Green Pupae Research Articles

Effect of constant and fluctuating low temperature on the survival of Tuta absoluta pupae.

Temperature is among the key factors impacting the establishment and spread of invasive pests. The tomato leafminer Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is one of the major pests attacking Solanaceae plants and is known to possess overwintering capacities. However, the cold hardiness of T. absoluta pupae is poorly documented. In this study, we investigated the effect of constant temperature and stepwise cooling on T. absoluta pupae under laboratory conditions. For this purpose, bioassays on pupal development under constant temperature (5°C) for 30, 60 and 90 days, and stepwise changes in temperature (11, 10 and 8°C; in this order every 30 days), were assessed. We found that exposure to 5°C for 30 and 60 days did not affect the post-cooling emergence time of adults compared to the control. Pupae completed their development after 60 days of cold exposure at 5°C, but more adults emerged after 30 than 60 days. Even though alive pupae were observed after 90 days of cold exposure at 5°C, no adults emerged. External colours of pupae depended on the duration of cold periods, and green pupae obtained after 30 and 60 days were found to be positively correlated with the emergence of adults. When pupae were kept at 11°C for 30 days, 47% emerged, and when the temperature was changed to 10, only 12% of pupae emerged for the period 31-60 days. However, the decrease of the temperature to 8°C yielded no emergence for the period 61-90 days. Our study provides useful information to better understand the population dynamics of overwintering T. absoluta, and to underpin the development of monitoring and control strategies for the pest.

Pupal colour–pupation substrate correlation in butterflies

AbstractThe pupal colour of many butterflies is plastic, being green or brown depending on the environment in which they develop. In most species with pupal colour plasticity (PCP), larvae that pupate on leaves tend to develop into green pupae whereas those that pupate off‐leaf (e.g., on the stem or soil) tend to become brown. This correlation between background and pupal colour has been hypothesized to protect the butterflies against predation by allowing them to match their background, although experimental evidence is scarce. Furthermore, not all butterflies exhibit PCP, and the extent to which substrate choice influences PCP remains unclear. We use a comparative approach involving multiple, closely related species varying in the degree of PCP to test predictions related to the correlation between pupal colour and pupation substrate. We studied PCP in response to larval density in five common satyrine species (Nymphalidae), two dimorphic and three monomorphic. As predicted, species with monomorphic green pupae preferred to pupate on leaves whereas those with monomorphic brown pupae preferred off‐leaf substrates. In species with dimorphic pupae, pupal colour correlated with pupation substrate. We also predicted that higher larval density induces a greater proportion of off‐leaf pupation in the dimorphic species, but not in the monomorphic species. However, this prediction was not supported. Overall, we found a strong across‐species correlation between pupation substrate choice and pupal colour plasticity, and our results support the idea that pupal colour plasticity can be an adaptation against predation.

Pupal Warning Coloration of Three Species of Cystidia (Lepidoptera: Geometridae: Ennominae) in Relation to Their Pupation Sites.

Many insects display a cryptic color to avoid detection by predators that search for prey by sight. However, some species with chemicals that predators dislike may display a warning color (aposematism) to predators. The predators can learn easier that the species is unsuitable as prey if the color is more conspicuous. Therefore, it is assumed that the acquisition of the warning color requires not only unpalatability, but also exposure of the color to predators and the ability of predators to recognize and learn it unpalatable. In the moths of the subfamily Ennominae, almost all of genera produce uniformly brown or green pupae, but the pupae of the genus Cystidia have conspicuous coloration of yellow background and black spots. In this study, to clarify whether the color of these pupae is the warning color or not, we compared the coloration, pupation site, and palatability among the three species of this genus: C. couaggaria, C. truncangulata, and C. stratonice. Learning by the predators was also examined using lizards as a potential predator of the moths. The results showed that all three species were repelled (unpalatable) by the lizards, and that repeated providing of the pupae to the lizards decreased their willingness to prey on them (probably due to learning). Pupation sites of C. couaggaria and C. truncangulata were located on the surface of branches and leaves high above the ground, whereas C. stratonice pupated in the space of leaves spun with course silk at lower site above the ground. Thus, the conspicuous coloration of pupal Cystidia is considered to be a warning color, but the pupae of C. stratonice are more blackish than those of the most closely related C. truncangulata. The pupal color of C. stratonice is likely to have a dual meaning as cryptic and warning colors. The dark colored pupa may be inconspicuous when hidden within the leaf space, but once detected by the predators, the yellow color of the pupa may function as a warning color.

