Identification of butterfly based on their shapes when viewed from different angles using an artificial neural network

Identification of butterfly species is essential because they are directly associated with crop plants used for human and animal consumption. However, the widely used reliable methods for butterfly identification are not efficient due to complicated butterfly shapes. We previously developed a novel shape recognition method that uses branch length similarity (BLS) entropy, which is a simple branching network consisting of a single node and branches. The method has been successfully applied to recognize battle tanks and characterize human faces with different emotions. In the present study, we used the BLS entropy profile (an assemble of BLS entropies) as an input feature in a feed-forward back-propagation artificial neural network to identify butterfly species according to their shapes when viewed from different angles (for vertically adjustable angle, θ = ± 10°, ± 20°, …, ± 60° and for horizontally adjustable angle, φ = ± 10°, ± 20°, …, ± 60°). In the field, butterfly images are generally captured obliquely by camera due to butterfly alignment and viewer positioning, which generates various shapes for a given specimen. To generate different shapes of a butterfly when viewed from different angles, we projected the shapes captured from top-view to a plane rotated through angles θ and φ. Projected shapes with differing θ and φ values were used as training data for the neural network and other shapes were used as test data. Experimental results showed that our method successfully identified various butterfly shapes. In addition, we briefly discuss extension of the method to identify more complicated images of different butterfly species.     

A novel approach to characterize branching network: Application to termite tunnel patterns

We defined a novel “branch length similarity” (BLS) entropy, S, on a simple network consisting of a single node and branches. This simple network is referred to as “unit branching network” (UBN) because UBNs are components of larger networks. As an application of BLS entropy, we considered the characterization of termite tunnel patterns because termite tunnel patterns can be broken down into a collection of simple units consisting of a single node and branches. These simple units correspond to UBNs. To this end, in additional to the entropy, we introduced the standard deviation (σ) of the difference in S between UBNs connected by a single tunnel branch. Forty simulated tunnel patterns were created for each of two termite species, Reticulitermes flavipes (Kollar) and Coptotermes formosanus Shiraki. These patterns were projected into <S>–σ phase space in order to assess their topological properties. This approach showed that for R. flavipes, their coordinates were relatively more clustered than those of C. formosanus. This result reflected that these two species were differently constrained by emergent property resulting from simple worker's tunneling behavior. We believe that the approach proposed in this study can be a useful tool to explore termite tunnel systems, but not limited to termite system.     

Identification of butterfly species with a single neural network system

Growing interest in conservation and biodiversity increased the demand for accurate and consistent identification of biological objects, such as insects, at the level of individual or species. Among the identification issues, butterfly identification at the species level has been strongly addressed because it is directly connected to the crop plants for human food and animal feed products. However, so far, the widely-used reliable methods were not suggested due to the complicated butterfly shape. In the present study, we propose a novel approach based on a back-propagation neural network to identify butterfly species. The neural network system was designed as a multi-class pattern classifier to identify seven different species. We used branch length similarity (BLS) entropies calculated from the boundary pixels of a butterfly shape as the input feature to the neural network. We verified the accuracy and efficiency of our method by comparing its performance to that of another single neural network system in which the binary values (0 or 1) of all pixels on an image shape are used as a feature vector. Experimental results showed that our method outperforms the binary image network in both accuracy and efficiency.     

Over-expression of Ultrabithorax alters embryonic body plan and wing patterns in the butterfly Bicyclus anynana

