An extract from the frass of swallowtail butterfly (Papilio machaon) larvae inhibits HCT116 colon cancer cell proliferation but not other cancer cell types.

BACKGROUND: The frass of several herbivorous insect species has been utilised as natural medicines in Asia; however, the metabolite makeup and pharmaceutical activities of insect frass have yet to be investigated. Oligophagous Papilionidae insects utilise specific kinds of plants, and it has been suggested that the biochemicals from the plants may be metabolised by cytochrome P450 (CYP) in Papilionidae insects. In this study, we extracted the components of the frass of Papilio machaon larvae reared on Angelica keiskei, Oenanthe javanica or Foeniculum vulgare and examined the biological activity of each component. Then, we explored the expression of CYP genes in the midgut of P. machaon larvae and predicted the characteristics of their metabolic system. RESULTS: The components that were extracted using hexane, chloroform or methanol were biochemically different between larval frass and the host plants on which the larvae had fed. Furthermore, a fraction obtained from the chloroform extract from frass of A. keiskei-fed larvae specifically inhibited the cell proliferation of the human colon cancer cell line HCT116, whereas fractions obtained from the chloroform extracts of O. javanica- or F. vulgare-fed larval frass did not affect HCT116 cell viability. The metabolites from the chloroform extract from frass of A. keiskei-fed larvae prevented cell proliferation and induced apoptosis in HCT116 cells. Next, we explored the metabolic enzyme candidates in A. keiskei-fed larvae by RNA-seq analysis. We found that the A. keiskei-fed larval midgut might have different characteristics from the O. javanica- or F. vulgare-fed larval metabolic systems, and we found that the CYP6B2 transcript was highly expressed in the A. keiskei-fed larval midgut. CONCLUSIONS: These findings indicate that P. machaon metabolites might be useful as pharmaceutical agents against human colon cancer subtypes. Importantly, our findings show that it might be possible to use insect metabolic enzymes for the chemical structural conversion of plant-derived compounds with complex structures.

Bioinspired, vertically stacked, and perovskite nanocrystal-enhanced CMOS imaging sensors for resolving UV spectral signatures.

Imaging and identifying target signatures and biomedical markers in the ultraviolet (UV) spectrum is broadly important to medical imaging, military target tracking, remote sensing, and industrial automation. However, current silicon-based imaging sensors are fundamentally limited because of the rapid absorption and attenuation of UV light, hindering their ability to resolve UV spectral signatures. Here, we present a bioinspired imaging sensor capable of wavelength-resolved imaging in the UV range. Inspired by the UV-sensitive visual system of the Papilio xuthus butterfly, the sensor monolithically combines vertically stacked photodiodes and perovskite nanocrystals. This imaging design combines two complementary UV detection mechanisms: The nanocrystal layer converts a portion of UV signals into visible fluorescence, detected by the photodiode array, while the remaining UV light is detected by the top photodiode. Our label-free UV fluorescence imaging data from aromatic amino acids and cancer/normal cells enables real-time differentiation of these biomedical materials with 99% confidence.

Long-term spatial memory across large spatial scales in Heliconius butterflies.

Locating food in heterogeneous environments is a core survival challenge. The distribution of resources shapes foraging strategies, imposing demands on perception, learning and memory, and associated brain structures. Indeed, selection for foraging efficiency is linked to brain expansion in diverse taxa, from primates1 to Hymenopterans2. Among butterflies, Heliconius have a unique dietary adaptation, actively collecting and feeding on pollen, providing a source of essential amino acids as adults, negating reproductive senescence and facilitating an extended longevity3. Several lines of evidence suggest that Heliconius learn the spatial location of pollen resources within an individual's home range4, and spatial learning may be more pronounced at these large spatial scales. However, experimental evidence of spatial learning in Heliconius, or any other butterfly, is so far absent. We therefore tested the ability of Heliconius to learn the spatial location of food rewards at three ecologically-relevant spatial scales, representing multiple flowers on a single plant, multiple plants within a locality, and multiple localities. Heliconius were able to learn spatial information at all three scales, consistent with this ability being an important component of their natural foraging behaviour.

