Pharate Pupal Stage Research Articles

Arylalkylamine N-acetyltransferase 1 gene (TcAANAT1) is required for cuticle morphology and pigmentation of the adult red flour beetle, Tribolium castaneum

In the insect cuticle tanning pathway (sclerotization and pigmentation), the enzyme arylalkylamine N-acetyltransferase (AANAT) catalyzes the acetylation of dopamine to form N-acetyldopamine (NADA), which is one of the major precursors for quinone-mediated tanning. In this study we characterized and investigated the function of TcAANAT1 in cuticle pigmentation of the red flour beetle, Tribolium castaneum. We isolated a full length TcAANAT1 cDNA that encodes a protein of 256 amino acid residues with a predicted GCN5-related acetyltransferase domain containing an acetyl-CoA binding motif. TcAANAT1 transcripts were detected at all stages of development with lowest expressions at the embryonic and pharate pupal stages. We expressed and purified the encoded recombinant TcAANAT1 protein (rTcAANAT1) that exhibited highest activity at slightly basic pH values (for example, pH 7.5 to 8.5 using dopamine as the substrate). In addition, rTcAANAT1 acts on a wide range of substrates including tryptamine, octopamine and norepinephrine with similar substrate affinities with Km values in the range of 0.05–0.11 mM except for tyramine (Km = 0.56 mM). Loss of function of TcAANAT1 caused by RNAi had no effect on larval and pupal development. The tanning of pupal setae, gin traps and urogomphi proceeded normally. However, the resulting adults (∼70%) exhibited a roughened exoskeletal surface, separated elytra and improperly folded hindwings. The body wall, elytra and veins of the hindwing of the mature adults were significantly darker than those of control insects probably due to the accumulation of dopamine melanin. A dark pigmentation surrounding the bristles located on the inter-veins of the elytron was evident primarily because of the underlying darkly pigmented trabeculae that partition the dorsal and ventral layers of the elytron. These results support the hypothesis that TcAANAT1 acetylates dopamine and plays a role in development of the morphology and pigmentation of T. castaneum adult cuticle.

Chitin is a necessary component to maintain the barrier function of the peritrophic matrix in the insect midgut

In most insects, the peritrophic matrix (PM) partitions the midgut into different digestive compartments, and functions as a protective barrier against abrasive particles and microbial infections. In a previous study we demonstrated that certain PM proteins are essential in maintaining the PM's barrier function and establishing a gradient of PM permeability from the anterior to the posterior part of the midgut which facilitates digestion (Agrawal et al., 2014). In this study, we focused on the effects of a reduction in chitin content on PM permeability in larvae of the red flour beetle, Tribolium castaneum. Oral administration of the chitin synthesis inhibitor diflubenzuron (DFB) only partially reduced chitin content of the larval PM even at high concentrations. We observed no nutritional effects, as larval growth was unaffected and neutral lipids were not depleted from the fat body. However, the metamorphic molt was disrupted and the insects died at the pharate pupal stage, presumably due to DFB's effect on cuticle formation. RNAi to knock-down expression of the gene encoding chitin synthase 2 in T. castaneum (TcCHS-2) caused a complete loss of chitin in the PM. Larval growth was significantly reduced, and the fat body was depleted of neutral lipids. In situ PM permeability assays monitoring the distribution of FITC dextrans after DFB exposure or RNAi for TcCHS-2 revealed that PM permeability was increased in both cases. RNAi for TcCHS-2, however, led to a higher permeation of the PM by FITC dextrans than DFB treatment even at high doses. Similar effects were observed when the chitin content was reduced by feeding DFB to adult yellow fever mosquitos, Aedes aegypti. We demonstrate that the presence of chitin is necessary for maintaining the PM's barrier function in insects. It seems that the insecticidal effects of DFB are mediated by the disruption of cuticle synthesis during the metamorphic molt rather than by interfering with larval nutrition. However, as DFB clearly affects PM permeability, it may be suitable to increase the efficiency of pesticides targeting the midgut.

A multicopper oxidase-related protein is essential for insect viability, longevity and ovary development.