In insects, forewings and hindwings usually have different shapes, sizes, and color patterns. A variety of RNAi experiments across insect species have shown that the hox gene Ultrabithorax (Ubx) is necessary to promote hindwing identity. However, it remains unclear whether Ubx is sufficient to confer hindwing fate to forewings across insects. Here, we address this question by over-expressing Ubx in the butterfly Bicyclus anynana using a heat-shock promoter. Ubx whole-body over-expression during embryonic and larvae development led to body plan changes in larvae but to mere quantitative changes to adult morphology, respectively. Embryonic heat-shocks led to fused segments, loss of thoracic and abdominal limbs, and transformation of head limbs to larger appendages. Larval heat-shocks led to reduced eyespot size in the expected homeotic direction, but neither additional eyespots nor wing shape changes were observed in forewings as expected of a homeotic transformation. Interestingly, Ubx was found to be expressed in a novel, non-characteristic domain – in the hindwing eyespot centers. Furthermore, ectopic expression of Ubx on the pupal wing activated the eyespot-associated genes spalt and Distal-less, known to be directly repressed by Ubx in the fly?s haltere and leg primordia, respectively, and led to the differentiation of black wing scales. These results suggest that Ubx has been co-opted into a novel eyespot gene regulatory network, and that it is capable of activating black pigmentation in butterflies.     

Wnt signaling underlies evolution and development of the butterfly wing pattern symmetry systems

Most butterfly wing patterns are proposed to be derived from a set of conserved pattern elements known as symmetry systems. Symmetry systems are so-named because they are often associated with parallel color stripes mirrored around linear organizing centers that run between the anterior and posterior wing margins. Even though the symmetry systems are the most prominent and diverse wing pattern elements, their study has been confounded by a lack of knowledge regarding the molecular basis of their development, as well as the difficulty of drawing pattern homologies across species with highly derived wing patterns. Here we present the first molecular characterization of symmetry system development by showing that WntA expression is consistently associated with the major basal, discal, central, and external symmetry system patterns of nymphalid butterflies. Pharmacological manipulations of signaling gradients using heparin and dextran sulfate showed that pattern organizing centers correspond precisely with WntA, wingless, Wnt6, and Wnt10 expression patterns, thus suggesting a role for Wnt signaling in color pattern induction. Importantly, this model is supported by recent genetic and population genomic work identifying WntA as the causative locus underlying wing pattern variation within several butterfly species. By comparing the expression of WntA between nymphalid butterflies representing a range of prototypical symmetry systems, slightly deviated symmetry systems, and highly derived wing patterns, we were able to infer symmetry system homologies in several challenging cases. Our work illustrates how highly divergent morphologies can be derived from modifications to a common ground plan across both micro- and macro-evolutionary time scales.     

Taxonomy of Mechanitis (F.) (Lepidoptera: Nymphalidae) from the West Colombian Andes: an Integrative Approach

Species identification in the butterfly genus Mechanitis (F.) (Lepidoptera: Nymphalidae) becomes difficult when it is based only on wing color patterns, a common practice in butterfly taxonomy. Difficulties in Mechanitis taxonomy are related to the widespread mimicry and polymorphism among species belonging to this genus. Species recognition and inventories of Mechanitis genus in geographic areas as the Andean region of Colombia are of particular interest and the use of more than one character for taxonomic identification is desirable. In this study, we included morphological, ecological, and mitochondrial DNA data to identify the occurring species in this region. Species of Mechanitis were studied from ecological, morphological, and molecular perspectives considering host plant identification, oviposition behavior, and life cycles under laboratory conditions. Immature morphology, patterns of wing color, and genital structures of adults were also studied. The genetic barcoding region of the cytochrome oxidase I mitochondrial gene was sequenced and used to verify the limits between species previously defined by the other characters and to validate its usefulness for species delimitation in this particular genus. The integrative approach combining independent datasets successfully allowed species identification as compared to the approach based on a single dataset. Three well-differentiated species were found in the studied region, Mechanitis menapis (Hewitson), Mechanitis polymnia (Linnaeus), and Mechanitis lysimnia (Fabricius). New valuable characters that could improve taxonomic identification in this genus are considered.     

