[Mechanism of action and clinical trial results of Lecanemab (Leqembi® 200†|mg, 500†|mg for Intravenous Infusion), a novel treatment for Alzheimer's disease].

Lecanemab is a humanized monoclonal antibody directed against human soluble amyloid-ß aggregates. It was developed for the treatment of early Alzheimer's disease (mild cognitive impairment or mild dementia stage of Alzheimer's disease). Among the amyloid-ß (Aß) involved in Alzheimer's disease, Lecanemab selectively binds to the highly neurotoxic Aß protofibrils, and is thought to reduce Aß protofibrils and amyloid plaques (Aß plaques) in the brain. The efficacy and safety of Lecanemab in early Alzheimer's disease were investigated in an international Phase II placebo-controlled study (Study 201) and an international Phase III placebo-controlled study (Study 301). Both studies included Japanese subjects. Lecanemab was given accelerated approval in the United States in January 2023, followed by traditional approval in July 2023. In Japan, it was approved for "control of the progression of mild cognitive impairment or mild dementia stage of Alzheimer's disease" in September 2023, and was added to the NHI drug price list in December 2023.

Male-killing virus in a noctuid moth Spodoptera litura.

A female-biased sex ratio is considered advantageous for the cytoplasmic elements that inhabit sexually reproducing organisms. There are numerous examples of bacterial symbionts in the arthropod cytoplasm that bias the host sex ratio toward females through various means, including feminization and male killing. Recently, maternally inherited RNA viruses belonging to the family Partitiviridae were found to cause male killing in moths and flies, but it was unknown whether male-killing viruses were restricted to Partitiviridae or could be found in other taxa. Here, we provide compelling evidence that a maternally inherited RNA virus, Spodoptera litura male-killing virus (SlMKV), selectively kills male embryos of the tobacco caterpillar Spodoptera litura, resulting in all-female broods. SlMKV injected into uninfected S. litura can also be inherited maternally and causes male killing. SlMKV has five genomic segments encoding seven open reading frames, has no homolog of known male-killing genes, and belongs to an unclassified group of arthropod-specific viruses closely related to Tolivirales. When transinfected into larvae, both male and female recipients allow SlMKV to proliferate, but only males die at the pupal stage. The viral RNA levels in embryonic and pupal male killing suggest that the mechanism of male killing involves the constitutive expression of viral products that are specifically lethal to males, rather than the male-specific expression of viral products. Our results, together with recent findings on male-killing partiti-like viruses, suggest that diverse viruses in arthropods tend to acquire male killing independently and that such viruses may be important components of intragenomic conflict in arthropods.

Cell-based analysis reveals that sex-determining gene signals in Ostrinia are pivotally changed by male-killing Wolbachia.

Wolbachia, a maternally transmitted bacterium, shows male-killing, an adaptive phenotype for cytoplasmic elements, in various arthropod species during the early developmental stages. In lepidopteran insects, lethality of males is accounted for by improper dosage compensation in sex-linked genes owing to Wolbachia-induced feminization. Herein, we established Ostrinia scapulalis cell lines that retained sex specificity per the splicing pattern of the sex-determining gene doublesex (Osdsx). We found that Wolbachia transinfection in male cell lines enhanced the female-specific splice variant of Osdsx (OsdsxF ) while suppressing the male-specific variant (OsdsxM ), indicating that Wolbachia affects sex-determining gene signals even in vitro. Comparative transcriptome analysis isolated only two genes that behave differently upon Wolbachia infection. The two genes were respectively homologous to Masculinizer (BmMasc) and zinc finger-2 (Bmznf-2), male-specifically expressed sex-determining genes of the silkworm Bombyx mori that encode CCCH-type zinc finger motif proteins. By using cultured cells and organismal samples, OsMasc and Osznf-2 were found to be sex-determining genes of O. scapulalis that are subjected to sex-specific alternative splicing depending upon the chromosomal sex, developmental stage, and infection status. Overall, our findings expound the cellular autonomy in insect sex determination and the mechanism through which sex is manipulated by intracellular selfish microbes.

Lipid droplets in the pheromone glands of bombycids: Effects of larval diet on their size and pheromone titer.

