation and physical stimulation on the feeding behaviour of the larvae of the silkworm, Bombyx mori, J. Insect Physiol. 52 (2006) 807–815.
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