Complementary roles of hummingbirds and bees: Pollen pickup, pollen deposition, and fruit production in the distylous Palicourea rigida.

PREMISE: Distyly is a condition in which individual plants in a population express two floral morphs, L- and S-morph, characterized by reciprocal placements of anthers and stigmas between morphs. The function of distyly requires that pollinators collect pollen from L- and S-morphs on different parts along their bodies to then deposit it on the stigmas of the opposite morph, known as legitimate pollination. However, different pollinator groups might differ in the ability to transfer pollen legitimately. METHODS: We investigated patterns of pollen pickup along the body of different functional groups (hummingbirds and bees) using preserved specimens to analyze their role in the reproductive success of Palicourea rigida. We measured pollen deposition on the body of pollinators, on stigmas, and fruit production after a single visit. RESULTS: Pollen from L- and S-flowers appeared segregated on different body parts of the hummingbird and bee used in the study. S-pollen was deposited primarily on the proximal regions (near the head), and L-pollen was placed in the distal regions (tip of the proboscis and bill). Hummingbirds were more efficient at legitimate pollination than bees, particularly to S-stigmas. However, fruit formation after single visits by both pollinators was similar. CONCLUSIONS: The morphology of distylous flowers allows the segregated placement of L-and S-pollen on different body parts of the animal specimens used, facilitating the promotion of legitimate pollen transfer, an observation consistent between the two functional pollinator groups. Also, the results show that full fruit set requires more than one visit.

Characterizing each step of pollination in Phlox drummondii reveals that a single butterfly species predominates in the pollinator assemblage.

PREMISE: A central goal of pollination biology is to connect plants with the identity of their pollinator(s). While predictions based on floral syndrome traits are extremely useful, direct observation can reveal further details of a species' pollination biology. The wildflower Phlox drummondii has a floral syndrome consistent with pollination by Lepidoptera. We tested this prediction using empirical data. METHODS: We observed each step of pollination in P. drummondii. First, we observed 55.5 h of floral visitation across the species range. We used temporal pollinator exclusion to determine the contribution of diurnal and nocturnal pollination to reproductive output. We then quantified P. drummondii pollen transfer by the dominant floral visitor, Battus philenor. Finally, we tested the effect of B. philenor visitation on P. drummondii reproduction by quantifying fruit set following single pollinator visits. RESULTS: Battus philenor is the primary pollinator of P. drummondii. Pollination is largely diurnal, and we observed a variety of lepidopteran visitors during the diurnal period. However, B. philenor was the most frequent visitor, representing 88.5% of all observed visits. Our results show that B. philenor is an extremely common visitor and also an effective pollinator by demonstrating that individuals transfer pollen between flowers and that a single visit can elicit fruit set. CONCLUSIONS: Our data are consistent with the prediction of lepidopteran pollination and further reveal a single butterfly species, B. philenor, as the primary pollinator. Our study demonstrates the importance of empirical pollinator observations, adds to our understanding of pollination mechanics, and offers a specific case study of butterfly pollination.

Geography shapes the microbial community in Heliconius butterflies.

Heliconius butterflies are an ideal organism for studying ecology, behavior, adaptation, and speciation. These butterflies can be found in various locations and habitats in Central and South America, where they encounter and interact with different sources of pollen, nectar, and host plants. However, there is limited knowledge on how geographic and habitat variations affect the microbiota of these insects, and whether microbial associates play a role in their ability to exploit different habitats. To date, research on the microbial communities associated with Heliconius has mainly focused on host phylogenetic signal in microbiomes or microbiome characterization in specific communities of butterflies. In this study, we characterized the microbiomes of several species and populations of Heliconius from distant locations that represent contrasting environments. We found that the microbiota of different Heliconius species is taxonomically similar but vary in abundance. Notably, this variation is associated with a major geographic barrier-the Central Cordillera of Colombia. Additionally, we confirmed that this microbiota is not associated with pollen-feeding. Therefore, it seems likely that geography shapes the abundance of microbiota that the butterfly carries, but not the taxonomic diversity of the microbial community. Based on the current evidence, the bacterial microbiota associated with Heliconius does not appear to play a beneficial role for these butterflies.