Differential Involvement of Hedgehog Signaling in Butterfly Wing and Eyespot Development

Butterfly eyespots may have evolved from the recruitment of pre-existent gene circuits or regulatory networks into novel locations on the wing. Gene expression data suggests one such circuit, the Hedgehog (Hh) signaling pathway and its target gene engrailed (en), was recruited from a role in patterning the anterior-posterior insect wing axis to a role patterning butterfly eyespots. However, while Junonia coenia expresses hh and en both in the posterior compartment of the wing and in eyespot centers, Bicyclus anynana lacks hh eyespot-specific expression. This suggests that Hh signaling may not be functioning in eyespot development in either species or that it functions in J. coenia but not in B. anynana. In order to test these hypotheses, we performed functional tests of Hh signaling in these species. We investigated the effects of Hh protein sequestration during the larval stage on en expression levels, and on wing size and eyespot size in adults. Hh sequestration led to significantly reduced en expression and to significantly smaller wings and eyespots in both species. But while eyespot size in B. anynana was reduced proportionately to wing size, in J. coenia, eyespots were reduced disproportionately, indicating an independent role of Hh signaling in eyespot development in J. coenia. We conclude that while Hh signaling retains a conserved role in promoting wing growth across nymphalid butterflies, it plays an additional role in eyespot development in some, but not all, lineages of nymphalid butterflies. We discuss our findings in the context of alternative evolutionary scenarios that led to the differential expression of hh and other Hh pathway signaling members across nymphalid species.     

Phenotypic plasticity in the range-margin population of the lycaenid butterfly <Emphasis Type="Italic">Zizeeria maha</Emphasis>

Background  

Many butterfly species have been experiencing the northward range expansion and physiological adaptation, probably due to climate warming. Here, we document an extraordinary field case of a species of lycaenid butterfly, Zizeeria maha, for which plastic phenotypes of wing color-patterns were revealed at the population level in the course of range expansion. Furthermore, we examined whether this outbreak of phenotypic changes was able to be reproduced in a laboratory.     

The population biology and ecology of the Homerus swallowtail, <Emphasis Type="Italic">Papilio (Pterourus) homerus</Emphasis>, in the Cockpit Country,Jamaica

The Homerus swallowtail, Papilio (Pterourus) homerus (Lepidoptera: Papilionidae), is an endangered species of butterfly endemic to Jamaica. As the largest species of the genus Papilio in the world and the largest butterfly in the Western Hemisphere, this rare butterfly once inhabited most of Jamaica but has now dwindled into two tiny populations: an eastern population, found where the Blue Mountains and John Crow Mountains merge, and a western population in the Cockpit Country. The present research focused on the previously unstudied Cockpit Country population of P. homerus; most previous information about this species is derived from studies of the eastern population. The purpose was to estimate the size of the remaining population in the Cockpit Country using MRR protocols, while making observations to better understand its ecology. Sampling consisted of carefully netting the butterfly, marking a permanent ink number on the wing (metallic Sharpie® marker), and recording winglength, wing condition, time, and sex. The population was found to be very small, estimated at fewer than 50 flying individuals. Many observations were made about the ecology of the species. These new data suggest a conservation plan is strongly needed, coupled with a breeding program to increase numbers of this extraordinary butterfly.     

Spatial patterns of correlated scale size and scale color in relation to color pattern elements in butterfly wings

Complex butterfly wing color patterns are coordinated throughout a wing by unknown mechanisms that provide undifferentiated immature scale cells with positional information for scale color. Because there is a reasonable level of correspondence between the color pattern element and scale size at least in Junonia orithya and Junonia oenone, a single morphogenic signal may contain positional information for both color and size. However, this color–size relationship has not been demonstrated in other species of the family Nymphalidae. Here, we investigated the distribution patterns of scale size in relation to color pattern elements on the hindwings of the peacock pansy butterfly Junonia almana, together with other nymphalid butterflies, Vanessa indica and Danaus chrysippus. In these species, we observed a general decrease in scale size from the basal to the distal areas, although the size gradient was small in D. chrysippus. Scales of dark color in color pattern elements, including eyespot black rings, parafocal elements, and submarginal bands, were larger than those of their surroundings. Within an eyespot, the largest scales were found at the focal white area, although there were exceptional cases. Similarly, ectopic eyespots that were induced by physical damage on the J. almana background area had larger scales than in the surrounding area. These results are consistent with the previous finding that scale color and size coordinate to form color pattern elements. We propose a ploidy hypothesis to explain the color–size relationship in which the putative morphogenic signal induces the polyploidization (genome amplification) of immature scale cells and that the degrees of ploidy (gene dosage) determine scale color and scale size simultaneously in butterfly wings.     