In addition to the blend ratio, the quantity of sex pheromone components secreted by female moths may affect the efficient attraction of conspecific males. The present study using the silkmoth Bombyx mori, which has bombykol as its pheromone component, demonstrated that pheromone titer, body weight, and lipid droplet (LD) diameter in the pheromone gland were affected by the larval diet. Although the artificial diet contained approximately 11-fold more total fatty acids than mulberry leaf, the pheromone titer in the group fed the artificial diet (group AD) was approximately 2-fold higher than that of the group fed mulberry (group M). The diameter of LDs, which store the pheromone-precursor fatty acyl, E10,Z12-16:Acyl, was also larger in the AD group. The relatively small increase in sex pheromone titer by feeding on a fatty-acid-rich diet may be partly attributable to the storage of excess precursors in the LDs. We detected LDs in the pheromone glands of Trilocha varians, the closest non-congener of B. mori available in Bombycidae. T. varians uses bombykal and bombykyl acetate as sex pheromone components, which are biosynthesized via the same precursor fatty acyl as that of B. mori. The presence of LDs in T. varians suggests that the storage and mobilization mechanisms of the pheromone precursor fatty acyl via LDs may be conserved in bombycids.

Oviposition in the onion fly Delia antiqua (Diptera: Anthomyiidae) is socially facilitated by visual cues.

Ovipositional decisions in herbivorous insects may be affected by social information from conspecifics. Social facilitation of oviposition has been suggested for the onion fly Delia antiqua. In the current study, we found that D. antiqua oviposition was unequal between paired oviposition stations of equal quality and that more eggs were laid on an oviposition station baited with decoy flies than on the control. The increased oviposition toward the decoys continued over time >8 h. When decoys were placed upside down, the number of eggs laid did not differ between the decoy and control sides of oviposition stations, suggesting that social facilitation of oviposition is mediated by visual cues. Based on these findings, mechanisms of social facilitation of oviposition in D. antiqua were discussed.

Pheromonal activities of the bombykol isomer, (10E,12E)-10,12-hexadecadien-1-ol, in the pheromone gland of the silkmoth Bombyx mori.

Bombykol (EZ) is the single component of the female sex pheromone in the silkmoth Bombyx mori. EZ alone evokes full courtship behaviors from conspecific males; however, its geometric isomer (EE) was consistently detected in the pheromone glands (PG) of 16 B. mori strains and a field population of the wild silkmoth Bombyx mandarina, which also uses EZ as the single pheromone component. We investigated the pheromonal activities of EE using a commercial hybrid strain of B. mori, Kinshu × Showa. The behavioral assay demonstrated that a 104-105-fold higher dose of EE than EZ was able to elicit behavioral responses from males. To elucidate whether the trace contaminant of EZ in the EE standard is responsible for these responses, we examined the responses of male antennae to EE using a gas chromatograph-electroantennographic detector system (GC-EAD). The EE, at high doses elicited marginal responses from the male antennae. We next examined antennal and behavioral responses of B. mori whose BmOR1 gene, which is responsible for the reception of bombykol, was knocked out. The knockout of BmOR1 resulted in the complete loss of antennal and behavioral responses to EE and EZ, demonstrating that if EE itself is active, it induces these responses via the incidental stimulation of BmOR1, not via the stimulation of EE-specific receptors. The existence of EE in the PG of B. mori and B. mandarina is discussed from the viewpoints of pheromone biosynthesis and the evolution of pheromone communication systems.

CYPs in different families are involved in the divergent regio-specific epoxidation of alkenyl sex pheromone precursors in moths.

In moth species that utilize alkenyl sex pheromones, the epoxidation of alkenes confers further diversity on the chemical structures of pheromone components. Hc_epo1 (CYP341B14), the first pheromone gland (PG)-specific epoxidase identified from the fall webworm Hyphantria cunea (Erebidae), specifically epoxidizes the Z9 double bond in the triene precursor, (3Z,6Z,9Z)-3,6,9-henicosatriene (Z3,Z6,Z9-21:H). In the present study, we identified a novel PG-specific epoxidase, As_epo1, from the Japanese giant looper Ascotis selenaria (Geometridae), which secretes cis-3,4-epoxy-(6Z,9Z)-6,9-nonadecadiene (epo3,Z6,Z9-19:H) as the main sex pheromone component. A functional assay using the Sf9 insect cell line-baculovirus expression system showed that As_epo1 specifically epoxidizes the Z3 double bond in the pheromone precursor triene, (3Z,6Z,9Z)-3,6,9-nonadecatriene (Z3,Z6,Z9-19:H). As_epo1 also Z3-specifically epoxidized a triene with a longer carbon chain, Z3,Z6,Z9-21:H, which does not occur in this species. A phylogenetic analysis indicated that As_epo1 belonged to the CYP340 family, not the CYP341 family to which Hc_epo1 belongs. These results suggest that moth PG-specific epoxidases with divergent regio-specificities have evolved independently.