RELATIONSHIP BETWEEN PUPAL COLOUR AND SEX RATIO OF THE COMMON MORMON, Papilio polytes romulus CRAMER, 1775 (INSECTA: LEPIDOPTERA), SRI LANKA

A free ranging female common Mormon (Papilio polytes romulus) butterfly was observed laying eggs on a Citrus aurantiifolia (Family: Rutaceae) plant in a home garden in Battaramulla. 16 eggs were collected and reared in the lab. The duration and measurements of each stage were recorded. After eclosion the sex and wingspan of all adult butterflies were recorded and they were released back to the wild. Pupae were observed in two colours: green and brown. Out of 16 pupae, 12 were green (75%) and four were brown (25%). The four brown pupae gave rise to two males (50%) and two females (50%). The 12 green pupae gave rise to 10 males (83%) and two females (17%). The sex ratios regardless of the pupal colour showed male dominance with 12 males (75%) and four females (25%). All four females were of the romulus form. A strong relationship between pupal colour and substrate texture was observed, but no relationship between pupal colour, sex and sex ratios was seen.

Pupal colour plasticity in the butterfly Catopsilia pomona (Lepidoptera: Pieridae)

Abstract Pupal colour plasticity (PCP) in many lepidopterans can be adaptive by helping pupae match their background colours. Studies on PCP, most of which have been laboratory-based, have largely used human assessment of colour to categorize pupae as green or brown. This binary categorization limits the understanding of finer pupal colour variations and their function. We conducted a study of PCP in the butterfly Catopsilia pomona by comparing laboratory-reared and wild populations. Considering pupal colour as a continuous variable, we showed that a large proportion of the pupae matched the colours of their substrates, with leaf-borne pupae tending to be greener and off-leaf pupae browner. Pupal colour also responded to the leaf substrate’s finer colour variations, highlighting the importance of treating pupal colour as a continuous variable. Compared to the wild population, the laboratory population had more green pupae on off-leaf substrates. Our study thus illustrates that caution should be used when extrapolating the results from laboratory-based studies to the natural world. In leaf-borne wild pupae, pupation position on the leaf, the thickness of the midrib where pupation occurred and the leaf’s length influenced the pupal colour. Our study underscores the need for further research on PCP as a background-matching strategy in light of predation.

Partially coupled reaction norms of pupal colour and spot size in a butterfly

Many species cope with heterogeneous environments through adaptive phenotypic plasticity, and plasticity of body colour is a widely used means of minimizing predation risk. Pupal colour in many butterflies is a plastic trait sensitive to pupation substrate, with green and brown pupae primarily formed on leaf and off-leaf substrates respectively. This is thought to be an example of adaptive phenotypic plasticity because the green and brown pupae are better camouflaged against their respective backgrounds. We tested whether pupal ‘spots’—paired circular markings on the anterior abdominal pupal segments of many butterflies—are also phenotypically plastic. Using experiments on the tropical satyrine butterfly Mycalesis mineus, we confirm that pupal spot size is plastic. Because it is known that pupation substrate influences pupal colour in this species, we tested whether substrate also influences pupal spot size. Further, we explored whether there was a correlation between pupal colour and spot size. We tested the effects of pupation substrate and pupal colour on both absolute spot size and spot size relative to body size. Pupal spot size (absolute and relative) was correlated with both pupal colour and pupation substrate. Brown pupae had larger spots, and green pupae formed off-leaf had intermediate sized spots compared to green pupae formed on leaves and brown pupae, the latter of which always formed off-leaf. Thus, the reaction norm of pupal spot size is partially coupled with both pupal colour and pupation substrate in this species. Since pupation substrate in turn influences pupal colour, the correlated traits of pupal colour and pupal spot could comprise a single functional suite, suggesting a developmental link. We speculate that pupal spots may function against predation, but further studies are needed to understand their possible adaptive significance.