Sexual dimorphism and intraspecific variation in wing size and shape of Tongeia fischeri (Lepidoptera: Lycaenidae)

Wing morphological variations are described here for the lycaenid butterfly Tongeia fischeri. A landmark‐based geometric morphometric approach based on wing venation of 197 male and 187 female butterflies collected in Japan was used to quantify wing size and shape variations between sexes and among populations. Sexual dimorphism in wing size and shape was detected. Females had significantly larger wings than males, while males showed a relatively elongated forewing with a longer apex and narrower wing tornus in comparison to females. Intraspecific variations in wing morphology among populations were revealed for the wing shape, but not wing size. Distinct wing shape differences were found in the vein intersections area around the distal part of the discal cell where median veins originated in the forewing and around the origin of the CU1 vein in the hindwing. In addition, phenotypic relationships inferred from wing shape variations grouped T. fischeri populations into three groups, reflecting the subspecies classification of the species. The spatial variability and phenotypic relationships between conspecific populations of T. fischeri detected here are generally in agreement with the previous molecular study based on mitochondrial and nuclear sequences, suggesting the presence of a phylogenetic signal in the wing shape of T. fischeri, and thus having taxonomic implications.     

Evolutionary pattern of the forewing shape in the Neotropical genus of jumping plant-lice (Hemiptera: Psylloidea: <Emphasis Type="Italic">Russelliana</Emphasis>)

Geometric morphometric and phylogenetic analyses, applied to 43 species of Russelliana, shed light on the evolution of insect wing shape. Unconstrained and constrained ordination techniques are introduced to detect patterns of the forewing shape variation within genus. Results show a high congruence between forewing shape variation and host-plant preference supporting monophyly of most phylogenetic groups in Russelliana. Reconstruction of the ancestral forewing state shows its similarity to a forewing shape of Solanaceae feeding species defined as ancestors by the phylogenetic study supporting a hypothesis as to a primary association of Russelliana with Solanaceae. In contrast to some other comparative studies on insect wing shape, results of the present study reveal a strong correlation between variation of forewing shape in Russelliana and its phylogeny. Potential influence of vicariant events and host shifts on the evolution of forewing shape is discussed.     

Unique wing scale photonics of male Rajah Brooke’s birdwing butterflies

Background

Ultrastructures in butterfly wing scales can take many shapes, resulting in the often striking coloration of many butterflies due to interference of light. The plethora of coloration mechanisms is dazzling, but often only single mechanisms are described for specific animals.

Results

We have here investigated the male Rajah Brooke’s birdwing, Trogonoptera brookiana, a large butterfly from Malaysia, which is marked by striking, colorful wing patterns. The dorsal side is decorated with large, iridescent green patterning, while the ventral side of the wings is primarily brown-black with small white, blue and green patches on the hindwings. Dense arrays of red hairs, creating a distinct collar as well as contrasting areas ventrally around the thorax, enhance the butterfly’s beauty. The remarkable coloration is realized by a diverse number of intricate and complicated nanostructures in the hairs as well as the wing scales. The red collar hairs contain a broad-band absorbing pigment as well as UV-reflecting multilayers resembling the photonic structures of Morpho butterflies; the white wing patches consist of scales with prominent thin film reflectors; the blue patches have scales with ridge multilayers and these scales also have centrally concentrated melanin. The green wing areas consist of strongly curved scales, which possess a uniquely arranged photonic structure consisting of multilayers and melanin baffles that produces highly directional reflections.