Functional characterization of the epoxidase gene, Li_epo1 (CYP341B14), involved in generation of epoxyalkene pheromones in the mulberry tiger moth Lemyra imparilis.

Epoxidation of alkenes derived from essential fatty acids is a key step in the biosynthesis of sex pheromones in moth species that utilize alkenyl sex pheromones. The position of the epoxy ring in the pheromone molecule differs depending on the species, thereby conferring diversities on sex pheromones. To date, only one pheromone gland (PG)-specific epoxidase, Hc_epo1 (CYP341B14), has been reported. Hc_epo1, which was identified from an arctiid moth Hyphantria cunea, catalyzes the epoxidation of a double bond at position 9 of the triene, Z3,Z6,Z9-21:H. In the present study, we investigated the PG-specific epoxidase from another arctiid, the mulberry tiger moth Lemyra imparilis, in order to verify whether cytochrome P450 in the CYP341B subfamily, to which Hc_epo1 belongs to, is responsible for the epoxidation of pheromone precursors at position 9 in moths other than H. cunea. A fragment of the Hc_epo1 homolog was amplified from cDNA prepared from the PG of L. imparilis by PCR with degenerate primers. The deduced amino acid sequence of the subsequently cloned homolog, Li_epo1, showed 88.5% identity to Hc_epo1. A functional assay using the Sf9 insect cell line-baculovirus expression system showed that Li_epo1 exhibited epoxidase activity with high selectivity to the double bond at position 9 of two trienes, Z3,Z6,Z9-21:H and Z3,Z6,Z9-23:H, precursors of epoxy diene sex pheromone components in L. imparilis.

In vivo functional characterisation of pheromone binding protein-1 in the silkmoth, Bombyx mori.

Male moths detect sex pheromones emitted by conspecific females with high sensitivity and specificity by the olfactory sensilla on their antennae. Pheromone binding proteins (PBPs) are highly enriched in the sensillum lymph of pheromone sensitive olfactory sensilla and are supposed to contribute to the sensitivity and selectivity of pheromone detection in moths. However, the functional role of PBPs in moth sex pheromone detection in vivo remains obscure. In the silkmoth, Bombyx mori, female moths emit bombykol as a single attractive sex pheromone component along with a small amount of bombykal that negatively modulates the behavioural responses to bombykol. A pair of olfactory receptor neurons, specifically tuned to bombykol or bombykal, co-localise in the trichodeum sensilla, the sensillum lymph of which contains a single PBP, namely, BmPBP1. We analysed the roles of BmPBP1 using BmPBP1-knockout silkmoth lines generated by transcription activator-like effector nuclease-mediated gene targeting. Electroantennogram analysis revealed that the peak response amplitudes of BmPBP1-knockout male antennae to bombykol and bombykal were significantly reduced by a similar percentage when compared with those of the wild-type males. Our results indicate that BmPBP1 plays a crucial role in enhancing the sensitivity, but not the selectivity, of sex pheromone detection in silkmoths.

Silkworms suppress the release of green leaf volatiles by mulberry leaves with an enzyme from their spinnerets.

In response to herbivory, plants emit a blend of volatile organic compounds that includes green leaf volatiles (GLVs) and terpenoids. These volatiles are known to attract natural enemies of herbivores and are therefore considered to function as an indirect defense. Selection should favor herbivores that are able to suppress these volatile emissions, and thereby make themselves less conspicuous to natural enemies. We tested this possibility for silkworms, which were observed to leave secretions from their spinnerets while feeding on mulberry leaves. When we ablated the spinnerets of silkworms, no secretions were observed. Leaves infested by intact silkworms released smaller amounts of GLVs than leaves infested by ablated silkworms, indicating that the spinneret secretion suppressed GLV production. This difference in GLV emissions was also reflected in the behavioral response of Zenillia dolosa (Tachinidae), a parasitoid fly of silkworms. The flies laid fewer eggs when exposed to the volatiles from intact silkworm-infested leaves than when exposed to the volatiles from ablated silkworm-infested leaves. We identified a novel enzyme in the secretion from the spinneret that is responsible for the GLV suppression. The enzyme converted 13(S)-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid, an intermediate in the biosynthetic pathway of GLVs, into its keto-derivative in a stereospecific manner. Taken together, this study shows that silkworms are able to feed on mulberry in a stealthy manner by suppressing GLV production with an enzyme in secretions of their spinnerets, which might be a countermeasure against induced indirect defense by mulberry plants.

