Molecular phylogeny, laboratory rearing, and karyotype of the bombycid moth, Trilocha varians

Abstract

This study describes the molecular phylogeny, laboratory rearing, and karyotype of a bombycid moth, Trilocha varians (F. Walker) (Lepidoptera: Bombycidae), which feeds on leaves of Ficus spp. (Rosales: Moraceae). The larvae of this species were collected in Taipei city, Taiwan, and the Ryukyu Archipelago (Ishigaki and Okinawa Islands, Japan). Molecular phylogenetic analyses revealed that T. varians belongs to the subfamily Bombycinae, thus showing a close relationship to the domesticated silkworm Bombyx mori (L.), a lepidopteran model insect. A laboratory method was developed for rearing T. varians and the time required for development from the embryo to adult was determined. From oviposition to adult emergence, the developmental zero was 10.47 °C and total effective temperature was 531.2 day-degrees, i.e., approximately 30 days for one generation when reared at 28 °C. The haploid of T. varians consisted of n = 26 chromosomes. In highly polyploid somatic nuclei, females showed a large heterochromatin body, indicating that the sex chromosome system in T. varians is WZ/ZZ (female/male). The results of the present study should facilitate the utilization of T. varians as a reference species for B. mori, thereby leading to a greater understanding of the ecology and evolution of bombycid moths.

Figure 1.

Phylogenetic analysis of Bombycoidea using nuclear DDC gene sequences. The topology shows Bayesian inference tree using program MrBayes 3.1.2. Lacosoma chiridota and Plagodis fervidaria were designated as out-group taxa. Numbers indicate bootstrap values from NJ (top left) and MP (top right) analyses, and posterior probabilities from Bayesian analysis (bottom). Only bootstrap values > 50% and Bayesian posterior probabilities > 0.5 are shown. Branch lengths represent nucleotide substitutions per site. Trilocha varians is shown as a box, and the subfamily Bombycinae is shown as a grey dashed box. Accession numbers are indicated in parentheses. Asterisks indicate subgroups of Bombycidae as defined by Minet (1994). High quality figures are available online.

Figure 2.

Geographical map showing sampling localities and phylogenetic analysis of Bombycini using mitochondrial COI gene sequences. (A) Larvae of Trilocha varians were collected in Taipei, Ishigaki Island, and Okinawa Island. (B) The topology shows Bayesian inference tree using program MrBayes 3.1.2. Apatelodes torrefacta and Antheraea pernyi were used as out-group taxa. Numbers indicate bootstrap values from NJ (top left) and MP (top right) analyses, and posterior probabilities from Bayesian analysis (bottom). Only bootstrap values > 50% and Bayesian posterior probabilities > 0.5 are shown. Branch lengths represent nucleotide substitutions per site. The sequences of Trilocha varians were 100% identical among samples from Taipei, Ishigaki Island, and Okinawa Island. Localities and accession numbers are indicated in parentheses. Bombyx huttoni (DQ415451) appeared to be erroneously registered by Arunkumar et al. (2006) as Theophila religiosa(e)^* in GenBank. ^* The history of Theophila religiosae (Helfer, 1837) is complicated. Moore (1858) and Hutton (1864) treated Bombyx huttoni Westwood, 1847 as a junior synonym of Theophila religiosae. However, Dierl (1979) inaccurately designated a specimen of the current Triuncina religiosae as the neotype of this specific epithet, religiosae, as the type specimen of Theophila religiosae was lost. Thus, the scientific name of Bombyx huttoni was revived instead of Theophila religiosae. High quality figures are available online.

Figure 3.

Immature stages of Trilocha varians. (A) Newly laid eggs of Trilocha varians. (B) Eggs one day before hatching. The head capsule is visible through the transparent eggshell. (C) Newly-hatched larva. (D) Third instar larva on a leaf of Ficus microcarpa. (E) Fifth instar larvae. (F) Last instar and spinning larvae in a 430 mL plastic cup. (G) Cocoon on a leaf of ficus microcarpa. (H) Male pupa. (I, J) Ventral views of abdomen of female (I) and male (J) pupae. The solid arrow indicates the suture on the ninth abdominal segment of the female, and the dashed arrow indicates the absence of the suture in the male. High quality figures are available online.

Figure 4.

Trilocha varians adults. (A) Female and (B) male adults of Trilocha varians. (C) A female (arrowhead) laid eggs on the cover and wall of a plastic cup (arrows). High quality figures are available online.

Figure 5.

Karyotype of Trilocha varians. Chromosome preparations of (A) male and (B) female Trilocha varians larvae, showing 26 bivalent chromosomes in both sexes. High quality figures are available online.

Figure 6.

Sex heterochromatin body of Trilocha varians females. (A) Nuclei prepared from the sucking stomach of adult females showing a deeply strained SB (arrows). (B) Male nuclei lacking SB. Preparations from three individuals of each sex are shown. Scale bar indicates 100 µm. High quality figures are available online.