Conclusion

Rajah Brooke’s birdwing employs a variety of structural and pigmentary coloration mechanisms to achieve its stunning optical appearance. The intriguing usage of order and disorder in related photonic structures in the butterfly wing scales may inspire novel optical materials as well as investigations into the development of these nanostructures in vivo.

     

Mutants highlight the modular control of butterfly eyespot patterns

SUMMARY The eyespots on butterfly wings are thought to be serially homologous pattern elements. Yet eyespots differ greatly in number, shape, color, and size, within and among species. To what extent do these serially homologues have separate developmental identities, upon which selection acts to create diversity? We examined x‐ray–induced mutations for the eyespots of the nymphalid butterfly Bicyclus anynana that highlight the modular control of these serially homologous wing pattern elements. These mutations reduce or eliminate individual eyespots, or groups of eyespots, with no further effect on the wing color pattern. The collection of mutants highlights a greater potential developmental repertoire than that observed across the genus Bicyclus. We studied in detail one such mutation, of codominant effect, that causes the elimination of two adjacent eyespots on the ventral hindwing. By analyzing the expression of genes known to be involved in eyespot formation, we found an alteration in the differentiation of the “organizing” cells at the eyespot's center. No such cells differentiate in the wing subdivisions lacking the two eyespots in the mutants. We propose several developmental models, based on wing compartmentalization in Drosophila, that provide the first framework for thinking about the molecular evolution of butterfly wing pattern modularity.     

Population demography of alpine butterflies: Boloria pales and Boloria napaea (Lepidoptera: Nymphalidae) and their specific adaptations to high mountain environments

High mountain ecosystems are extreme habitats, and adaptation strategies to this ecosystem are still poorly understood in most groups. To unravel such strategies, we performed a MRR study in the Hohe Tauern National Park (Salzburg, Austria) with two nymphalid butterfly species, Boloria pales and B. napaea. We analysed their population structure over one flight period by studying the development of population size and wing wear. B. pales had more individuals and a higher survival probability than B. napaea; the sensitivity to extreme weather conditions or other external influences was higher in B. napaea. We only observed proterandry in B. pales. Imagines of both species survived under snow for at least some days. Additionally, we observed a kind of risk-spreading, in that individuals of both species, and especially B. pales, have regularly emerged throughout the flight period. This emergence pattern divided the population's age structure into three phases: an initial phase with decreasing wing quality (emergence > mortality), followed by an equilibrium phase with mostly constant average wing condition (emergence = mortality) and a final ageing phase with strongly deteriorating wing condition (mortality » emergence). Consequently, neither species would likely become extinct because of particularly unsuitable weather conditions during a single flight period. The observed differences between the two species suggest a better regional adaptation of B. pales, which is restricted to high mountain systems of Europe. In contrast, the arctic-alpine B. napaea might be best adapted to conditions in the Arctic and not the more southern high mountain systems. However, this needs to be examined during future research in the Arctic.     

REACTION NORMS OF MORPHOLOGICAL TRAITS IN DROSOPHILA: ADAPTIVE SHAPE CHANGES IN A STENOTHERM CIRCUMTROPICAL SPECIES?

Reaction norms of wing length, thorax length, and ovariole number were studied according to growth temperature in the circumtropical Drosophila ananassae, and compared to similar data from the cosmopolitan D. melanogaster. In the two species convex reaction norms were observed, but they were not parallel and sometimes exhibited intersections either at high (wing) or at low (thorax) temperature. On average, D. ananassae may be considered as a species with a bigger thorax but shorter wings than D. melanogaster. The shapes of reaction norms were analyzed and compared after quadratic polynomial adjustments. Significant differences were observed, in several cases between polynomial parameters, and in all cases between characteristic points that is, Maximum Value (MV) and Temperature of Maximum Value (TMV). The wing/thorax ratio may also be considered as a specific trait related to wing loading. Major differences were observed between the two species for the mean value and the shape of the response curves of this trait. The main observation of this work was however a shift of TMVs for wing and thorax length and ovariole number in D. ananassae toward higher temperatures. These variations in the reaction norms corresponded to a shift in the species thermal range, suggesting that temperature adaptation was accompanied by a modification of the shape of the response curves.     