Settlement of Stenotic Site and Enhancement of Risk Factor Load for Atherosclerosis in Left Anterior Descending Coronary Artery by Myocardial Bridge.

OBJECTIVE: The aim of this study was to investigate the influence of a myocardial bridge (MB) on atherosclerosis development in the left anterior descending artery of the normal heart and the importance of traditional risk factors (RFs). An additional objective was to determine the correlation between intimal thickening and luminal narrowing. APPROACH AND RESULTS: The left anterior descending artery from 150 autopsied hearts was treated with formalin perfusion fixation, and each left anterior descending artery was serially cross-sectioned. The intima-media and luminal stenosis ratios were examined using computer-assisted histomorphometry. The luminal stenosis ratio was closely correlated with the intima-media ratio (r=0.792; P<0.001). When an MB was present, the luminal stenosis ratios proximal to the MB in the RF (+) group were significantly greater than those in the RF (-) group (P=0.022 by a multiple comparison test), but there were no differences between the RF (+) and RF (-) groups when an MB was absent. In addition, the site of the greatest stenosis in the MB (+) RF (+) group was 2.5 cm proximal to the MB entrance. Multivariate analyses indicated that age was an independent factor for luminal stenosis ratios ≥50% and 60% (P=0.002 and 0.029, respectively). Furthermore, the presence of an MB plus RFs was an independent factor for a luminal stenosis ratio ≥70% (P=0.037). CONCLUSIONS: An MB enhances left anterior descending artery atherosclerosis development at a site proximal to the MB entrance, particularly in subjects who have some RFs.

Conservation and lineage-specific rearrangements in the GOBP/PBP gene complex of distantly related ditrysian Lepidoptera.

General odorant binding proteins (GOBPs) and pheromone binding proteins (PBPs) form a monophyletic subfamily of insect odorant binding proteins (OBPs) specific for Lepidoptera, butterflies and moths. The GOBP/PBP genes include six subgroups (GOBP1-2, PBP-A-D) previously reported to form a complex arrayed in a conserved order in representative moths (superfamily Bombycoidea) and butterflies (Nymphalidae). Although our knowledge of lepidopteran genomes has increased greatly recently, the structure of the GOBP/PBP complex has been studied only for species that represent limited lineages of the highly diverged Ditrysia. To understand the evolution of this functionally important gene complex, we determined 69-149 kb genomic sequences that include GOBP2 and five PBP genes in three Ostrinia moths (Pyraloidea), O. nubilalis, O. furnacalis, and O. latipennis, using bacterial artificial chromosome (BAC) and fosmid clones. The structure of the GOBP2/PBP gene cluster was well conserved despite the different sex pheromone composition utilized by the three moths. Five expressed PBP genes in Ostrinia moths were the result of two duplications of PBP-A genes. Surprisingly, an allele containing a fusion gene between tandemly arrayed PBP-A genes was observed in O. nubilalis. We also revealed duplication and intra-chromosomal translocation of the GOBP1 gene in P. xylostella by fluorescence in situ hybridization (FISH) analysis. Additionally, we compared the structure of the GOBP/PBP gene complex of seventeen species covering six superfamilies and twelve families of the lepidopteran clade, Ditrysia, and found the gene order was basically conserved despite the frequent occurrence of lineage-specific gains, losses, inversions and translocations of these genes, compared with their neighboring genes. Our findings support the hypothesis that the structure of the GOBP/PBP gene complex was already established in the common ancestor of Ditrysia.

Effects of mating on host selection by female small white butterflies Pieris rapae (Lepidoptera: Pieridae).