Typical multicopper oxidases (MCOs) have ten conserved histidines and one conserved cysteine that coordinate four copper atoms. These copper ions are required for oxidase activity. During our studies of insect MCOs, we discovered a gene that we named multicopper oxidase-related protein (MCORP). MCORPs share sequence similarity with MCOs, but lack many of the copper-coordinating residues. We identified MCORP orthologs in many insect species, but not in other invertebrates or vertebrates. We predicted that MCORPs would lack oxidase activity due to the absence of copper-coordinating residues. To test this prediction, we purified recombinant Tribolium castaneum (red flour beetle) MCORP and analyzed its enzymatic activity using a variety of substrates. As expected, no oxidase activity was detected. To study MCORP function in vivo, we analyzed expression profiles of TcMCORP and Anopheles gambiae (African malaria mosquito) MCORP, and assessed RNAi-mediated knockdown phenotypes. We found that both MCORPs are constitutively expressed at a low level in all of the tissues we analyzed. Injection of TcMCORP dsRNA into larvae resulted in 100% mortality prior to adult eclosion, with death occurring mainly during the pharate pupal stage or late pharate adult stage. Injection of TcMCORP dsRNA into pharate pupae resulted in the death of approximately 20% of the treated insects during the pupal to adult transition and a greatly shortened life span for the remaining insects. In addition, knockdown of TcMCORP in females prevented oocyte maturation and, thus, greatly decreased the number of eggs laid. These results indicate that TcMCORP is an essential gene and that its function is required for reproduction. An understanding of the role MCORP plays in insect physiology may help to develop new strategies for controlling insect pests.

Hormonal control of pupal coloration in the painted lady butterfly Vanessa cardui

Pupae of the painted lady butterfly Vanessa cardui exhibit pupal color polyphenism consisting of white, dark and intermediate types. We investigated environmental factors affecting pupal coloration and the physiological mechanisms underlying the control of pupal color polyphenism in this species. Over 80% of larvae reared at 16 °C developed into pupae of dark types, whereas over 82% of larvae at 32 °C developed into pupae of white types irrespective of long/short-day photoperiod conditions. When mature larvae reared at 32 °C were ligatured between thoracic and abdominal parts at three different pharate pupal stages, all of the head–thoracic parts developed into white pupae regardless of pupal stage, but all abdominal parts ligatured at the early pharate pupal stage only developed into dark pupae. These results indicate that temperature during larval stages is an important element affecting pupal coloration as an environmental cue in V. cardui, and that a factor(s) inducing white pupae is released from head–thoracic parts under conditions of high temperature. Additionally, when ligatured abdomens destined to develop into dark pupae were treated with crude extracts prepared from the central nervous system, all of the ligatured abdomens developed into white pupae at a level dependent on dose and pupal stage. These results suggest that the factor inducing white pupae is a key molecule controlling pupal color polyphenism in V. cardui.

Ontophylogenetic character analysis of Diaphanes (Coleoptera: Lampyridae) and extrapolation to the broader Coleoptera

A new diagnostic approach called an “ontophylogenetic character analysis” is introduced that can determine whether differences between larviform lampyrids are due to change in the degree of metamorphic elaboration or somatic gene mutation. When performed on five species of larviform female Diaphanes, D. citrinus Olivier 1911, D. cheni Jeng 2001, D. flavilateris Jeng & Yang 2001, D. nubilus Jeng & Lai 2001, and D. formosus Olivier 1910 are determined to be, consecutively in that order, more and more imaginal. It is concluded that each is quitting the pharate pupal stage consecutively later and not finishing the full metamorphic program experienced by the male because the endocrine system is arresting elaboration at those points. They then move on to the pupa, pharate adult, and teneral adult stages to render imagos that are variously hypermorphic or neotenic relative to each other. The same character analysis is extrapolated to the broader Coleoptera, with the finding that hypermorphosis and neoteny were in operation during cladogenesis of the Order.

Properties of Orange-Pupa-Inducing Factor (OPIF) in the swallowtail butterfly, Papilio xuthus L.

