Pieris Species Research Articles

Oviposition strategies in Pieridae butterflies and the role of an egg-killing plant trait therein.

Most herbivorous insects are host-plant specialists that evolved detoxification mechanisms to overcome their host plant's toxins. In the evolutionary arms-races between Pieridae butterflies and Brassicaceae plants, some plant species have evolved another defence against the pierids: egg-killing. Underneath the eggs, leaves develop a so-called hypersensitive response (HR)-like cell death. Whether some butterflies have evolved oviposition strategies to counter-adapt against egg-killing remains to be studied. In this study, we assessed the oviposition site location of Pieridae butterflies on their natural host plants. We described the plant tissue on which we located the eggs of the most common Pieridae in the Netherlands: Gonepteryx rhamni, Anthocharis cardamines, Pieris rapae, P. napi, P. brassicae and P. mannii. Additionally, we assessed expression of HR-like cell death in response to the deposited butterfly eggs. We found that both A. cardamines and G. rhamni mainly oviposited on the floral stem and the branch, respectively, and oviposited on host plants from lineages not expected to kill pierid eggs. Accordingly, no HR-like cell death was seen. All Pieris eggs found were located on leaves of their host, the only tissue found to express HR-like cell death. Furthermore, each Pieris species was found to at least occasionally oviposit on Brassica nigra. This was the only plant species in this survey that expressed HR-like cell death in response to the eggs of P. rapae, P. napi and P. brassicae. Our observations demonstrate that HR-like cell death remains an effective defence strategy against these Pieris species and as such did not find evidence for the hypothesized counterstrategies. Surveying certain key species and disentangling the micro-evolution of oviposition strategies within a species would allow us to further investigate potential counter-adaptations that evolved against HR-like cell death. This study provides the basis for further investigation of potential counter-adaptations to egg-killing defences.

Molecular identification and genetic diversity of ten Pierid butterflies based on mitochondrial COI gene

The IUCN Bangladesh has documented a total of 31 species belonging to the Pieridae family in Bangladesh. There is currently limited knowledge regarding the genetic diversity and molecular relationships among these butterflies. The present investigation involved the generation of mitochondrial COI (mtCOI) gene sequences for ten species of butterflies belonging to the Pierid family. After that, BLAST analysis showed 96% to 100% similarity, and then the sequences were submitted to NCBI’s GenBank. MEGA10 and BioEdit were used to identify a 584 bp COI gene fragment with 179 variable sites and 128 parsimony-informative sites. The COI sequences' average base compositions were 38.56% T, 17.12% C, 30.18% A, and 14.12% G. While a significant AT bias (68.74%) existed among the Pierid species. The genetic distance between ten Pierid species was assessed using the Kimura 2-Parameter (K2P) algorithm, and the nucleotide differences ranged from 0.00172% to 0.24261%. Afterwards, the Maximum Likelihood (ML) method was used to construct a phylogenetic tree using ten sequences of Pierid species. These species belongs to two subfamilies, Pierinae and Coliadinae in Bangladesh. On the tree, the Pierinae subfamily of Pieridae formed a paraphyletic arrangement whereas the Coliadinae subfamily was shown to be monophyletic. The data analyses support the following relationships between the two subfamilies as follows: [(Appias + Leptosia) + (Pieris + Appias) + (Delias) + (Eurema + Catopsilia) + (Pareronia)]. This supported the theory that Pierinae and Coliadinae are sister taxa. Nonetheless, Pareronia hippia remains outside the main Pierinae group, requiring further study to resolve this issue. Finally, these studies generated ten mtCOI gene sequences that have the potential to serve as valuable references for the accurate identification of Pierid species. In addition, this result could be used in the future to reveal the subfamily relationships within the Pieridae taxonomic classification. Bangladesh J. Zool. 51(3): 289-299, 2023

Bumblebees and butterflies in green structure elements in Malmö, Sweden

Within the BiodiverCity project in Malmö (Sweden), green structure elements were implemented aiming to support biodiversity and enhancing amenity in denser parts of the city. In this study, bumblebee and butterfly abundances and species richness were studied in 20 green structure elements in three areas in Malmö. The investigated green structure elements consisted of objects established within the BiodiverCity project and other green structure elements in their surroundings (e.g. road verges, lawns and flowerbeds). Observed bumblebee and butterfly abundances and species richness was generally low. In total, 528 bumblebees of eight species were recorded, with 97% of all bumblebees belonging to two species, Bombus lapidarius and B. terrestris/lucorum. A total of 154 butterflies from 10 species were detected, most commonly Pieris species. There were no significant differences in mean individual or species numbers (bumblebees and butterflies) between green structure elements designed to support biodiversity and conventional ones. Bumblebee species richness and abundance were positively correlated with mean cover of flowering vegetation and mean number of flowering plant species. Butterfly species richness and abundance were positively correlated with mean number of flowering plant species and size of the green structure. Observations of flower visits showed that the two groups of insects had different preferences. The results of this study show that enhancing bumblebee and butterfly diversity and abundances in densely built city areas can be challenging. To support bumblebees and butterflies more successfully, the habitat requirements of these insect groups need to be better considered from the beginning in the design of green structure elements.