Molecular Mechanisms Underlying Pupal Protective Color Switch in Papilio polytes Butterflies

Mimicry is a survival strategy in which organisms deceive their predators by imitating other organisms or the surrounding environment. One example of this involves pupal color polymorphism, which is widely observed in butterflies and moths. It has been suggested that the pupal colors of Papilio butterflies are selected according to the tactile stimulation experienced before pupation. Specifically, larvae crawling on smooth leaves become green pupae, but those crawling on rough stems become brown pupae. These protective colors fit with the surrounding environment. However, the detailed molecular mechanisms underlying pupal polymorphism have generally remained unknown. To reveal these mechanisms, we first established control over the green and brown pupal coloration in Papilio polytes under laboratory conditions and clarified temporal and spatial changes of pupal pigments under both conditions. We also analyzed the expression of coloration genes during the pre-pupal stage and at pupation in the epidermis under the green and brown conditions, by RNA sequencing and quantitative RT-PCR. These analyses revealed that the brown pupal color is regulated mainly by melanin synthesis genes, tyrosine hydroxylase (TH) and laccase 2. In contrast, the green pupal color was suggested to be formed mainly through the expression of both multiple bilin binding protein (BBP)-related genes responsible for blue pigmentation and multiple juvenile hormone binding protein (JHBP)-related genes responsible for yellow pigmentation. Electroporation-mediated RNAi showed that the knockdown of TH or laccase 2 blocked the brown pupal coloration, and that the knockdown of BBP- or JHBP-related genes caused yellow or blue coloration in the green-conditioned pupae, respectively, supporting the above hypothesis. We here report how genes involved in the pupal coloration of P. polytes are regulated, which sheds light on the evolutionary process of pupal protective colors among Lepidoptera.

Pupal colour dimorphism in a desert swallowtail (Lepidoptera: Papilionidae) is driven by changes in food availability, not photoperiod

1. The swallowtail butterfly Battus polydamas archidamas Boisduval, 1936, exhibits polyphenism for pupal coloration (green and brown). It is distributed across arid regions with winter rains and is monophagous on Aristolochia plants, which emerge after the winter rains and dry out the during summer. Thus, day length does not covary positively with host plant productivity. It was hypothesised that pupal colour was driven by food availability, not photoperiod. The benefits of pupal coloration matching the colour of pupation sites in terms of field survival were also investigated to evaluate the adaptive value of pupa colour.2. Larvae were reared under a factorial array of two photoperiods (LD 10:14 h and LD 14:10 h) and two food availability regimes (leaves ad libitum and available every other day) to assess the frequency of green and brown pupae. Field survival of green and brown pupae was quantified in three commonly used habitats that differ in background coloration (cacti, rocks and shrubs).3. Food availability determined pupal colour. Larvae in the ad libitum regime resulted mostly in green pupae, while those with restricted food were mostly brown. In contrast, photoperiod did not influence pupal colour. Survival probability of pupae placed on cacti was higher than those placed on rocks and shrubs, and the lowest predation risk across habitats was for green pupae on cacti.4. Food availability plays a major role in the seasonal polyphenism for pupal colour of specialist butterflies inhabiting arid environments with winter rains.

Pupal colour plasticity in a tropical butterfly, Mycalesis mineus (Nymphalidae: Satyrinae).