Diapause pupae of the swallowtail butterfly Papilio xuthus L. exhibit diapause-green, orange and brownish-orange color polymorphism. Development of orange pupae involves a neuroendocrine factor inducing orange pupa (Orange-Pupa-Inducing Factor, OPIF), which is secreted from the head–thoracic region during late pharate pupal stages, in particular from the ganglia of short-day animals located posteriorly from the second thoracic ganglion 2 (TG 2). This report describes certain properties of OPIF using bioassays involving ligated abdomens of short-day pharate pupae. Localization of OPIF in the central nervous system of short-day larvae indicated that it was present predominantly in TG 2, thoracic ganglion 3 (TG 3) and abdominal ganglion 1 (AG 1) complexes. OPIF activity in TG 2,3–AG 1 complexes was over two times higher than in the more posteriorely located ganglia. The developmental profile of OPIF in last instar short-day larvae revealed that OPIF activity in larval ganglia posterior to TG 2 became gradually higher as larval growth proceeded, suggesting that OPIF might be accumulated in TG 2,3–AG 1–7 complexes as larvae prepare for pupal molting. Furthermore, ligated abdomens of short-day larvae developed into pupae of an orange type when a 2% NaCl extract containing OPIF prepared from TG 2,3–AG 1–7 complexes of long-day larvae was injected into ligated abdomens of short-day pharate pupae, indicating that OPIF is also present in long-day larvae. Additionally, a biochemical investigation using gel filtration chromatography showed that the molecular weight of OPIF was about 10 kDa.

Induction of dauer pupae by fenoxycarb in the silkworm, Bombyx mori

Topical application of fenoxycarb (1 μg per animal) at 129 or 132 h of the fifth instar larvae of the silkworm, Bombyx mori, did not induce morphological abnormalities in the pupal stage, but these animals became dauer (permanent) pupae. This condition of B. mori and the endocrine events leading to permanent pupae are discussed in this work. Application of fenoxycarb at 132 h of the fifth instar elicited a high ecdysteroid titre in the pharate pupal stage and a steadily high ecdysteroid titre in the pupal stage. The fenoxycarb-induced permanent pupae had non-degenerating prothoracic glands that secreted low amounts of ecdysteroid and did not respond to recombinant prothoracicotropic hormone (rPTTH) late in the pupal stage. The Bombyx PTTH titre in the haemolymph, determined by a time-resolved fluoroimmunoassay, was lower than that of controls at the time of pupal ecdysis, but higher than controls later in the pupal stage in fenoxycarb-treated animals. After application of fenoxycarb, its haemolymph level, measured by ELISA, reached a peak at pupal ecdysis, then remained low. These results suggest that the fenoxycarb-mediated induction of permanent pupae is only partially a brain-centred phenomenon. It also involves alterations in the hormonal interplay that govern both the initiation of pupal–adult differentiation and changes in the steroidogenic pathway of the prothoracic glands of B. mori.

Acceleration of Pupal-Adult Development by Fenoxycarb in the Silkworm, Bombyx mori

Treatment by fenoxycarb (> 0.1 μg/animal) during a short developmental period in the pharate pupal stage of the silkworm, Bombyx mori, induced alterations in the normal pupal-adult development. All fenoxycarb-treated animals showed accelerated development and precocious adult cuticle deposition very early in the pupal stage. Various internal and external organs of the developing adults underwent abnormal differentiation and were extensively malformed. Consequently, pupal-adult development and adult eclosion behaviour were disturbed. Similar effects were induced by injections of high doses (≥ 4 μg/animal) of 20-hydroxyecdysone (20E) only around the time of pupal ecdysis. Treatment with 1 μg of fenoxycarb in the pharate pupal stage did not affect haemolymph ecdysteroid titers early in the pupal stage. Our results, therefore, suggest that fenoxycarb, a potent juvenile hormone mimic, can imitate the developmental effects of excess 20E and behave as an ecdysteroid mimic after its application during the short developmental period of the pharate pupal stage of B. mori. These results shed light on the timely and coordinated increase of endogenous juvenile hormone titer before pupal ecdysis.

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

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.