Diverse flower-visiting responses among pollinators to multiple weather variables in buckwheat pollination.

Response diversity to environmental change among species is important for the maintenance of ecosystem services, but response diversity to changes in multiple environmental parameters is largely unexplored. Here, we examined how insect visitations to buckwheat flowers differ among species groups in response to changes in multiple weather variables and landscape structures. We found differences in responses to changes in weather conditions among insect taxonomic groups visiting buckwheat flowers. Beetles, butterflies, and wasps were more active in sunny and/or high-temperature conditions, whereas ants and non-syrphid flies showed the opposite pattern. When looking closely, the different response pattern among insect groups was itself shown to be different from one weather variable to another. For instance, large insects were responsive to temperatures more than small insects while smaller insects were responsive to sunshine duration more than large insects. Furthermore, responses to weather conditions differed between large and small insects, which agreed with the expectation that optimal temperature for insect activity depends on body size. Responses to spatial variables also differed; large insects were more abundant in fields with surrounding forests and mosaic habitats, whereas small insects were not. We suggest that response diversity at multiple spatial and temporal niche dimensions should be a focus of future studies of the biodiversity-ecosystem service relationships.

Polyhydroxy Acids as Fabaceous Plant Components Induce Oviposition of the Common Grass Yellow Butterfly, Eurema Mandarina.

The common grass yellow butterfly, Eurema mandarina is a Fabaceae-feeding species, the females of which readily oviposit on Albizia julibrissin and Lespedeza cuneata in mainland Japan. We previously demonstrated that the methanolic leaf extracts of these plants, and their highly polar aqueous fractions strongly elicit female oviposition. Furthermore, the three subfractions obtained by ion-exchange chromatographic separation of the aqueous fraction have been found to be less effective alone, but synergistically stimulate female oviposition when combined. This indicates that female butterflies respond to multiple compounds with different acidity. We have previously identified d-pinitol from the neutral/amphoteric subfractions and glycine betaine from the basic subfractions as oviposition stimulants of E. mandarina. The present study aimed to identify active compounds in the remaining acidic subfractions of A. julibrissin and L. cuneata leaf extracts. GC-MS analyses of trimethylsilyl-derivatized samples revealed the presence of six compounds in the acidic subfractions. In bioassays using these authentic chemicals, erythronic acid (EA) and threonic acid (TA) were moderately active in eliciting oviposition responses in E. mandarina, with their d-isomers showing slightly higher activity than their l-isomers. Female responsiveness differed between d-EA and l-TA, the major isomers of these compounds in plants, with the response to d-EA reaching a plateau at concentrations above 0.005% and that to l-TA peaking at a concentration of 0.01%. The natural concentrations of d-EA and l-TA in fresh A. julibrissin and L. cuneata leaves were sufficient to stimulate oviposition. Furthermore, mixing 0.001% d-EA or 0.001% l-TA, to which females are mostly unresponsive, with 0.1% d-pinitol resulted in a synergistic enhancement of the oviposition response. These findings demonstrate that E. mandarina females utilize both polyhydroxy acids, EA and TA, as chemical cues for oviposition.

Testing hypotheses of a coevolutionary key innovation reveals a complex suite of traits involved in defusing the mustard oil bomb.

Coevolutionary interactions are responsible for much of the Earth's biodiversity, with key innovations driving speciation bursts on both sides of the interaction. One persistent question is whether macroevolutionary traits identified as key innovations accurately predict functional performance and selection dynamics within species, as this necessitates characterizing their function, investigating their fitness consequences, and exploring the selection dynamics acting upon them. Here, we used CRISPR-Cas9 mediating nonhomologous end joining (NHEJ) in the butterfly species Pieris brassicae to knock out and directly assess the function and fitness impacts of nitrile specifier protein (NSP) and major allergen (MA). These are two closely related genes that facilitate glucosinolate (GSL) detoxification capacity, which is a key innovation in mustard feeding Pierinae butterflies. We find NSP and MA are both required for survival on plants containing GSLs, with expression differences arising in response to variable GSL profiles, concordant with detoxification performance. Importantly, this concordance was only observed when using natural host plants, likely reflecting the complexity of how these enzymes interact with natural plant variation in GSLs and myrosinases. Finally, signatures of positive selection for NSP and MA were detected across Pieris species, consistent with these genes' importance in recent coevolutionary interactions. Thus, the war between these butterflies and their host plants involves more than the mere presence of chemical defenses and detoxification mechanisms, as their regulation and activation represent key components of complex interactions. We find that inclusion of these dynamics, in ecologically relevant assays, is necessary for coevolutionary insights in this system and likely others.