Mating might significantly affect the host selection behaviors of phytophagous insects. Here, we investigated the post-mating changes in behavioral and antennal responses of Pieris rapae (Lepidoptera: Pieridae) females to host plant volatiles. In two-choice bioassays using artificial plant models, mated females visited the model scented with synthetic blends (15-, 9-, or 6-components) of cabbage plant volatiles more frequently than the unscented control, whereas virgin females did not exhibit this preference. Because single compounds and the 3-component blend did not induce preferential visiting, mated females apparently utilized complex odor blends as their host-finding cue. Moreover, 2- to 4-day-old mated females visited the models, scented and unscented, more frequently than did their virgin counterparts. Therefore, mating enhanced the host-finding behavior of young females and their responsiveness to plant volatiles. Gas chromatography-electroantennographic detector analysis revealed that eight of the 15 compounds in the cabbage plant volatiles elicited responses from female antennae. However, post-mating and age-dependent changes in antennal responses were not detected. Because female peripheral (antennal) sensitivity to volatiles remained practically unchanged after emergence, post-mating changes in host selection might be attributed to changes in the central nervous system.

piggyBac- and phiC31 integrase-mediated transgenesis in Drosophila prolongata.

The development of transgenesis systems in non-model organisms provides a powerful tool for molecular analysis and contributes to the understanding of phenomena that are not observed in model organisms. Drosophila prolongata is a fruit fly that has unique morphology and behavior not found in other Drosophila species including D. melanogaster. In this study, we developed a phiC31 integrase-mediated transgenesis system for D. prolongata. First, using piggyBac-mediated transgenesis, 37 homozygous attP strains were established. These strains were further transformed with the nosP-Cas9 vector, which was originally designed for phiC31-mediated transgenesis in D. melanogaster. The transformation rate varied from 0% to 3.4%. Nine strains with a high transformation rate of above 2.0% were established, which will serve as host strains in future transformation experiments in D. prolongata. Our results demonstrate that genetic tools developed for D. melanogaster are applicable to D. prolongata with minimal modifications.

Differences between gastric signet-ring cell carcinoma and poorly differentiated adenocarcinoma: A comparison of histopathologic features determined by mucin core protein and trefoil factor family peptide immunohistochemistry.

We investigated differences between the pathological features of gastric signet-ring cell carcinoma (sig) and poorly differentiated adenocarcinoma (por) by examining the expressions of the trefoil factor family peptides (TFFs) and mucin core proteins (MUCs). Ninety-seven tissues of 97 gastric cancer patients were selected for this study. After gastrectomy, the major histopathologic types were determined to be sig, solid-type poorly differentiated adenocarcinoma (por1), non-solid type poorly differentiated adenocarcinoma (por2), and well-differentiated tubular adenocarcinoma (tub1). We evaluated the prevalence of positive staining for MUCs (MUC5AC and MUC2) and TFFs (TFF1 and TFF3) and assessed the correlation between MUCs and TFFs in each histopathological type. The rate of MUC2 expression significantly differed between sig and por2 (50.0% vs 11.7%, P = 0.011). TFF3 expression in sig significantly differed from TFF3 expression in both por2 (100% vs 17.6%, P < 0.0001) and por1 (100% vs 33.3%, P = 0.0004). MUC5AC and TFF1 expressions were significantly correlated in por1 (r = 0.705, P = 0.002), por2 (r = 0.535, P = 0.0009), and tub1 (r = 0.470, P = 0.0034), while MUC2 and TFF3 expressions were significantly correlated only in sig (r = 0.593, P = 0.040). The expression and correlation patterns of the TFFs and MUCs suggest that the histopathologic features of gastric sig differ from those of por.

Comparative analysis of the brain transcriptome in a hyper-aggressive fruit fly, Drosophila prolongata.

Aggressive behavior is observed in many animals, but its intensity differs between species. In a model animal of genetics, Drosophila melanogaster, genetic basis of aggressive behavior has been studied intensively, including transcriptome analyses to identify genes whose expression level was associated with intra-species variation in aggressiveness. However, whether these genes are also involved in the evolution of aggressiveness among different species has not been examined. In this study, we performed de novo transcriptome analysis in the brain of Drosophila prolongata to identify genes associated with the evolution of aggressiveness. Males of D. prolongata were hyper-aggressive compared with closely related species. Comparison of the brain transcriptomes identified 21 differentially expressed genes in males of D. prolongata. They did not overlap with the list of aggression-related genes identified in D. melanogaster, suggesting that genes involved in the evolution of aggressiveness were independent of those associated with the intra-species variation in aggressiveness in Drosophila. Although females of D. prolongata were not aggressive as the males, expression levels of the 21 genes identified in this study were more similar between sexes than between species.

Morphology and physiology of antennal lobe projection neurons in the hawkmoth Agrius convolvuli.