Structure and spatial expression of the Manduca sexta MSCP14.6 cuticle gene

The genomic and cDNA sequences of the MSCP14.6 gene § § This gene was previously referred to as LCP-14.6 (Riddiford et al., 1986; Rebers et al., 1987). Since its expression is not confined to larval stages, the nomenclature has been changed to MSCP14.6 (MS=Manduca sexta; CP14.6=14.6 kD cuticular protein. , which encodes a Manduca sexta cuticle protein, have been determined. The genomic sequence presented here includes 1428 N of 5′ flanking DNA, 1587 N of transcribed DNA, and 465 N of 3′ flanking DNA. The intron/exon boundaries mapped by comparison of these sequences were confirmed by primer extension and S1 nuclease mapping. Prior work showed that the RNA produced by this gene is regulated both temporally and spatially during larval, pupal, and adult development, and also showed that both 20-hydroxyecdysone (20E) and juvenile hormone (JH) are important in controlling the amount of RNA present in the epidermis (Riddiford et al., 1986). In situ hybridizations show that MSCP14.6 is expressed throughout the epidermis in larvae and is spatially restricted in pharate pupal and pharate adult stages. The results presented here delimiting the transcription unit and 5' flanking DNA of the MSCP14.6 gene will facilitate further research to determine the DNA elements necessary for regulation of gene expression.

Isolations of eupyrene sperm bundles and apyrene spermatozoa from seminal fluid of the silkmoth, Bombyx mori

Fractions of eupyrene sperm bundles and apyrene spermatozoa were separated by centrifugation of the seminal fluid of the vesicula seminalis of normal 1-day-old male moths of the silkworm, Bombyx mori, on a discontinuous gradient of Percoll in silkworm Ringer at 25°C. On similar centrifugation of the seminal fluid of 1-day-old males that had been kept at 32°C only during the pharate-pupal period, eupyrene bundles were obtained in lower yield, and apyrene spermatozoa were recovered in poor yield. Thus, maintenance at 32°C during the pharate-pupal stage seemed to cause not only marked decrease in production of apyrene spermatozoa, but also some decrease in production of eupyrene spermatozoa. Apyrene spermatozoa separated from the seminal plasma were not activated by treatment with only a serine protease such as trypsin or initiatorin, which is present in the prostatic secretion. Both an endopeptidase and a cyclic nucleotide, cAMP or cGMP were required for their activation, and the further addition of 2-oxoglutarate enhanced their mortality. No glycogen phosphorylase activity was detected in either the isolated apyrene spermatozoa or the eupyrene sperm bundles.

Some Aspects of the Developmental Biology of Blaps kollari (Coleoptera: Tenebrionidae) Under Laboratory Conditions

The study focused on the influence of an artificial diet on the health, fecundity, duration and pattern of development, and survival rates of the beetle, Blaps kollari Seidlitz. The breeding period was confined mainly to the cooler months during which females laid their eggs; the largest number per individual was 650. Sixty-two percent of the eggs that matured in 9 d produced viable first-instar larvae. Voracious feeding from second instar onward produced an exponential weight gain; a coefficient of 400 from second to terminal eighth instar. In isolation, instar mortality was small; larvae consumed exuviae when not removed, and communal life allowed considerable cannibalism. Full larval development was quite rapid, lasting only 80 d, and terminated in a prolonged semidormant, nonfeeding pharate-pupal stage; a majority entered this stage after the seventh instar. Last-instar larvae were susceptible to a bacterial infection. Mortality and morbidity increased during pharate-pupal stage and deformity occurred also during pupal and adult molts. About 68% of pharate-pupal to pupal molts were normal. The pupal stadium averaged 15 d, during the latter half of which rudiments of the sternal gland, a characteristic of the males, appeared between sternites two and three. A majority of the pupal-to-adult molts resulted in deformed individuals. Experimental groups allowed cannibalism, and exuvial feeding fared better at this molt. A total of 8.5% healthy normal adults was obtained from all groups taken together. The pharate-pupal and newly emerged adult phases were accompanied by a significant weight loss. Within 1 mo after adult feeding was initiated, weight exceeded the maximum attained by last-instar larvae and plateaued within 2 mo.

