Capsid Proteins are Necessary for Replication of a Parvovirus

Despite tight genetic compression, viral genomes are often organized in functional gene clusters, a modular structure that might favor their evolvability. This has greatly facilitated biotechnological developments, such as the recombinant Adeno-Associated Virus (AAV) systems for gene therapy. Following this lead, we endeavored to engineer the related insect parvovirus Junonia coenia densovirus (JcDV) to create addressable vectors for insect pest biocontrol. To enable safer manipulation of capsid mutants, we translocated the non-structural ( ns ) gene cluster outside the viral genome. To our dismay, this yielded a virtually non-replicable clone. We linked the replication defect to an unexpected modularity breach, as ns translocation truncated the overlapping 3’ UTR of the capsid transcript ( vp ). We found that native vp 3’UTR is necessary to high VP production, but that decreased expression do not adversely impact the expression of NS proteins, which are known replication effectors. As nonsense vp mutations recapitulate the replication defect, VP proteins appear directly implicated in the replication process. Our findings suggest intricate replication-encapsidation couplings that favor maintenance of genetic integrity. We discuss possible connections with an intriguing cis-packaging phenomenon previously observed in parvoviruses, whereby capsids preferentially package the genome from which they were expressed. IMPORTANCE Densoviruses could be used as biological control agents to manage insect pests. Such applications require in depth biological understanding and associated molecular tools. However, the genomes of these viruses remain hard to manipulate due too poorly tractable secondary structures at their extremities. We devised a construction strategy that enable precise and efficient molecular modifications. Using this approach, we endeavored to create a split clone of the Junonia coenia densovirus (JcDV) that can be used to safely study the impact of capsid mutations on host specificity. Our original construct proved to be non-functional. Fixing this defect led us to uncover that capsid proteins and their correct expression are essential for continued rolling-hairpin replication. This points to an intriguing link between replication and packaging, which might be shared with related viruses. This serendipitous discovery illustrates the power of synthetic biology approaches to advance our knowledge of biological systems.

Classification of color features in butterflies using the Support Vector Machine (SVM)

Research in digital images is expanding widely and includes several sectors. One sector currently being carried out research is in insects; specifically, butterflies are used as a dataset. A total of 890 types of butterflies divided into ten classes were used as a dataset and classified based on color. Ten types of butterflies include Danaus plexippus, Heliconius charitonius, Heliconius erato, Junonia coenia, Lycaena phlaeas, Nymphalis antiopa, Papilio cresphontes, Pieris rapae, Vanessa atalanta, Vanessa cardui. The process of extracting color features on butterfly wings uses the RGB method to become HSV color space with color quantization (CQ). The purpose of adding CQ is that the computation process is carried out faster without reducing the image's information. In the color feature extraction process, the image is converted into 3-pixel sizes and normalized. The process of normalizing the dataset has the aim that the value ranges in the dataset have the same value. The 890 butterfly dataset was classified using the Support Vector Machine (SVM) method. Based on this research process, the accuracy of the 256x160 pixel size is 72%, the 420x315 pixel is 75%, and the 768x576 pixel is 75%. The test results on a system with a 768x576 pixel get the highest results with a precision value of 74.6%, a recall of 72%, and an f-measure of 73.2% Keywords—image processing; classification; butterflies; color features; features extraction

Ultrabithorax Is a Micromanager of Hindwing Identity in Butterflies and Moths

The forewings and hindwings of butterflies and moths (Lepidoptera) are differentiated from each other, with segment-specific morphologies and color patterns that mediate a wide range of functions in flight, signaling, and protection. The Hox gene Ultrabithorax (Ubx) is a master selector gene that differentiates metathoracic from mesothoracic identities across winged insects, and previous work has shown this role extends to at least some of the color patterns from the butterfly hindwing. Here we used CRISPR targeted mutagenesis to generate Ubx loss-of-function somatic mutations in two nymphalid butterflies (Junonia coenia, Vanessa cardui) and a pyralid moth (Plodia interpunctella). The resulting mosaic clones yielded hindwing-to-forewing transformations, showing Ubx is necessary for specifying many aspects of hindwing-specific identities, including scale morphologies, color patterns, and wing venation and structure. These homeotic phenotypes showed cell-autonomous, sharp transitions between mutant and non-mutant scales, except for clones that encroached into the border ocelli (eyespots) and resulted in composite and non-autonomous effects on eyespot ring determination. In the pyralid moth, homeotic clones converted the folding and depigmented hindwing into rigid and pigmented composites, affected the wing-coupling frenulum, and induced ectopic scent-scales in male androconia. These data confirm Ubx is a master selector of lepidopteran hindwing identity and suggest it acts on many gene regulatory networks involved in wing development and patterning.