Chromosome-Level Assemblies of the Pieris mannii Butterfly Genome Suggest Z-Origin and Rapid Evolution of the W Chromosome.

The insect order Lepidoptera (butterflies and moths) represents the largest group of organisms with ZW/ZZ sex determination. While the origin of the Z chromosome predates the evolution of the Lepidoptera, the W chromosomes are considered younger, but their origin is debated. To shed light on the origin of the lepidopteran W, we here produce chromosome-level genome assemblies for the butterfly Pieris mannii and compare the sex chromosomes within and between P. mannii and its sister species Pieris rapae. Our analyses clearly indicate a common origin of the W chromosomes of the two Pieris species and reveal similarity between the Z and W in chromosome sequence and structure. This supports the view that the W in these species originates from Z-autosome fusion rather than from a redundant B chromosome. We further demonstrate the extremely rapid evolution of the W relative to the other chromosomes and argue that this may preclude reliable conclusions about the origins of W chromosomes based on comparisons among distantly related Lepidoptera. Finally, we find that sequence similarity between the Z and W chromosomes is greatest toward the chromosome ends, perhaps reflecting selection for the maintenance of recognition sites essential to chromosome segregation. Our study highlights the utility of long-read sequencing technology for illuminating chromosome evolution.

Hypersensitive-like response in Brassica plants is specifically induced by molecules from egg-associated secretions of cabbage white butterflies

Plants perceive and respond to herbivore insect eggs. Upon egg deposition on leaves, a strong hypersensitive response (HR)-like cell death can be activated leading to egg desiccation and/or dropping. In Brassica spp., including many crops, the HR-like mechanism against eggs of cabbage white butterflies (Pieris spp.) is poorly understood. Using two Brassica species, the crop B. rapa and its wild relative B. nigra, we studied the cellular and molecular plant response to Pieris brassicae eggs and characterized potential insect egg-associated molecular patterns (EAMPs) inducing HR-like cell death. We found that eggs of P. brassicae induced typical hallmarks of early immune responses, such as callose deposition, production of reactive oxygen species and cell death in B. nigra and B. rapa leaf tissue, also in plants that did not express HR-like cell death. However, elevated levels of ethylene production and upregulation of salicylic acid-responsive genes were only detected in a B. nigra accession expressing HR-like cell death. Eggs and egg wash from P. brassicae contains compounds that induced such responses, but the eggs of the generalist moth Mamestra brassicae did not. Furthermore, wash made from hatched Pieris eggs, egg glue, and accessory reproductive glands (ARG) that produce this glue, induced HR-like cell death, whereas washes from unfertilized eggs dissected from the ovaries or removal of the glue from eggs resulted in no or a reduced response. This suggests that there is one or multiple egg associated molecular pattern (EAMP) located in the egg glue a that teresponse in B. nigra is specific to Pieris species. Lastly, our results indicate that the EAMP is neither lipidic nor proteinaceous. Our study expands the knowledge on the mechanism of Brassica-Pieris-egg interaction and is a step closer toward identification of EAMPs in Pieris egg glue and corresponding receptor(s) in Brassica.

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.

Plant poisoning in goats

As herbivores goats are at risk of plant poisoning. Poisoning may occur if goats are accidentally fed plant cuttings or through contamination of feed, but as goats are agile and inquisitive they can escape from their enclosure and encounter poisonous plants in gardens or woodland. They may also eat plants they would normally avoid if other forage is unavailable. Poisoning with plants containing grayanotoxins (Pieris and Rhododendron species) or cyanogenic glycosides (e.g cherry laurel, Prunus laurocerasus) is very common in goats. Other plants that have caused poisoning in goats include yew (Taxus species), oak (Quercus species), hemlock (Conium maculatum), leylandii (× Hesperotropsis leylandii), box (Buxus sempervirens) and plants containing cardiac glycosides such as oleander (Nerium oleander) or pyrrolizidine alkaloids such as ragwort (Jacobaea vulgaris). Sudden death is frequently the first sign of plant toxicosis in livestock. Management of plant poisoning in goats is supportive including removal from exposure, and providing analgesia, rehydration and potentially a rumenotomy for plant exposure in valuable animals.