Relationship between the ecdysteroid titres of a host and those of its parasite

Haemolymph ecdysteroids of larvae and pupae of the fruit fly Anastrepha suspensa and its endoparasitic wasp Biosteres (=Opius) longicaudatus were measured by radioimmunoassay. The ability of parasite first instar larvae to sequester and metabolize ecdysone from an artificial medium was also evaluated. From the third larval instar until the pharate pupal stage of parasitized hosts ecdysteroid titres increased from 30.9 ± 1.8 to 224 ± 5.3 pg/μl. Titres of the corresponding unparasitized stages ranged from 56 ± 9.3 to 280 ± 6.1 pg/μl. In both parasitized and unparasitized individuals titres fell sharply on the day when apolysis and pupal cuticle synthesis occurred. They increased again as pupal development progressed. Ecdysteroid levels of the parasite increased during the first larval instar and remained unchanged until the parasite's third instar. The levels fell in the newly moulted parasite fourth instar but thereafter increased sharply until the larval-pupal moult 2 days later. The predominant free ecdysteroid in parasitized and healthy A. suspensa pharate pupae was ecdysone while in the fourth instar parasite it was 20-OH-ecdysone. Parasites in vitro were able to take up [ 3H]ecdysone from a medium and convert it to 20-OH-ecdysone. This suggests that they may be able to sequester ecdysteroids from the hosts' haemolymph.

Comparative studies on ecdysone metabolism between mature larvae and pharate pupae in the fleshfly, Sarcophaga peregrina

AbstractMetabolites of radioactive ecdysone or 20‐hydroxyecdysone in larvae and pharate pupae of Sarcophaga peregrina were separated and identified by using thin‐layer chromatography, high‐performance liquid chromatography, and chemical methods. At the larval stage ecdysone was metabolized to biologically less active ecdysteroids predominantly through 20‐hydroxyecydsone, at the pharate pupal stage, to other ecdysteroids which were tentatively identified as 26‐hydroxyecdysone, 3‐epi‐26‐hydroxyecdysone, and 3‐epi‐20,26‐dihydroxyecdysone. Ecdysteroid acids were found in the polar metabolites during pharate pupal‐pupal transformation, but scarcely detected in the larval metabolites. These acids were presumed to be ecdysonoic acid, 20‐hydroxyecdysonoic acid, and their epimers. The conjugates of ecdysteroid that released the free ecdysteroids by enzymatic hydrolysis were produced more in larvae than in pupae, whereas the very polar ecdysteroids that were not affected by the enzyme were found more in pupae. Therefore, there are different metabolic pathways of ecdysone between these two successive developmental stages, and the alteration of the metabolic pathway may serve as one of the important factors in a regulatory mechanism of molting hormone activity which is responsible for normal development of this insect.

Temporal storage of kynurenine and 3-OH-kynurenine in the fat body of metamorphosingEphestia kühniella

During the pharate pupal stage a massive accumulation of kynurenine and 3-OH-kynurenine is observed in the fat body ofEphestia kuhniella. By injection it can be demonstrated that this organ is capable of sequestering at least 3-OH-kynurenine, the dominating tryptophan metabolite inEphestia. It is suggested that the fat body reduces a possibly harmfull excess of tryptophan metabolites at the beginning of metamorphosis. These sequestered metabolites provide a precursor depot for ommochrome synthesis in later development.

Delayed inhibition of host development by the nonparalyzing venoms of parasitic wasps

The nonparalyzing venoms of two unrelated parasitic wasps, Eulophus larvarum and Clinocentrus gracilipes, in separate ways cause delayed yet well-defined arrests in their hosts' development, apparently by disrupting events normally under endocrine control. The venom of E. larvarum (for which species host acceptance and oviposition behavior is described) prevents apolysis in its host after a depressed feeding period. The venom employed by C. gracilipes switches the host to a pharate pupal stage irrespective of its larval instar.

Effects of l-canavanine on arginine catabolism in manduca sexta (sphingidae: lepidoptera)

1. 1. Hemolymph ornithine concentrations in tobacco horn worm larvae fed a 2.5 mM l -canavanine plus 25 mM l -arginine-supplemented artificial diet (CAAM) were higher than those in larvae fed diets supplemented with 2.5 mM canavanine (CAV), 25 mM arginine (ARG), or controls (CON). 2. 2. Ornithine concentrations in CAV-treated larvae were significantly greater than the control or ARG treatment, but less than the CAAM treatment during the latter part of the wandering larval stage and during the pharate pupal stage. 3. 3. Urea concentrations were greater during the active feeding stage with the CAAM- and ARG-treated larvae having significantly higher levels than control or CAV-treated larvae. 4. 4. Urea concentrations in all treatments never exceeded 36.5% of the ornithine concentration. 5. 5. Canavanine concentrations were higher in CAV-treated larvae than in CAAM-treated larvae. 6. 6. During active feeding, arginine concentrations for all treatments were similar, but were lower in CAV- and CAAM-treated larvae during the pharate pupal stage.