Emperors, admirals and giants, zebras, tigers and woolly bears: casting a broader net in exploring heparin effects on Lepidoptera wing patterns

Background: Studies of heparin effects on Lepidoptera wing patterns have been restricted to a small number of species. I report observations from experiments on a broader range of taxa, including first results from swallowtails, tiger moths and microlepidoptera. Methods: Heparin injections were made in prepupae and pupae of Junonia coenia (common buckeyes), Agraulis vanillae (gulf fritillaries), Heliconius charithonia (zebra longwings), Asterocampa clyton (tawny emperors), Danaus plexippus (monarchs), Vanessa atalanta (red admirals); Heraclides cresphontes (giant swallowtails), Pterourus troilus (spicebush swallowtails), Protographium marcellus (zebra swallowtails), Battus polydamas (polydamas swallowtails); Hypercompe scribonia (giant leopard moths), Estigmene acrea (acrea moths), Hyphantria cunea (fall webworm moths), Utetheisa ornatrix (ornate bella moths); Glyphodes sibillalis (mulberry leaftier). Results: Heparin sometimes altered the entire pattern in a dramatic way, sometimes caused changes locally. In buckeyes, the previous heparin study conducted on pupae was compared to injections made at a prepupal stage. In gulf fritillaries, zebra longwings and tawny emperors, the dramatic changes occurred throughout their wings, while in monarchs, changes were restricted to wing margins. Changes achieved in red admirals, show that heparin action is unrelated to the original color. In swallowtails, transformations were restricted to border system, indicating higher levels of stability and compartmentalization of wing patterns. In mulberry leaftier, changes were restricted to the marginal bands. In tiger moths, elongation of black markings led to merging of spots; in the ornate bella moth, it was accompanied by an expansion of the surrounding white bands, and results were compared to the effects of colder temperatures. Conclusions: Using pharmaceutical intervention demonstrates that there are many similarities and some very significant differences in the ways wing patterns are formed in different Lepidoptera lineages. By creating a range of variation one can demonstrate how one pattern can easily evolve into another, aiding in understanding of speciation and adaptation processes.

Emperors, admirals and giants, zebras, tigers and woolly bears: casting a broader net in exploring heparin effects on Lepidoptera wing patterns

Background: Studies of heparin effects on Lepidoptera wing patterns have been restricted to a small number of species. I report observations from experiments on a broader range of taxa, including first results from swallowtails, tiger moths and microlepidoptera. Methods: Heparin injections were made in prepupae and pupae of Junonia coenia (common buckeyes), Agraulis vanillae (gulf fritillaries), Heliconius charithonia (zebra longwings), Asterocampa clyton (tawny emperors), Danaus plexippus (monarchs), Vanessa atalanta (red admirals); Heraclides cresphontes (giant swallowtails), Pterourus troilus (spicebush swallowtails), Protographium marcellus (zebra swallowtails), Battus polydamas (polydamas swallowtails); Hypercompe scribonia (giant leopard moths), Estigmene acrea (acrea moths), Hyphantria cunea (fall webworm moths), Utetheisa ornatrix (ornate bella moths); Glyphodes sibillalis (mulberry leaftier). Results: Heparin sometimes altered the entire pattern in a dramatic way, sometimes caused changes locally. In buckeyes, the previous heparin study conducted on pupae was compared to injections made at a prepupal stage. In gulf fritillaries, zebra longwings and tawny emperors, the dramatic changes occurred throughout their wings, while in monarchs, changes were restricted to wing margins. Changes achieved in red admirals, show that heparin action is unrelated to the original color. In swallowtails, transformations were restricted to border system, indicating higher levels of stability and compartmentalization of wing patterns. In mulberry leaftier, changes were restricted to the marginal bands. In tiger moths, elongation of black markings led to merging of spots; in the ornate bella moth, it was accompanied by an expansion of the surrounding white bands, and results were compared to the effects of colder temperatures. Conclusions: Using pharmaceutical intervention demonstrates that there are many similarities and some very significant differences in the ways wing patterns are formed in different Lepidoptera lineages. By creating a range of variation one can demonstrate how one pattern can easily evolve into another, aiding in understanding of speciation and adaptation processes.