Morphological integration and modularity in Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) hind wings

The morphological integration of the hind wings of the western corn rootworm Diabrotica virgifera virgifera LeConte was investigated to get a better insight of the undergone by this invasive species. Geometric morphometric methods were used to test two modularity hypotheses associated with the wing development and function (hypothesis H1: anterior/posterior or H2: distal/proximal wing parts). Both hypotheses were rejected and the results showed the integrated behavior of the hind wings of D. v. virgifera. The hypothesized modules do not represent separate units of variation, so in a similar fashion as exhibited by the model species Drosophila melanogaster, the hind wings of D. v. virgifera act as a single functional unit. The moderate covariation strength found between anterior and posterior and distal and proximal parts of the hind wing of D. v. virgifera confirms its integrated behavior. We conclude that the wing shape shows internal integration, which could enable flexibility and thus enhance flight maneuverability. This study contributes to the understanding of morphological integration and modularity on a non-model organism. Additionally, these findings lay the groundwork for future flight performance and biogeographical studies on how wing shape and size vary across the endemic and expanded/invaded range in the USA and Europe infested with D. v. virgifera.     

Time-Varying Wing-Twist Improves Aerodynamic Efficiency of Forward Flight in Butterflies

Insect wings can undergo significant chordwise (camber) as well as spanwise (twist) deformation during flapping flight but the effect of these deformations is not well understood. The shape and size of butterfly wings leads to particularly large wing deformations, making them an ideal test case for investigation of these effects. Here we use computational models derived from experiments on free-flying butterflies to understand the effect of time-varying twist and camber on the aerodynamic performance of these insects. High-speed videogrammetry is used to capture the wing kinematics, including deformation, of a Painted Lady butterfly (Vanessa cardui) in untethered, forward flight. These experimental results are then analyzed computationally using a high-fidelity, three-dimensional, unsteady Navier-Stokes flow solver. For comparison to this case, a set of non-deforming, flat-plate wing (FPW) models of wing motion are synthesized and subjected to the same analysis along with a wing model that matches the time-varying wing-twist observed for the butterfly, but has no deformation in camber. The simulations show that the observed butterfly wing (OBW) outperforms all the flat-plate wings in terms of usable force production as well as the ratio of lift to power by at least 29% and 46%, respectively. This increase in efficiency of lift production is at least three-fold greater than reported for other insects. Interestingly, we also find that the twist-only-wing (TOW) model recovers much of the performance of the OBW, demonstrating that wing-twist, and not camber is key to forward flight in these insects. The implications of this on the design of flapping wing micro-aerial vehicles are discussed.     

Weighted relative entropy for phylogenetic tree based on 2-step Markov Model

The degree of similarity of DNA sequences can be concluded according to the comparison of DNA sequences, which helps to speculate their relationship in respect of the structure, function and evolution. In this paper, we introduce the fundamental of the weighted relative entropy based on 2-step Markov Model to compare DNA sequences. The DNA sequence, consisted of four characters A, T, C, G, can be considered as a Markov chain. By taking state space I = {A, T, C, G} and describe the DNA sequences with 2-step transition probability matrix we can get the eigenvalue of the DNA sequence to define the similarity metric. Therefore, we find a new method to compare the DNA sequences, which is used to classify chromosomes DNA sequences obtained from 30 species. The phylogenetic tree built by the alignment-free method of the distance matrix resulted from the weighted relative entropy has clearer and more accurate division.