Ecdysteroids and diapause in pupae of Heliothis punctiger

Most pupae of H. punctiger enter diapause when reared at 19°C, 12L:12D. When pharate pupae were treated for only 12 hr at 28°C about 50% developed at 19°C. The proportion of non-diapausing pupae increased as the temperature at which the pharate pupal stage was spent increased. The quantity of injected 20-hydroxyecdysone necessary to promote development in diapausing pupae varied from about 1 μg g −1 soon after pupation to about 4 μg g −1 after 50 days. It fell somewhat after 150 days. Removing brains from non-diapausing pupae showed that the brain secreted its hormone at the time of pupation (or just before). However, if the pupae were kept at 19°C development did not occur unless the brain remained in situ for at least 20 hr at 28°C. Implanting brains from non-diapausing pupae into diapausing ones had no measurable effect. These results may be explained by postulating that the prothoracic gland is ‘activated’ by exposure to high temperature, but that it reverts to inactivity over a period at 19°C. The ‘active’ gland must then be stimulated by brain hormone for a long period to trigger secretion of its hormone, which results in development. Diapause is thus the result of the failure of the prothoracic gland to secrete.

Dependence of the parasitoid Gonia cinerascens on the hormones of its lepidopterous hosts

Development of first instar larvae of Gonia cinerascens, which rest in the muscles of host caterpillars, is triggered by the release of the host's ecdysteroids when the juvenile hormone is absent. Ecdysteroids act on the parasitoid directly and at the same time induce physiological and biochemical changes in the host, which are indispensable for the parasitoid's development. These changes do not occur when metamorphosis of the host is suppressed with the juvenile hormone. Normally the parasitoids initiate development at the larval-pupal transformation of the host, but under experimental conditions, they do so whenever a high ecdysteroid titre is coupled with the proper internal environment in the host, that is in decapitated caterpillars, isolated host abdomens, and when implanted into host pupae. Activated parasitoids moult into the second instar and migrate to the exuvial space of the host; this migratory behaviour is also triggered by ecdysteroids and may be induced experimentally in the first instar parasitoids. Unknown clues direct the migrating parasitoids under the wings and appendages of the host pharate pupal stage. The second instar parasitoids, which anchor to the integument of the host pupae, apparently develop independently of the host's hormones: they can produce third instar larvae, pupae, and adult flies when cultured in vitro.

Some adaptations between Danaus plexippus and its food plant, with notes on Danaus chrysippus and Euploea core (Insecta: Lepidoptera)

Behaviour of the egg‐laying Monarch in captivity suggests that the concentration and quality of cardiac glycosides in the food plant are not important oviposition cues. The presence of eggs (as previously noted by Urquhart, 1960) and larvae feeding on the food plant, act as mild deterrents.The butterfly's emetic potency (see Table XIII(a)) can sometimes surpass that of the leaves of the host plant itself. Unidentified factors, providing the internal plant environment, are more important as cardiac glycoside storage stimulants than either the quantity or quality of the cardenolides present. In the laboratory D. plexippus oviposited preferentially on a plant with relatively low cardiac glycoside content, but which produced the most powerfully protected (emetic) adult.Metabolic changes during the pharate pupal stage, but also, in the case of Euploea core, in the larval fifth instar, rather than larval sequestration, may account for the major increase or decrease in butterfly toxicity compared with that of the food plant.Temperature does not affect the storage of cardenolides except indirectly by altering metabolic rate. There is no evidence to support the concept that current “physiological cost” of cardenolide storage is high. Like the toad, this butterfly can be assumed to have evolved an enzvmatic system well adjusted to the presence of cardenolides in its bodv tissues.