Lepidopteran insects have provided excellent study systems for understanding adaptive phenotypic plasticity. Although there are a few well-studied examples of adult plasticity among tropical butterflies, our understanding of plasticity of larval and pupal stages is largely restricted to temperate butterflies. The environmental parameters inducing phenotypic plasticity and the selective pressures acting on phenotypes are likely to differ across tropical and temperate climate regimes. We tested the influence of relative humidity (RH), a prominent yet under-appreciated tropical climatic component, along with pupation substrate, larval development time, pupal sex and weight in determining pupal colour in the tropical satyrine butterfly Mycalesis mineus. Pupae of this butterfly are either brown or green or very rarely intermediate. Larvae were reared at high (85%) and low (60%) RH at a constant temperature. Proportions of green and brown pupae were expected to vary across low and high RH and pupation substrates in order to enhance crypsis. Brown pupae were more common at low RH than at high RH, as predicted, and developed faster than green pupae. Pupal colour was correlated with pupation substrate. Choice of pupation substrate differed across RH treatments. It is unclear whether pupal colour influences substrate selection or whether substrate influences pupal colour. Our study underscores the need for further work to understand the basis of pupal plasticity in tropical butterflies.

Observations on the biology of Afrotropical Hesperiidae (Lepidoptera) principally from Kenya. Part 3. Pyrginae: Celaenorrhinini

Partial life histories for 15 species and two subspecies of Afrotropical Celaenorrhinini (Hesperiidae: Pyrginae) are described and illustrated: Celaenorrhinus plagiatus Berger, C. undescribed nr. dargei Berger, C. proxima proxima (Mabille), C. proxima maesseni Berger, C. zanqua Evans, C. humbloti (Mabille), C. sanjeensis Kielland, C. galenus opalinus Butler, C. galenus biseriata Butler, C. handmani Collins & Congdon, Eretis umbra maculifera Mabille & Boullet, E. lugens (Rogenhofer), Sarangesa phidyle (Walker), S. motozi (Wallengren), S. haplopa Swinhoe, S. maculata (Mabille) and Triskelionia compacta (Evans). The published information on other species of the tribe is summarised, and generalisations are made for the tribe in Africa. All species of Celaenorrhinus, Eretis and Sarangesa spp. reared to date use Acanthaceae as food plants, but T. compacta feeds on a Fabaceae and Ortholexis hollandi Druce feeds on an Apocynaceae. Three groups of Celaenorrhinus spp. are recognised based on their pupae: Group 1 having brown pupae with cephalic projections, Group 2 having green pupae, with no cephalic projections, and a very long proboscis sheath, and a third group based on published illustrations of the life history of C. mokeezi (Wallengren), in which the pupa is generally covered with white powder, and has just a short blunt frontal projection. Loxolexis Karsch is returned to synonomy with Katreus Watson, stat.rev. and Ortholexis Karsh stat rev. is reinstated for the species which had been placed in Loxolexis: O. holocausta (Mabille) comb. nov., O. dimidia (Holland) comb. nov. and O. hollandi Druce comb. rev.

Diapause dynamics, seasonal phenology, and pupal color dimorphism of Papilio polyxenes in southern Florida, USA

AbstractThe seasonal phenology, pupal diapause, and pupal color dimorphism of a Papilio polyxenes Fabr. (Lepidoptera: Papilionidae: Papilionini) population from subtropical southern Florida, USA, were studied. Adult eclosion from field‐collected individuals indicated that this population is primarily active from the late dry season through the wet season (March–September). Most pupae entering diapause in April eclosed the same year but one pupa eclosed the following February. In laboratory tests, larval rearing under short days (L10:D14) induced 88.6% pupal diapause. Diapause responses to relatively longer (L12:D12–L16:D8) photoperiods ranged from 35.1–51.5%, and were statistically equivalent. These results indicate greater variability in the photoresponse of subtropical P. polyxenes compared to temperate populations that generally show little diapause under long‐day conditions and complete diapause under short days. The proportions of diapause pupae and brown pupal color, among broods reared at photoperiods ≥L12:D12, were both normally distributed. Diapause pupae were sensitive to photoperiods from L10:D14 to L16:D8. Diapause duration was inversely related to the photophase length to which the pupae were continuously exposed. Four generations of selection, at L10:D14, for non‐diapause reduced pupal diapause from 88.6 to 20.6%, indicating the existence of considerable additive genetic variance for this trait. Selection for non‐diapause increased the percentage of green pupae from 21.8 to 58.8% and decreased the incidence of diapause in both green and brown pupae. Brown pupal color and liability to diapause are linked, but this linkage is weaker than that documented in temperate populations. The overriding effect of short days (L10:D14) inducing only brown diapause pupae did not occur in southern Florida P. polyxenes.

