Staying in touch: how highly specialised moth pollinators track host plant phenology in unpredictable climates

Background For specialised pollinators, the synchrony of plant and pollinator life history is critical to the persistence of pollinator populations. This is even more critical in nursery pollination, where pollinators are obligately dependant on female host plant flowers for oviposition sites. Epicephala moths (Gracillariidae) form highly specialised nursery pollination mutualisms with Phyllanthaceae plants. Several hundred Phyllanthaceae are estimated to be exclusively pollinated by highly specific Epicephala moths, making these mutualisms an outstanding example of plant–insect coevolution. However, there have been no studies of how Epicephala moths synchronise their activity with host plant flowering or persist through periods when flowers are absent. Such knowledge is critical to understanding the ecology and evolutionary stability of these mutualisms. We surveyed multiple populations of both Breynia oblongifolia (Phyllanthaceae) and it’s Epicephala pollinators for over two years to determine their phenology and modelled the environmental factors that underpin their interactions. Results The abundance of flowers and fruits was highly variable and strongly linked to local rainfall and photoperiod. Unlike male flowers and fruits, female flowers were present throughout the entire year, including winter. Fruit abundance was a significant predictor of adult Epicephala activity, suggesting that eggs or early instar larvae diapause within dormant female flowers and emerge as fruits mature. Searches of overwintering female flowers confirmed that many contained pollen and diapausing pollinators. We also observed diapause in Epicephala prior to pupation, finding that 12% (9/78) of larvae emerging from fruits in the autumn entered an extended diapause for 38–48 weeks. The remaining autumn emerging larvae pupated directly without diapause, suggesting a possible bet-hedging strategy. Conclusions Epicephala appear to use diapause at multiple stages in their lifecycle to survive variable host plant phenology. Furthermore, moth abundance was predicted by the same environmental variables as male flowers, suggesting that moths track flowering through temperature. These adaptations may thereby mitigate against unpredictability in the timing of fruiting and flowering because of variable rainfall. It remains to be seen how widespread egg diapause and pre-pupal diapause may be within Epicephala moths, and, furthermore, to what degree these traits may have facilitated the evolution of these highly diverse mutualisms.

Expression Patterns of Three Important Hormone Genes and Respiratory Metabolism in Antheraea pernyi during Pupal Diapause under a Long Photoperiod

The Chinese oak silkworm is commonly used in pupal diapause research. In this study, a long photoperiod was used to trigger pupal diapause termination. Genes encoding three hormones, namely prothoracicotropic hormone (PTTH), ecdysis triggering hormone (ETH), and eclosion hormone (EH), were studied. Additionally, ecdysteroids (mainly 20-hydroxyecdysone, 20E) were quantified by HPLC. Pupal diapause stage was determined by measuring respiratory intensity. The pupae enter a low metabolic rate, which starts approximately 1 month after pupal emergence. ApPTTH expression showed a small increase at 14 days and then a larger increase from 35 days under the long photoperiod treatment. A similar pattern was observed for the titer of 20E in the hemolymph. However, ApETH expression later increased under the long photoperiod treatment (42 days) just before eclosion. Moreover, ApEH expression increased from 21 to 35 days, and then decreased before ecdysis. These results suggest that hormone-related gene expression is closely related to pupal development. Our study lays a foundation for future diapause studies in A. pernyi.

Bioactive Secondary Metabolites of Wild Antheraea mylitta Silkworm Cocoons

The wild silkworm Antheraea mylitta is grown and cultivated in several parts of India ranging from Bihar to West Bengal and several parts of Telangana. The wild silkworm rearing has been a source of income for the tribal populations who rely on it as income source; the intervention of government agencies has increased the cultivation. Our research involves understanding the secondary metabolites in the silkworm Cocoons and elucidating how the pupa survives the harsh environment during pupal diapause of the insect. We have realized the role of insect repellent compounds and other metabolites and their interaction with the insect. Wild silkworm Cocoons are the specialized natural structures constructed by Antheraea mylitta silkworms. They are the protein composites of sericin and fibroin as a structural material. The silkworm cocoons are presumed to be evolved structures through the course of evolution over millions of years. This chapter focuses on Biophysical analysis of chemical compounds, proteins and other secondary metabolites traced in the Wild Antheraea mylitta Tasar cocoons which are predicted to be the key factors to achieve the unique structural and chemical barriers to protect the pupa within the cocoons.

Comparative proteomics provides insights into diapause program of Bactrocera minax (Diptera: Tephritidae)

The Chinese citrus fly, Bactrocera minax, is a notorious univoltine pest that causes damage to citrus. B. minax enters obligatory pupal diapause in each generation to resist harsh environmental conditions in winter. Despite the enormous efforts that have been made in the past decade, the understanding of pupal diapause of B. minax is currently still fragmentary. In this study, the 20-hydroxyecdysone solution and ethanol solvent was injected into newly-formed pupae to obtain non-diapause- (ND) and diapause-destined (D) pupae, respectively, and a comparative proteomics analysis between ND and D pupae was performed 1 and 15 d after injection. A total of 3,255 proteins were identified, of which 190 and 463 were found to be differentially abundant proteins (DAPs) in ND1 vs D1 and ND15 vs D15 comparisons, respectively. The reliability and accuracy of LFQ method was validated by qRT-PCR. Functional analyses of DAPs, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network construction, were conducted. The results revealed that the diapause program of B. minax is closely associated with several physiological activities, such as phosphorylation, chitin biosynthesis, autophagy, signaling pathways, endocytosis, skeletal muscle formation, protein metabolism, and core metabolic pathways of carbohydrate, amino acid, and lipid conversion. The findings of this study provide insights into diapause program of B. minax and lay a basis for further investigation into its underlying molecular mechanisms.