Contribution to the knowledge of the genus Agalope Walker from mainland China, with descriptions of four new species (Lepidoptera, Zygaenidae, Chalcosiinae).

New information of the genus Agalope Walker, 1854 from mainland China is presented. Three new species of the Agalope pica species-group are described: A. haoi S.-Y. Huang sp. n. from Weixi County, Yunnan, A. chayuensis S.-Y. Huang Pan sp. n. from Chayu County, Southeastern Xizang and A. owadai S.-Y. Huang sp. n. from Bomi and Jiali Counties, Southeastern and Eastern Xizang. The little-known Agalope aurelia Oberthr, 1923 and A. lucia Oberthr, 1923 were rediscovered, with the male of the latter reported for the first time. Based on the newly discovered male, Agalope lucia is found to be a close relative of A. dejeani, hence it is excluded from the A. pica species-group and transferred to the A. bieti species-group. A new species of the Agalope hyalina species-group is also described: A. jianqingi S.-Y. Huang sp. n. from Pianma, Western Yunnan. Adults and genitalia of the aforementioned and related taxa are illustrated. An updated checklist of the genus is also provided.

Head and Tail Oxidized Terpenoid Esters from Androconia of Heliconius erato Butterflies.

Heliconius erato is a neotropical butterfly species that is part of a complex mimicry ring, with colorful wing patterns. For intraspecific communication, males use pheromones that are released from two different scent-emitting structures. Scent glands located near the abdominal claspers of males, containing antiaphrodisiac pheromones, release a highly complex mixture of compounds that is transferred to females during mating, rendering them unattractive to other males. On the other hand, androconia, scent-emitting scale areas on the wings of male butterflies, release a structurally more restricted set of compounds that likely serves an aphrodisiac role. We report here on two structurally related compounds that are the major androconial constituents, produced in high amounts and are not volatile due to their high molecular mass. Their structures were established by extensive analysis of mass, infrared, and NMR spectra, as well as microderivatization reactions of the natural extract. After establishing synthetic access, the compounds were unequivocally identified as two unusual head and tail oxidized terpenoids, (4E,8E,12E)-4,8,12-trimethyl-16-oxoheptadeca-4,8,12-trien-1-yl oleate (1) and stearate (2). Although behavioral assays are necessary to fully comprehend their role in the chemical communication of the species, hypotheses for their use by the butterflies are also discussed.

Insecticidal and Feeding Deterrent Effects of Kaempferia galanga L. and Amomum subulatum on Pieris rapae L. Larva.

<b>Background and Objective:</b> <i>Pieris rapae</i> L., is one of the most widespread and destructive pests of cruciferous plants. At present, synthetic chemical insecticide is still the main approach to control this pest despite several disadvantages to human health and the wildlife environment as well as biological resistance. To search for plants having insecticidal activity, the biological effects of two medicinal plants <i>Kaempferia galanga</i> L. and <i>Amomum subulatum </i>on <i>Pieris rapae</i> L., were investigated. <b>Materials and Methods:</b> The methanol extracts of dry rhizomes and fruits of <i>Kaempferia galanga</i> L. and <i>Amomum subulatum </i>were used to determine the mortality, feeding and oviposition deterrence of larvae and adult of <i>Pieris rapae</i> L. <b>Results:</b> <i>Kaempferia galanga</i> L. and <i>Amomum subulatum</i> exhibited insecticidal activity against <i>Pieris rapae</i> L., with LC₅₀ values of 2.11 and 11.80% (w/v), respectively. In the antifeedant test, <i>Kaempferia galanga</i> L., extract showed no significant difference with the control at the low concentration (0.5 and 1%). Whereas, with a concentration of 0.5%, <i>Amomum subulatum</i> extract demonstrated a high antifeedant effect on <i>Pieris rapae</i> L., larvae. In addition, plants treated with these two extracts reduced eggs laid by <i>Pieris rapae</i> L., in field conditions showing the oviposition deterrent properties. <b>Conclusion:</b> These results indicated that <i>Kaempferia galanga</i> L. and <i>Amomum subulatum </i>extracts have insecticidal substances against <i>Pieris rapae </i>L., which can be used for developing effective pesticides or/and oviposition deterrents for integrated pest management.