Spring poisoning hazards for pets

Warmer weather in the spring may encourage owners and their animals to spend more time outdoors where pets may be exposed to garden products and spring plants. Ingestion of common spring flowering plants, such as tulips, primrose, hyacinth, snowdrops, grape hyacinth, spring crocus, bluebells and daffodil, often cause gastrointestinal signs in pets. Severe poisoning is uncommon but there is a risk of more pronounced clinical signs in some cases and ingestion of plant bulbs may cause gastrointestinal obstruction. Similarly, ingestion of fertiliser may cause gastrointestinal signs and ingestion of bone meal is a potential obstruction risk. Plants containing grayanotoxins, such as Rhododendron and Pieris species cause gastrointestinal and potentially cardiac effects. Patio cleaners and disinfectants containing cationic detergents such as benzalkonium chloride or didecyldimethylammonium chloride, are a particular risk to pets, especially cats. They are usually exposed after walking over a treated surface and then grooming, presenting hours later with salivation, hyperthermia and oral injury. Treatment of pets with clinical signs after exposure to a spring poisoning hazard is supportive.

Impact of Artificial Diet on Development and Coloration in Zerene Cesonia

The development of artificial diets for Lepidopteran species is instrumental for the study of natural biological variation in controlled laboratory conditions. In this study, we test the ability to rear Zerene cesonia, a pierid species, on artificial diets containing varying amounts of two plant species: Dalea purpurea and Trifolium pratense. We evaluate the quality of each diet based on survivorship, developmental timing, and analyses of larval and adult coloration. This study concludes that diets that include the preferred host plant, D. purpurea, are best for rearing Z. cesonia based on survivorship and the ability to recapitulate the natural color variation observed in the wild.

Spring poisoning hazards

Poisoning in the spring may occur in livestock from exposure to glyphosate which is used prior to sowing of plant crops or from ingestion of poisonous plants. Glyphosate is of low toxicity but many products contain a carrier which is irritant to tissues. Plant poisoning may occur because other forage is unavailable and hungry animals will eat unpalatable toxic plants if other food sources are scare. Some plants such as bluebell (Hyacinoides species) and ransom (wild garlic, Allium urinsum) grow in profusion in the spring. Bluebells cause gastrointestinal and cardiac effects and Allium species cause anaemia. Some plants are more toxic in the spring as concentrations of toxic compounds are high compared with other times of the year. This is the case with hemlock (Conium maculatum) and water hemlock (Cicuta virosa). Both these plants cause neurological effects and water hemlock, in particular, causes very rapid onset of clinical signs. Spring flowering plants such as Rhododendron and Pieris species are commonly associated with poisoning, particularly in ruminants. Both these species contain grayanotoxins which cause gastrointestinal and cardiac effects. Access to areas where poisonous plants are known to grow should be restricted and good quality forage provided. Treatment options for management of poisoning in livestock are limited and, in some cases, the only sign of exposure is sudden death.

Cross infection of Pieris brassicae granulosis virus on other siblings of Pieris species

Pieris brassicae granulosis virus ( PibrGV ) has been found to infect yet another two siblings namely P. canidia Linn. & Pontia daplidice (= Pieris daplidice ) (Roeber) in addition to P. brassicae (Linn.). The susceptibility of P. canidia and P. daplidice has been established clearly in this work by inoculating them with PibrGV @ 5x10 4 occlusion bodies (OBs) per ml by the conventional leaf disc method. All the inoculated larvae exhibited typical symptoms of viral infection after 5-7 days of post infection. The above three species of the genus Pieris occur on cabbage and cauliflower plants at different periods in the valley region of Imphal and therefore, it is possible to control all the three species of Pieris using the same virus. LC 50 value of PibrGV on P. brassicae , P. canidia and P. daplidice was found to be 7.9x10 4 ; 10x10 6 ; and 6.3x10 6 OBs/ml and the LT 50 was 134.89; 138.03; 174.50 hours, respectively.

Molecular signatures of selection associated with host plant differences in Pieris butterflies.