Role of Larval Stemmata in Control of Pupal Color and Pupation Site Preference in Swallowtail Butterflies <I>Papilio troilus</I>, <I>Papilio polyxenes</I>, <I>Eurytides marcellus</I>, and <I>Papilio glaucus</I> (Lepidoptera: Papilionidae)

Swallowtail butterfly species (Lepidoptera: Papilionidae) dimorphic for pupal color produce cryptic brown and green pupae in response to environmental cues. The occurrence of perceptible and reliable cues about pupation site color is a necessary condition for the evolution of this adaptation. By comparing the pupal color responses of prepupal larvae blinded by cautery of the stemmata with prepupal larvae cauterized, but not blinded, we show that the larval eyes (stemmata) mediate the pupal color response. Using a similar comparison, we also show that the stemmata are involved in pupation site choice by prepupal larvae. Considered in light of previously published work on the spectral sensitivities of stemmatal photoreceptors, the absorption spectra of green vegetation and the effects of different colored surfaces on pupal color, our results suggest a model for the proximate control of pupal color involving the differential stimulation of green and blue stemmatal photoreceptors.

Interactions of environmental factors influencing pupal coloration in swallowtail butterfly Papilio xuthus

The swallowtail butterfly Papilio xuthus Linné [Lepidoptera: Papilionidae] exhibits pupal protective color polyphenism. Interactions of various environmental factors on pupal coloration were analyzed in non-diapausing individuals. Under sufficient light (200 lux), most pupating larvae became green pupae when the surface of the pupation site was smooth, while they became brown when the surface was rough. Tactile signals are the positive environmental factors causing induction of the brown pupal coloration. In dark boxes, the induction of the brown pupal coloration was easily induced even on a smooth surface, suggesting that light suppresses induction of brown coloration. Different colors of pupation sites did not affect pupal coloration under sufficient light. Environmental factors received during a critical period both before girdling and after girdling affected pupal coloration. When tactile signals received from rough surfaces reach threshold levels during pupation, brown pupal coloration is determined. Larvae reared under a daily periodicity of natural light formed a girdle at midnight, subsequently, the prepupae received strong daylight the following day. Under natural light most larvae produced brown pupae on rough surfaces and green pupae on smooth surfaces.

Two different sensory mechanisms for the control of pupal protective coloration in butterflies

The butterflies Graphium sarpedon nipponum Fruhstorfer and Papilio xuthus Linné show pupal protective color polymorphism, but the two species appear to have different sensory mechanisms for determining pupal coloration. When light was of sufficient illumination, the larvae of Graphium sarpedon became bright yellowish green pupae on white pupation boards and reddish brown pupae on black pupation boards. The pupal coloration thus strongly depended on the brightness of the pupation site. In addition, larvae became bright yellowish green pupae in complete darkness. From these results, measurement of the illumination suggested that pupal color is determined by the illuminant difference between incidence light from the dorsal direction and ventral light from a paper board; i.e., the sum of the reflected light of the board plus the penetrated light passing through the board. The illuminant difference required for reddish brown coloration was 40 lux or more. The optical signals received through the stemmata during a critical period before formation of the thorax garter (band string) were important for coloration. By contrast, in Papilio xuthus, successive tactile signals from a rough surfaced pupation site during a critical period before and after formation of the garter were important for determining brown pupal coloration.

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.