The neuronal pathways involved in the processing of sex pheromone information were investigated in the hawkmoth Agrius convolvuli (Lepidoptera: Sphingidae), which uses (E,E)-11,13-hexadecadienal (E11,E13-16:Ald) as the single sex pheromone component. We first clarified the anatomical organization of the antennal lobe of A. convolvuli. Subsequently, central neurons in the antennal lobe that responded to E11,E13-16:Ald were identified. The dendritic processes of these neurons were confined within a specific glomerulus (cumulus) in the antennal lobe. The axons of these neurons projected to the inferior lateral protocerebrum and mushroom body calyx. Although the anatomical organization and morphology of individual neurons in A. convolvuli were similar to other species in the superfamily Bombycoidea, the use of cumulus as the single pathway for sex pheromone information processing was characteristic to this species.

Inheritance Pattern of Female Receptivity in Drosophila prolongata.

Choosiness, or female receptivity to copulation, is a critical component in female preference for a mating partner, as it functions at the final step in the mating process by determining whether she accepts copulation with a given male. In spite of its importance in the evolutionary process of male traits via sexual selection, little is known about the genetic variation and inheritance pattern of female receptivity. Drosophila prolongata shows a unique courtship behavior, leg vibration, which increases female receptivity to copulation. In the present study, we analyzed variation in female receptivity and its inheritance pattern in isofemale strains of D. prolongata using leg vibration as an index. There was a significant difference in female receptivity among the strains examined. A high-receptivity phenotype was semi-dominantly expressed in F1 females of crosses between strains with low and high receptivity. Backcrossing F1 females to low-receptivity strains resulted in a lower level of receptivity, suggesting that the high-receptivity phenotype is controlled by multiple genes with epistatic interactions. These results indicate a genetic basis of female receptivity, shedding light on the evolutionary process of sexual selection in D. prolongata.

Sebaceous mantleoma (mantle adenoma): reappraisal of the myth of the problematic benign neoplasm with sebaceous mantle differentiation.

Few cases of a true benign neoplasm with sebaceous mantle differentiation have been reported, and little is known about this tumor. Herein, we present a rare case of the neoplasm called sebaceous mantleoma, along with a comparison of the histology and immunoprofile with those of normal sebaceous mantles. A pedunculated polyp occurred on the scalp of a 51-year-old woman. Histopathologically, the tumor showed lobulated epithelial-mesenchymal units that were separated from the normal dermis by clefts. The lesion was composed of cords and columns of basaloid cells containing a few mature sebocytes, with a focal connection to infundibulocystic structures as well as dense fibrotic or fibromyxoid stroma. Immunohistochemically, androgen receptor, estrogen receptor, and CD117 were partially positive for the tumor, and CD8 (C8/144B) and epithelial membrane antigen were focally positive. Additionally, cytokeratin 20-positive Merkel cells were individually admixed in the tumor nests as well as in normal sebaceous mantles. This case report reveals the characteristic histology and immunoprofile of this problematic benign neoplasm and helps to understand this entity.

Insights into How Longicorn Beetle Larvae Determine the Timing of Metamorphosis: Starvation-Induced Mechanism Revisited.

Larvae of holometabolous insects must determine the timing of their metamorphosis. How they determine this timing has only been studied in detail for a few insect species. In a few species of Coleoptera, starvation is known to be a cue for metamorphosis, leading to the formation of smaller adults (starvation-induced pupation, SiP). We investigated the occurrence of SiP in the beetle Psacothea hilaris. When P. hilaris larvae were starved late in the feeding phase of the last (5th) instar, they exhibited typical SiP characterized by constancy of the time from food deprivation to pupation (TTP) irrespective of the body weight upon food deprivation or the length of prior feeding. In contrast, when larvae were starved early in the feeding phase, TTP decreased by roughly 1 day as the feeding became 1 day longer. The change in the response to starvation was estimated to occur on day 5.9 in the last instar. A series of refeeding experiments suggested that whereas SiP occurred readily in the larvae starved in the late feeding phase, activation of SiP was suspended until day 5.9 in the larvae starved early in the feeding phase. When P. hilaris larvae were fed continuously, they eventually ceased feeding spontaneously and pupated. The time length between spontaneous cessation of feeding and pupation was approximately equal to the TTP in SiP. This suggests that the same mechanism was activated by food deprivation in the late feeding phase and by spontaneous cessation of ad libitum feeding.