Adaptive traits that enable organisms to conquer novel niches and experience subsequent diversification are ecologically and evolutionarily important. The larvae of Pieris butterflies express nitrile-specifier proteins (NSPs), a key innovation for overcoming the glucosinolate (GLS)-myrosinase-based defence system of their Brassicales host plants. Nitrile-specifier proteins are a member of the NSP-like gene family, which includes the major allergen (MA) protein, a paralog of NSP with a GLS-disarming function, and a single domain major allergen (SDMA) protein, whose function is unknown. The arms-race between GLS-based defences and the NSP-like gene family is suggested to mediate diversification in both Pierid butterflies and Brassicales plants. Here, we tested whether the expected strong selection on NSP-like gene family correlates with shifts in host plant spectra among Pierid butterflies. We combined feeding experiments using 25 Brassicaceae plants and five Pieris species with larval transcriptome data to investigate the patterns of selection acting on NSP-like gene family members. Although we observed significantly elevated nonsynonymous to synonymous substitution rate ratios in NSPs on branches associated with changes in patterns of host plant usage, no such pattern was observed in MAs or SDMAs. Furthermore, we found evidence for positive selection of NSP at a phylogenetic branch which reflects different host plant spectra. Our data indicate that the NSP-related gene members have evolved differently: NSPs have accumulated more amino acid changes in response to shifting preferences for host plants, whereas MAs and SDMAs appear to be more conserved. Further detailed functional assays of these genes would provide important insights to understand their role in the chemical arms-race between Pieris butterflies and their Brassicales host plants.

Interspecific Differences in the Larval Performance of Pieris Butterflies (Lepidoptera: Pieridae) Are Associated with Differences in the Glucosinolate Profiles of Host Plants.

The tremendous diversity of plants and herbivores has arisen from a coevolutionary relationship characterized by plant defense and herbivore counter adaptation. Pierid butterfly species feed on Brassicales plants that produce glucosinolates as a chemical deterrent against herbivory. In turn, the larvae of pierids have nitrile specifier proteins (NSPs) that are expressed in their gut and disarm glucosinolates. Pierid butterflies are known to have diversified in response to glucosinolate diversification in Brassicales. Therefore, each pierid species is expected to have a spectrum of host plants characterized by specific glucosinolate profiles. In this study, we tested whether the larval performance of different Pieris species, a genus in Pieridae (Lepidoptera: Pieridae), was associated with plant defense traits of putative host plants. We conducted feeding assays using larvae of three Pieris species and 10 species of the Brassicaceae family possessing different leaf physical traits and glucosinolate profile measurements. The larvae of Pieris rapae responded differently in the feeding assays compared with the other two Pieris species. This difference was associated with differences in glucosinolate profiles but not with variations in physical traits of the host plants. This result suggests that individual Pieris species are adapted to a subset of glucosinolate profiles within the Brassicaceae. Our results support the idea that the host ranges of Pieris species depend on larval responses to glucosinolate diversification in the host species, supporting the hypothesis of coevolution between butterflies and host plants mediated by the chemical arms race.

Host preference variation cannot explain microhabitat differentiation among sympatric Pieris napi and Pieris rapae butterflies

1. Often, closely related insect species feed on different host plant species, and the tremendous diversity of phytophagous insects is therefore attributed to host plant‐driven speciation. However, for most taxa, host use information comes from field observations of egg‐laying females or feeding caterpillars, which means that the underlying reason for a particular host‐affiliation is not easily determined.2. Therefore, it is often unclear whether an insect feeds on a certain host because it prefers that plant to alternative hosts, or because the host distribution overlaps with the habitat requirements of the insect.3. We ask to what extent a divergent host use in the field mirrors the host plant preferences of two closely related butterflies, Pieris napi and Pieris rapae (Pieridae). In nature, P. napi typically occurs in moister habitats than P. rapae.4. We scanned several microhabitats at a field site in Southern Sweden during multiple years, and collected Pieris eggs from three different plants, Cardamine pratensis (wet meadows), Barbarea vulgaris (drier micro‐habitats) and Alliaria petiolata (intermediate areas).5. As predicted, P. rapae eggs were more common than P. napi eggs on B. vulgaris, whereas all of the 358 individuals collected from C. pratensis were P. napi, indicating a divergence in host use between the Pieris species. However, under controlled laboratory conditions, both species had virtually identical oviposition preferences, laying eggs on all three plants, notably P. rapae also laying eggs on C. pratensis, indicating that habitat use, not plant preference, drives host plant use in nature.

Discovery of a rare and striking new pierid butterfly from Panama (Lepidoptera: Pieridae).

We here describe and name a distinctive new pierid species in the subfamily Pierinae, Catasticta sibyllae Nakahara, Padrón MacDonald, n. sp. from western Panama. Catasticta. sibyllae n. sp. is known from only two male specimens collected at two sites which are approximately 130 km apart in western Panama. This new species is the only species in the genus without markings in the median area of both surfaces of forewing and hindwing, and our molecular data suggest that the Peruvian species C. lisa Baumann Reissinger, 1969 is its sister species.