New taxa and new records of butterflies from Vietnam (Lepidoptera, Papilionoidea)nbsp.

A new species and eight new subspecies of Papilionoidea discovered in Vietnam between 2002 and 2020 are described and illustrated. The status of two taxa are revised. New taxa include Pieridae: Delias sanaca bidoupa Monastyrskii Vu subspec. nov. and Talbotia naganum aurelia Monastyrskii Vu subspec. nov.; Nymphalidae: Abrota ganga pulcheria Monastyrskii Vu, subspec. nov.; Bassarona recta consonensis Monastyrskii Vu, subspec. nov.; Pantoporia bieti aurantina Monastyrskii To subspec. nov.; Ragadia latifasciata cristata Monastyrskii Vu, subspec. nov.; Ragadia latifasciata crystallina Monastyrskii Vu, subspec. nov.; Faunis indistincta luctus Monastyrskii Vu subspec. nov. Aemona gialaica Monastyrskii, K. Saito Vu, spec. nov. The taxon infuscata Devyatkin Monastyrskii, previously described as the subspecies Aemona tonkinensis infuscata, was elevated to the species level, while the taxon critias (Ragadia critias Riley Godfrey) was reduced to a subspecies. Three Satyrinae species were recorded from Vietnam for the first time: Palaeonympha opalina Butler, 1871; Ypthima motschulskyi Bremer Grey, 1853; and Ragadia latifasciata Leech, 1891.

When the average hides the risk of Bt-corn pollen on non-target Lepidoptera: Application to Aglais io in Catalonia.

Field cultivation of Genetically Modified (GM) Bt-plants has a potential environmental risk toward non-target Lepidoptera (NTLs) larvae through the consumption of Bt-maize pollen. The Bt-maize Cry protein targeting Lepidoptera species detrimental to the crop is also expressed in pollen which is dispersed by wind and can thus reach habitats of NTLs. To better assess the current ecological risk of Bt-maize at landscape scales, we developed a spatially-explicit exposure-hazard model considering (i) the dynamics of pollen dispersal obtained by convolving GM plants emission with a dispersal kernel and (ii) a toxicokinetic-toxicodynamic (TKTD) model accounting for the impact of toxin ingestion on individual lethal effects. We simulated the model using real landscape observations in Catalonia (Spain): GM-maize locations, flowering dates, rainfall time series and larvae emergence date of the European peacock butterfly Aglais io. While in average, the additional mortality appears to be negligible, we show significant additional mortality at sub-population level, with for instance a mortality higher than 40% within the 10m for the 10% most Bt-sensitive individuals. Also, using Pareto optimality we capture the best trade-off between isolation distance and additional mortality: up to 50 m are required to significantly buffer Bt-pollen impact on NTLs survival at the individual level. Our study clears up the narrow line between diverging conclusions: those claiming no risk by only looking at the average regional effect of Bt on NTLs survival and those pointing out a significant threaten when considering the variability of individuals mortality.

Pollen feeding in Heliconius butterflies: the singular evolution of an adaptive suite.

