Katsuhiko Endo

Hormonal control of seasonal-morph determination in the small copper butterfly, Lycaena phlaeas daimio Seitz.

Abstract Spring and summer morphs of Lycaena phlaeas daimio Seitz. are characterized by a wing colour of red and reddish brown, respectively. When newly ecdysed pupae destined to be summer or intermediate morphs (90 or 10%) by larval exposure to long days (long-day pupae) were decapitated or decerebrated, more than half of the operated pupae developed into intermediate and spring morphs (48 and 7%). But, in pupae destined to be spring, intermediate, or summer morphs (72, 22 or 4%) (short-day pupae) these operations did not produce any significant changes in the seasonal morph. Brains excised from newly ecdysed long-day pupae were transplanted into the abdomen of decapitated short-day pupae of the same age. The implants changed most recipients into summer and intermediate morphs (46 and 36%). However, when the brains of short-day pupae were used, no significant changes occurred in the seasonal morph. When long-day or short-day pupae were treated with 20-hydroxyecdysone just after pupation, they produced more reddish wings than those of the untreated or saline-treated controls. When the application was followed by chilling, already known to induce the reddish morph, the effects of both treatments are cumulative so that more reddish adults developed. The results indicate that the brain of long-day pupae secretes a factor causing the wing to be brownish. In the absence (or low titre) of the factor, most short-day pupae develop into spring or intermediate morphs. Furthermore, ecdysteroids make the wing more reddish, when applied to newly ecdysed pupae.

Effect of Bombyx mori chitinase against Japanese pine sawyer (Monochamus alternatus) adults as a biopesticide.

Bombyx mori chitinase (Bm-CHI), with a molecular mass of 75 kDa, was investigated on the possibility that it can serve as a biocontrol agent against the adult Japanese pine sawyer (JPS), Monochamus alternatus (Coleoptera: Cerambycidae). Oral ingestion of purified chitinase at concentrations of 3 μM (11.25 μg/50 μl) and 0.3 μM (1.125 μg/50 μl) caused high mortality in JPS, a significant decrease in bark consumption, and, only in high concentration, a slight reduction of body weight. Fluorescence assays indicated that peritrophic membrane (PM) chitin is degraded by the action of orally ingested Bm-CHI at 3 μM concentration only. Scanning electron micrographs clearly indicated that the beetles that ingested Bm-CHI of the same high concentration had their PM perforated and disrupted, but ultrastructural studies showed that the ingested chitinase did not affect the midgut epithelium. These findings open up the possibility of using insect chitinase as a biopesticidal enzyme. It should have agronomic potential for insect control.

Hormonal control of seasonal morph determination in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

Abstract Seasonal morphs (spring and sumer forms) of Papilio xuthus L. are determined coincidentally with diapause and non-diapause in pupae by larval exposure to short days and long days respectively. The neuroendocrine principle underlying seasonal-morph determination was studied using surgical operations in P. xuthus . When recipient 0-day old or chilled diapause pupae were joined to donor 0-day old non-diapause pupae, the recipients developed into summer or intermediate morphs. When the same kinds of recipients used above were joined to 0-day old or chilled diapause pupae, there were no significant effects on the adult morph. In contrast, recipient non-diapause pupae all developed into summer morphs, regardless of groups of the type of donors. Furthermore, when the brain of 5th-instar larvae, pharate pupae or pupae, predetermined to be diapause, was transplanted into the abdomen of 0-day old, 30-day old or chilled diapause pupae, the recipients developed into summer or intermediate morphs. The results indicate that the brain of non-diapause pupae secretes a humoral factor producing the summer morph. In non-diapause pupae, the factor may be secreted at about the stage of larval-pupal ecdysis coincidentally with that of prothoracicotropic hormone.

A putative binding protein for lipophilic substances related to butterfly oviposition

A unique protein of 23 kDa (Jf23) was found in the tarsus of the female swallowtail butterfly, Atrophaneura alcinous. Jf23 has 38% identity with a bilin‐binding protein, which was found in the cabbage butterfly, Pieris brassicae, and which has two consensus sequences in common with the members of the lipocalin family, suggesting that it is a binding protein for lipophilic ligands. Western blot analysis showed that Jf23 was expressed only in the female, and not in the male. Electrophysiological response of the female tarsi was stimulated by methanolic extract of their host plant, Dutchmans pipe (Aristolochia debilis). The stimulated response was depressed by the presence of Jf23 antiserum. These results suggest that Jf23 is one of the chemosensory signaling proteins, which plays one or more roles in female butterfly oviposition.

Extraction and partial characterization of pupal-cuticle-melanizing hormone (PCMH) in the swallowtail butterfly, Papilio xuthus L. (Lepidoptera, Papilionidae)

Abstract The pupae of Papilio xuthus show green, brown, and orange color polymorphism. The color of the pupal body is determined by a hormone that produces brown coloration, a hormone called pupal-cuticlemelanizing hormone (PCMH) which is secreted from brain-suboesophageal ganglion and prothoracic ganglion (Br-SG-PG) complexes during the pharate pupal stage. PCMH was extracted with 2% NaCl from Br-SG-PG complexes of P. xuthus green pupae. When pharate pupae producing brown pupae (brown-pupa-producers) were ligatured between the thorax and abdomen with cotton thread, the head-thorax complex developed into the brown type and the abdomens into the green type. Ligatured abdomens treated with crude PCMH produced intermediates of green- and brown-pupae (melanization degree of grades 0–3), which was used to assay PCMH-activity. Extracts of Br-SG complexes from Bombyx mori adults also shifted the color of the pupal cuticle toward the brown type in ligatured P. xuthus abdomens of brown-pupa-producers. The molecular weight of the B. mori factor showing PCMH activity (PCMH-active factor) was estimated to be 3,000–4,000 Da by gel filtration. The PCMH-active factor is a hydrophobic peptide(s) that binds to a cation exchange resin at pH 6.9.