Genomic architecture of a genetically assimilated seasonal color pattern

Developmental plasticity allows genomes to encode multiple distinct phenotypes that can be differentially manifested in response to environmental cues. Alternative plastic phenotypes can be selected through a process called genetic assimilation, although the mechanisms are still poorly understood. We assimilated a seasonal wing color phenotype in a naturally plastic population of butterflies (Junonia coenia) and characterized three responsible genes. Endocrine assays and chromatin accessibility and conformation analyses showed that the transition of wing coloration from an environmentally determined trait to a predominantly genetic trait occurred through selection for regulatory alleles of downstream wing-patterning genes. This mode of genetic evolution is likely favored by selection because it allows tissue- and trait-specific tuning of reaction norms without affecting core cue detection or transduction mechanisms.

The Effect of Phenoloxidase Activity on Survival Is Host Plant Dependent in Virus-Infected Caterpillars

An important goal of disease ecology is to understand trophic interactions influencing the host–pathogen relationship. This study focused on the effects of diet and immunity on the outcome of viral infection for the polyphagous butterfly, Vanessa cardui Linnaeus (Lepidoptera: Nymphalidae) (painted lady). Specifically, we aimed to understand the role that larval host plants play when fighting a viral pathogen. Larvae were orally inoculated with the entomopathogenic virus, Junonia coenia densovirus (JcDV) (Parvovirididae: Densovirinae, Lepidopteran Potoambidensovirus 1) and reared on two different host plants (Lupinus albifrons Bentham (Fabales: Fabaceae) or Plantago lanceolata Linnaeus (Lamiales: Plantaginaceae)). Following viral infection, the immune response (i.e., phenoloxidase [PO] activity), survival to adulthood, and viral load were measured for individuals on each host plant. We found that the interaction between the immune response and survival of the viral infection was host plant dependent. The likelihood of survival was lowest for infected larvae exhibiting suppressed PO activity and feeding on P. lanceolata, providing some evidence that PO activity may be an important defense against viral infection. However, for individuals reared on L. albifrons, the viral infection had a negligible effect on the immune response, and these individuals also had higher survival and lower viral load when infected with the pathogen compared to the controls. Therefore, we suggest that host plant modifies the effects of JcDV infection and influences caterpillars’ response when infected with the virus. Overall, we conclude that the outcome of viral infection is highly dependent upon diet, and that certain host plants can provide protection from pathogens regardless of immunity.

Capsid Proteins are Necessary for Replication of a Densovirus

Despite tight genetic compression, viral genomes are often organized in functional gene clusters, a modular structure that might favor their evolvability. This has greatly facilitated biotechnological developments, such as the recombinant Adeno-Associated Virus (AAV) systems for gene therapy. Following this lead, we endeavored to engineer the related insect parvovirus Junonia coenia densovirus (JcDV) to create addressable vectors for insect pest biocontrol. To enable safer manipulation of capsid mutants, we translocated the non-structural (ns) gene cluster outside the viral genome. To our dismay, this yielded a virtually non-replicable clone. We linked the replication defect to an unexpected modularity breach, as ns translocation truncated the overlapping 3’ UTR of the capsid transcript (vp). We found that native vp 3’UTR is necessary to high VP production, but that decreased expression do not adversely impact the expression of NS proteins, which are known replication effectors. As nonsense vp mutations recapitulate the replication defect, VP proteins appear directly implicated in the replication process. Our findings suggest intricate replication-encapsidation couplings that favor maintenance of genetic integrity. We discuss possible connections with an intriguing cis-packaging phenomenon previously observed in parvoviruses, whereby capsids preferentially package the genome from which they were expressed.ImportanceDensoviruses could be used as biological control agents to manage insect pests. Such applications require in depth biological understanding and associated molecular tools. However, the genomes of these viruses remain hard to manipulate due too poorly tractable secondary structures at their extremities. We devised a construction strategy that enable precise and efficient molecular modifications. Using this approach, we endeavored to create a split clone of the Junonia coenia densovirus (JcDV) that can be used to safely study the impact of capsid mutations on host specificity. Our original construct proved to be non-functional. Fixing this defect led us to uncover that capsid proteins and their correct expression are essential for viral replication. This points to an intriguing link between replication and packaging, which might be shared we related viruses. This serendipitous discovery illustrates the power of synthetic biology approaches to advance our knowledge of biological systems.