Phenotypic variation in adult morphology and pupal colour within and among families of the speckled wood butterfly Pararge aegeria

Abstract.1. Larvae from eggs of fifteen wild‐caught speckled wood females were reared individually under common conditions (LD 18:6 h and 17 °C) in the laboratory.2. Pupal colour (green or brown) and the following adult morphological variables that are known or assumed to be related to behavioural variation (thermoregulation, flight, crypsis) were studied: size, relative thorax mass, area of yellow patches on the dorsal forewing, size of submarginal eyespots on the dorsal hindwing, colour of the dorsal thorax, dorsal basal wing, and dorsal distal wing.3. The results of the breeding experiment indicate significant differences in adult morphological traits among families, sexes, and pupal colour types.4. All adult morphological variables (except spot size and thorax colouration) differed significantly among families, suggesting genetic variation underlying the phenotypic variation. Heritabilities for these features were intermediate (0.38) to high (> 1).5. Apart from known aspects (e.g. size), novel aspects of the sexual dimorphism were found: females had paler thoraxes than males, which relates to higher abundance of fur on the thorax and hence to thermoregulatory differences.6. Green pupae produced larger individuals with a larger relative thorax mass than brown pupae. Green pupae produced adults with a paler basal wing colour in females, but not in males. These relationships are novel and suggest a trade‐off between juvenile and adult investment.

The evolution of environmentally‐cued pupal colour in swallowtail butterflies: natural selection for pupation site and pupal colour

1. Environmentally‐cued pupal colour in swallowtail butterflies has been hypothesized to evolve as a consequence of (a) the evolution of a preference for pupation sites above the ground that vary in colour and (b) natural selection for crypsis on such sites.2. This hypothesis was tested by comparing the field survival of green and brown Papilio polyxenes Fabr. pupae placed on green or brown pupation sites that were either above the ground on near the ground.3. Green pupae on green sites above the ground had a significantly higher probability of survival than did all other pupal colour and pupation site combinations.4. Pupae on sites above the ground were more likely to be preyed upon during the day, whereas those on sites near the ground were more likely to be preyed upon during the night, suggesting that variation in nocturnal and diurnal predation influences the evolution of pupation site preference.5. To the extent that diurnal predators use colour vision to locate prey, diurnal predation should favour environmentally‐cued pupal colour.

Pupation site preference and environmentally cued pupal colour dimorphism in the swallowtail butterfly Papilio polyxenes Fabr. (Lepidoptera: Papilionidae)

Environmentally cued polymorphisms are hypothesized to evolve when the environment is coarse-grained and different genotypes are unable to choose the habitats in which they are most fit. InPapilio polyxenes, which has an environmentally cued pupal colour dimorphism, there is genetic variation in both tendency to produce brown or green pupae and preference for green- or brown-inducing pupation sites, but the two traits are not correlated.

Pupation site preference and environmentally cued pupal colour dimorphism in the swallowtail butterflyPapilio polyxenesFabr. (Lepidoptera: Papilionidae)

Environmentally cued polymorphisms are hypothesized to evolve when the environment is coarse-grained and different genotypes are unable to choose the habitats in which they are most fit. InPapilio polyxenes, which has an environmentally cued pupal colour dimorphism, there is genetic variation in both tendency to produce brown or green pupae and preference for green- or brown-inducing pupation sites, but the two traits are not correlated.

Genetic variation for the rate of recruitment of novel insect prey into the diet of a bird

Two lines of quail (Cotumix coturnix japonicus) were selected over three generations for the speed of their response to the red colour form of the two-spot ladybird beede (Adalia bipunctata), an aposematic, semi-palatable prey insect. One line was bred for fast recruitment of die new prey into the diet and die odier for slow recruitment. Differences between the lines suggested additive genetic variation. The birds' response to insect prey with different colour patterns and toxicities (brown beetles, green butterfly pupae, melanic two-spot ladybirds and toxic seven-spot ladybirds) demonstrated that die selection involved a specific response to novel prey. The results are discussed in relation to predator strategy and the evolution of aposematism.