Environmental factors controlling seasonal morph determination in the small copper butterfly, Lycaena phlaeas daimio Seitz.

Abstract Environmental factors controlling seasonal morph determination and extension of the larval stages in Lycaena phlaeas daimio were studied in field observations and laboratory experiments, using criteria based on the numbers of red scales in the wing spots. Photoperiod and temperature conditions applied during the larval period and a low-temperature (5°C) introduced in pupal period were expected to be factors affecting the seasonal morph determination: the effects were confirmed by laboratory experiments. Furthermore, the length of the larval period extended by exposure to short days at 20°C, but not at 25°C, were found to show a clear correlation with the spring morph determination, but the correlation coefficient was negative in marked contrast to the situation in Polygonia and Papilio.

Neuroendocrine regulation of the development of seasonal forms of the Asian comma butterfly, Polygonia c-aureum L.

In the butterfly, Polygonia c‐aureum, development of seasonal forms controlled by the photoperiod and temperature was shown to involve a neuroendocrine system of the brain‐corpus cardiacum‐corpus allatum complex.

Separation of Bombyxin from a neuropeptide of Bombyx mori showing Summer-morph-producing Hormone (SMPH) activity in the Asian Comma Butterfly, Polygonia c-aureum L.

A neuropeptide from brain-suboesophageal ganglion (Br-SG) complexes of the silkmoth, Bombyx mori, shows summer-morph-producing hormone (SMPH) activity in the Asian comma butterfly, P. c-aureum. The SMPH-active peptide was extracted and demonstrated to be almost the same molecular size as bombyxin (4-5kD), a nueropeptide which shows prothoracicotropic hormone (PTTH) activity when assayed in vitro with prothoracic glands (PGs) of 4th-instar B. mori larvae in vitro. A Sephadex G-50 fraction of 3-8kD molecules prepared from Br-SG complexes of B. mori adults was applied to CM-, SP-, DEAE- or QAE- Toyoperal columns at pH 5.6 (or pH 6.9). The SMPH-activity could be separated from the PTTH-activity (or bombyxin) by subjecting a SMPH- and PTTH-active preparation of B. mori to anion-exchange chromatography at pH 6.9. By reversed-phase HPLC following an anion-exchange chromatography, SMPH-activity was recovered in two fractions of 40-45% acetonitril. Results demonstrate that the B. mori peptide showing the SMPH-activity in P. c-aureum is a different molecule than bombyxin.

Species-Specificity in the Action of Big and Small Prothoracicotropic Hormones (PTTHs) of the Swallowtail Butterflies, Papilio xuthus, P. machaon, P. bianor and P. helenus

Abstract To investigate whether four Papilio species, Papilio xuthus, P. machaon, P. bianor and P. helenus, have two molecular forms of the prothoracicotropic hormones (PTTHs), referred to as big- and small-PTTHs, the PTTHs were extracted and fractionated from their pupal brains. The activating ability of big- and small-PTTH fractions was examined by the in vitro assay using the prothoracic glands (PGs) of 2-day-old 5th-instar larvae of their own and several other papilionid species. Big- and small-PTTH fractions activated the larval PGs of their own species to increase the ecdysteroid secretion in vitro. The doses of small-PTTH fractions for activating the larval PGs were 8- to 10-times larger than those of big-PTTH fractions. The big- and small-PTTH fractions as well as those of P. machaon activated the PGs of 2-day-old 5thinstar larvae of several heterogeneous papilionids, but the activating ability did not always decrease with the distance of the genetic (or phylogenetic) relationships. The results indicate that P. machaon, P. bianor and P. helenus may have two molecular forms of the PTTHs, both of which activate the larval PGs of the same species in vitro as in the case of P. xuthus. The bigand small- PTTHs of P. xuthus as well as those of several other Papilio species may retain an ability to activate the 5th-instar larval PGs of numbers of heterogeneous papilionids in vitro.

Hormonal Control of the Orange Coloration of Diapause Pupae in the Swallowtail Butterfly, Papilio xuthus L. (Lepidoptera: Papilionidae)

Abstract Diapause pupae of Papilio xuthus show color polymorphism, represented by diapause-green, orange, and brownish-orange types that are each associated with specific pupation sites. We investigated the role of the site of pupation on the induction of the development of orange types (or brownish-orange types), and the endocrine mechanism underlying the control of color polymorphism in short-day pupae. All short-day larvae of the wandering stage developed into orange or brownish-orange type pupae when they were placed in rough-surfaced containers after gut-purge. Utilizing a pharate pupal ligation between the thorax and abdomen, the endocrine mechanism underlying the control of color polymorphism was shown to involve a head-thorax factor (Orange-Pupa-Inducing Factor: OPIF) that induced orange types in short-day pupae. OPIF was bioassayed using the ligated abdomens of short-day pharate pupae. OPIF was extractable with 2% NaCl solution from 5th-instar larval ganglia complexes following the mesothoracic complex (TG2,3-AG1–7), but it could not be extracted with either acetone or 80% ethanol solution. OPIF may not exist in the brains of day-0 pupae or in brain-subesophageal ganglion and prothoracic ganglion complexes of 5th-instar larvae. The short-day pharate pupae responded to OPIF in a dose-dependent manner.