Major evolutionary transitions can be triggered by behavioural novelty, and are often associated with 'adaptive suites', which involve shifts in multiple co-adapted traits subject to complex interactions. Heliconius butterflies represent one such example, actively feeding on pollen, a behaviour unique among butterflies. Pollen feeding permits a prolonged reproductive lifespan, and co-occurs with a constellation of behavioural, neuroanatomical, life history, morphological and physiological traits that are absent in closely related, non-pollen-feeding genera. As a highly tractable system, supported by considerable ecological and genomic data, Heliconius are an excellent model for investigating how behavioural innovation can trigger a cascade of adaptive shifts in multiple diverse, but interrelated, traits. Here, we synthesize current knowledge of pollen feeding in Heliconius, and explore potential interactions between associated, putatively adaptive, traits. Currently, no physiological, morphological or molecular innovation has been explicitly linked to the origin of pollen feeding, and several hypothesized links between different aspects of Heliconius biology remain poorly tested. However, resolving these uncertainties will contribute to our understanding of how behavioural innovations evolve and subsequently alter the evolutionary trajectories of diverse traits impacting resource acquisition, life history, senescence and cognition.

Observation of morphological abnormalities in silkworm pupae after feeding 137CsCl-supplemented diet to evaluate the effects of low dose-rate exposure.

Since the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident, morphological abnormalities in lepidopteran insects, such as shrinkage and/or aberration of wings, have been reported. Butterflies experimentally exposed to radiocesium also show such abnormalities. However, because of a lack of data on absorbed dose and dose-effect relationship, it is unclear whether these abnormalities are caused directly by radiation. We conducted a low dose-rate exposure experiment in silkworms reared from egg to fully developed larvae on a 137CsCl-supplemented artificial diet and estimated the absorbed dose to evaluate morphological abnormalities in pupal wings. We used 137CsCl at 1.3 × 103 Bq/g fresh weight to simulate 137Cs contamination around the FDNPP. Absorbed doses were estimated using a glass rod dosimeter and Monte Carlo particle transport simulation code PHITS. Average external absorbed doses were approximately 0.24 (on diet) and 0.016 mGy/day (near diet); the average internal absorbed dose was approximately 0.82 mGy/day. Pupal wing structure is sensitive to radiation exposure. However, no significant differences were observed in the wing-to-whole body ratio of pupae between the 137CsCl-exposure and control groups. These results suggest that silkworms are insensitive to low dose-rate exposure due to chronic ingestion of high 137Cs at a high concentration.

Heliconiini butterflies can learn time-dependent reward associations.

For many pollinators, flowers provide predictable temporal schedules of resource availability, meaning an ability to learn time-dependent information could be widely beneficial. However, this ability has only been demonstrated in a handful of species. Observations of Heliconius butterflies suggest that they may have an ability to form time-dependent foraging preferences. Heliconius are unique among butterflies in actively collecting pollen, a dietary behaviour linked to spatio-temporally faithful 'trap-line' foraging. Time dependency of foraging preferences is hypothesized to allow Heliconius to exploit temporal predictability in alternative pollen resources. Here, we provide the first experimental evidence in support of this hypothesis, demonstrating that Heliconius hecale can learn opposing colour preferences in two time periods. This shift in preference is robust to the order of presentation, suggesting that preference is tied to the time of day and not due to ordinal or interval learning. However, this ability is not limited to Heliconius, as previously hypothesized, but also present in a related genus of non-pollen feeding butterflies. This demonstrates time learning likely pre-dates the origin of pollen feeding and may be prevalent across butterflies with less specialized foraging behaviours.

Characterization of Limonoids Isolated from the Fruits of Melia toosendan and Their Antifeedant Activity against Pieris rapae.

The fruits of Melia toosendan Sieb. et Zucc. (Meliaceae) are a source of bioactive limonoids that can be used as effective pesticides. In this study, two novel limonoids, 6-acetylsendanal and 6-ketocinamodiol, were isolated together with fourteen known compounds, namely four protolimonoids, six trichilin-class limonoids, and four C-seco limonoids. The structures of the new compounds were determined by extensive spectroscopic analyses (HR-ESI-MS, UV, IR, 1D and 2D NMR). The bioassay results revealed that eleven of the extracted limonoids exhibited interesting antifeedant activities against the larvae of Pieris rapae with AFC50 values in the range of 0.11-1.79 mm. Particularly, mesendanin H, with an AFC50 value of 0.11 mm, exhibited a higher activity than the positive control toosendanin. Information on new bioactive limonoids may provide further insight into M. toosendan as a source of bioactive components.