Watching the days go by: asymmetric regulation of caterpillar development by changes in photoperiod

Many insects possess the plastic ability to either develop directly to adulthood, or enter diapause and postpone reproduction until the next year, depending on environmental cues (primarily photoperiod) that signal the amount of time remaining until the end of the growth season. These two developmental pathways often differ in co-adapted life history traits, e.g. with slower development and larger size in individuals headed for diapause. The developmental timing of these differences may be of adaptive importance: if pathways diverge late, the scope for phenotypic differences is smaller, whereas if pathways diverge early, the risk is higher of expressing a maladaptive phenotype if the selective environment changes. Here we explore the effects of changes in photoperiodic information during life on pupal diapause and associated life history traits in the butterfly Pararge aegeria. We find that both pupal diapause and larval development rate are asymmetrically regulated: while exposure to long days late in life (regardless of earlier experiences) was sufficient to produce nondiapause development and accelerate larval development accordingly, more prolonged exposure to short days was required to induce diapause and slow down pre-diapause larval development. While the two developmental pathways diverged early in development, development rates could be partially reversed by altered environmental cues. Meanwhile, pathway differences in body size were more inflexible, despite emerging late in development. Hence, in P. aegeria several traits are regulated by photoperiod, along subtly different ontogenies, into an integrated phenotype that strikes a balance between flexibility and phenotype-environment matching.

Characterization of an Ecdysteroid-Regulated 16 kDa Protein Gene in Chinese Oak Silkworm, Antheraea pernyi (Lepidoptera: Saturniidae)

A large number of ecdysteroid-regulated 16 kDa proteins (ESR16s) of insects have been isolated and annotated in GenBank; however, knowledge on insect ESR16s remain limited. In the present study, we characterized an ecdysteroid-regulated 16 kDa protein gene isolated in Chinese oak silkworm, Antheraea pernyi Guérin-Méneville (‘ApESR16’ in the following), an important silk-producing and edible insect. The obtained cDNA sequence of ApESR16 is 1,049 bp, harboring an open reading frame of 441 bp that encodes a polypeptide of 146 amino acids. CD-search revealed that ApESR16 contains the putative cholesterol/lipid binding sites on conserved domain Npc2_like (Niemann–Pick type C-2) belonging to the MD-2-related lipid-recognition superfamily. Sequence comparison revealed that ApESR16 exhibits 51–57% identity to ESR16s of lepidopteran insects, 36–41% identity to ESR16 or NPC2a of nonlepidopteran insects, and 28–32% identity to NPC2a of vertebrates, indicating a high sequence divergence during the evolution of animals. Phylogenetic analysis found that the used sequences were divided into two groups corresponding to vertebrates and invertebrates, and the used insect sequences were also well clustered according to their families. The A. pernyi ESR16 mRNA is expressed during all four developmental stages and in all tested tissues. Injection of 20-hydroxyecdysone (20-E) into A. pernyi diapausing pupae triggering diapause termination induced upregulation of ESR16 mRNA compared to the diapausing pupae, with the highest expression level at day 2 in the ovaries but day 12 in the fat body. Our results suggested that ApESR16 might be a diapause-related gene and plays a vital role in the pupal diapause of A. pernyi.

A rapidly evolved shift in life history timing during ecological speciation is driven by the transition between developmental phases

For insect species in temperate environments, seasonal timing is often governed by the regulation of diapause, a complex developmental program that allows insects to weather unfavorable conditions and synchronize their lifecycles with available resources. Diapause consists of a series of phases that govern initiation, maintenance, and termination of this developmental pathway. The evolution of insect seasonal timing depends in part on how these phases of diapause development and post-diapause development interact to affect variation in phenology. Here, we dissect the physiological basis of a recently evolved phenological shift in Rhagoletis pomonella (Diptera: Tephritidae), a model system for ecological divergence. A recently derived population of R. pomonella shifted from specializing on native hawthorn fruit to earlier fruiting introduced apples, resulting in a 3-4 week shift in adult emergence timing. We tracked metabolic rates of individual flies across post-winter development to test which phases of development may act either independently or in combination to contribute to this recently evolved divergence in timing. Apple and hawthorn flies differed in a number of facets of their post-winter developmental trajectories. However, divergent adaptation in adult emergence phenology in these flies was due almost entirely to the end of the pupal diapause maintenance phase, with post-diapause development having a very small effect. The relatively simple underpinnings of variation in adult emergence phenology suggest that further adaptation to seasonal change in these flies for this trait might be largely due to the timing of diapause termination unhindered by strong covariance among different components of post-diapause development.Data accessibilityAll data (in the form of tables of all metabolic rate measurements for all individual flies in the study) will be available on DRYAD when the manuscript is published.