Developmental dynamics of butterfly wings: real-time in vivo whole-wing imaging of twelve butterfly species

Colour pattern development of butterfly wings has been studied from several different approaches. However, developmental changes in the pupal wing tissues have rarely been documented visually. In this study, we recorded real-time developmental changes of the pupal whole wings of 9 nymphalid, 2 lycaenid, and 1 pierid species in vivo, from immediately after pupation to eclosion, using the forewing-lift method. The developmental period was roughly divided into four sequential stages. At the very early stage, the wing tissue was transparent, but at the second stage, it became semi-transparent and showed dynamic peripheral adjustment and slow low-frequency contractions. At this stage, the wing peripheral portion diminished in size, but simultaneously, the ventral epithelium expanded in size. Likely because of scale growth, the wing tissue became deeply whitish at the second and third stages, followed by pigment deposition and structural colour expression at the fourth stage. Some red or yellow (light-colour) areas that emerged early were “overpainted” by expanding black areas, suggesting the coexistence of two morphogenic signals in some scale cells. The discal spot emerged first in some nymphalid species, as though it organised the entire development of colour patterns. These results indicated the dynamic wing developmental processes common in butterflies.

Warming neutralizes host-specific competitive advantages between a native and invasive herbivore

Although native-invasive species interactions have become a common mechanism shaping ecosystems, whether these interactions shift under warming remains unclear. To investigate how warming may affect native and invasive species separately and together (intraspecific and interspecific competition, respectively) and whether any warming impact is resource dependent, we examined the performance of two competing herbivores (native Pieris canidia and invasive P. rapae) on two common host plants under three temperature settings (control, 3 °C, and 6 °C warming using environmental chambers). The results revealed that warming benefited the development and growth of both Pieris under intraspecific competition, but the benefits were host-plant dependent. Notably, the native or invasive Pieris gained an advantage from interspecific competition (host-plant dependent), but warming neutralized the competitive advantages of either Pieris species. These findings raise the possibility that warming-induced shifts in competitive status may become a crucial mechanism shaping ecosystems worldwide, because most ecosystems are challenged by species invasion and warming. Moreover, this study revealed a discrepancy in species thermal performance between intra- and interspecific competition. Therefore, to predict native-invasive species competition under warming, current thermal performance applications should use species performance curves derived from interspecific rather than intraspecific competition studies (although the latter is more readily available).

Morphological differences of three Pieris species in Hokkaido

Morphological differences of three Pieris species in Hokkaido

Description of a new species of the genus Pieris (Lepidoptera Pieridae) from North Yunnan, China

Description of a new species of the genus Pieris (Lepidoptera Pieridae) from North Yunnan, China

β-Cyanoalanine Synthases and Their Possible Role in Pierid Host Plant Adaptation.

Cyanide is generated in larvae of the glucosinolate-specialist Pieris rapae (Lepidoptera:Pieridae) upon ingestion of plant material containing phenylalanine-derived glucosinolates as chemical defenses. As these glucosinolates were widespread within ancient Brassicales, the ability to detoxify cyanide may therefore have been essential for the host plant shift of Pierid species from Fabales to Brassicales species giving rise to the Pierinae subfamily. Previous research identified β-cyanoalanine and thiocyanate as products of cyanide detoxification in P. rapae larvae as well as three cDNAs encoding the β-cyanoalanine synthases PrBSAS1-PrBSAS3. Here, we analyzed a total of eight species of four lepidopteran families to test if their cyanide detoxification capacity correlates with their feeding specialization. We detected β-cyanoalanine synthase activity in gut protein extracts of all six species tested, which included Pierid species with glucosinolate-containing host plants, Pierids with other hosts, and other Lepidoptera with varying food specialization. Rhodanese activity was only scarcely detectable with the highest levels appearing in the two glucosinolate-feeding Pierids. We then amplified by polymerase chain reaction (PCR) 14 cDNAs encoding β-cyanoalanine synthases from seven species. Enzyme characterization and phylogenetic analysis indicated that lepidopterans are generally equipped with one PrBSAS2 homolog with high affinity for cyanide. A second β-cyanoalanine synthase which grouped with PrBSAS3 was restricted to Pierid species, while a third variant (i.e., homologs of PrBSAS1), was only present in members of the Pierinae subfamily. These results are in agreement with the hypothesis that the host shift to Brassicales was associated with the requirement for a specialized cyanide detoxification machinery.