Genome-wide identification and gene-editing of pigment transporter genes in the swallowtail butterfly Papilio xuthus

Background Insect body coloration often functions as camouflage to survive from predators or mate selection. Transportation of pigment precursors or related metabolites from cytoplasm to subcellular pigment granules is one of the key steps in insect pigmentation and usually executed via such transporter proteins as the ATP-binding cassette (ABC) transmembrane transporters and small G-proteins (e.g. Rab protein). However, little is known about the copy numbers of pigment transporter genes in the butterfly genomes and about the roles of pigment transporters in the development of swallowtail butterflies. Results Here, we have identified 56 ABC transporters and 58 Rab members in the genome of swallowtail butterfly Papilio xuthus. This is the first case of genome-wide gene copy number identification of ABC transporters in swallowtail butterflies and Rab family in lepidopteran insects. Aiming to investigate the contribution of the five genes which are orthologous to well-studied pigment transporters (ABCG: white, scarlet, brown and ok; Rab: lightoid) of fruit fly or silkworm during the development of swallowtail butterflies, we performed CRISPR/Cas9 gene-editing of these genes using P. xuthus as a model and sequenced the transcriptomes of their morphological mutants. Our results indicate that the disruption of each gene produced mutated phenotypes in the colors of larvae (cuticle, testis) and/or adult eyes in G0 individuals but have no effect on wing color. The transcriptomic data demonstrated that mutations induced by CRISPR/Cas9 can lead to the accumulation of abnormal transcripts and the decrease or dosage compensation of normal transcripts at gene expression level. Comparative transcriptomes revealed 606 ~ 772 differentially expressed genes (DEGs) in the mutants of four ABCG transporters and 1443 DEGs in the mutants of lightoid. GO and KEGG enrichment analysis showed that DEGs in ABCG transporter mutants enriched to the oxidoreductase activity, heme binding, iron ion binding process possibly related to the color display, and DEGs in lightoid mutants are enriched in glycoprotein binding and protein kinases. Conclusions Our data indicated these transporter proteins play an important role in body color of P. xuthus. Our study provides new insights into the function of ABC transporters and small G-proteins in the morphological development of butterflies.

Destination of apyrene sperm following migration from the bursa copulatrix in the monandrous swallowtail butterfly Byasa alcinous

Most male lepidopterans produce fertile eupyrene sperm and non-fertile apyrene sperm, both of which are transferred to the female in a spermatophore during mating. Apyrene sperm outnumbers eupyrene sperm and both sperm types migrate from the bursa copulatrix to the spermatheca after mating. While eupyrene sperm are maintained in the spermatheca until oviposition, the number of apyrene sperm decreases with time. It is unclear whether apyrene sperm disappear from all sperm storage organs in females because both sperm types are often observed in the spermathecal gland. To investigate this, the numbers of both sperm types were estimated in the spermatheca and spermathecal gland of female Byasa alcinous (a monandrous butterfly) 6, 12, 48, 96, and 192 h after mating terminated. Apyrene sperm arrived in the spermatheca earlier than eupyrene sperm; however, some eupyrene and apyrene sperm migrated to the spermathecal gland from the spermatheca at almost the same time. The number of apyrene sperm reached a peak 12 h after the termination of mating and then decreased with time in both the spermatheca and spermathecal gland. Our results suggest that the role of apyrene sperm might be completed early after arriving in the spermatheca of B. alcinous.

Batesian mimicry has evolved with deleterious effects of the pleiotropic gene doublesex

Dimorphic female-limited Batesian mimicry in the swallowtail butterfly Papilio polytes is regulated by the supergene locus H, harbouring the mimetic (H) and non-mimetic (h) doublesex (dsx) gene. In the present study, we demonstrated that dsx-H negatively affects the number of eggs laid, hatching rate, larval survival rate, and adult lifespan. When crossed with hh males, the number of eggs laid of mimetic females (genotype HH) was lower than that of non-mimetic females (hh). Moreover, hh and Hh females laid fewer eggs when crossed with HH males. The hatching and larval survival rates were lower when both female and male parents harboured dsx-H. The adult lifespan of HH females was shorter than that of hh females, while it was similar in males regardless of the genotype. These findings suggest the presence of a cost–benefit balance of Batesian mimicry, which is evolved to avoid predation but is accompanied by physiological deficits, in this species.

Deciphering Novel Antimicrobial Peptides from the Transcriptome of Papilio xuthus

An insect’s innate immune system is the front line of defense against many invading microorganisms. One of the important components of this defense system is antimicrobial peptides (AMPs). Papiliocin is a well-studied antimicrobial peptide (AMP) isolated from the swallowtail butterfly, Papilio xuthus, and it was previously reported to be effective against Gram-positive bacteria, Gram-negative bacteria, and fungi, particularly in drug resistant Gram-negative bacteria. Hence, we aimed to identify novel AMPs from Papilio xuthus using its transcriptome. We immunized the swallowtail butterfly with Escherichia coli, Staphylococcus aureus, Candida albicans, and the total RNA was isolated. De novo transcriptome assembly and functional annotations were conducted, and AMPs were predicted using an in-silico pipeline. The obtained 344,804,442 raw reads were then pre-processed to retrieve 312,509,806 (90.6%) total clean reads. A total of 38,272 unigenes were assembled with the average length of 1010 bp. Differential gene expression analysis identified 584 and 1409 upregulated and downregulated genes, respectively. The physicochemical, aggregation, and allergen propensity were used as filtration criteria. A total of 248 peptides were predicted using our in-house pipeline and the known AMPs were removed, resulting in 193 novel peptides. Finally, seven peptides were tested in vitro and three peptides (Px 5, 6, and 7) showed stronger antimicrobial activity against Gram-negative bacteria and yeast. All the tested peptides were non-allergens. The identified novel AMPs may serve as potential candidates for future antimicrobial studies.

Comparison of flower-visiting behaviour of bumblebees and swallowtail butterflies to the Japanese azalea (Rhododendron japonicum)

Examination of the efficacy of pollinators that are not morphologically matched to a flower’s shape can deepen our understanding of the main pollinators of a plant species. In central Japan, Rhododendron japonicum is visited much more frequently by bumblebees than by butterflies although its flower shape is more suited for pollination by large butterflies. Here, we observed flower-visiting behaviour of Bombus diversus queens and the alpine black swallowtail butterfly, Papilio maackii, and compared their efficacy as pollinators. Papilio maackii always touched the stigma during a flower visit, whereas B. diversus queens did so during just 54% of their flower visits. As bumblebees visit neighboring flowers of a R. japonicum tree in sequence not like Papilio maackii, we hypothesized that they deliver self pollen to R. japonicum so that the fruit set would be low. However, the fruit set in a bumblebee-dominated area was 72.5%, significantly higher than that by hand-pollination with self pollen (31.4%). This suggests that R. japonicum can obtain sufficient fruits by B. diversus queens although its flower shape morphologically matches to pollination by swallowtail butterflies.