Coexpression of Three Odorant-Binding Protein Genes in the Foreleg Gustatory Sensilla of Swallowtail Butterfly Visualized by Multicolor FISH Analysis

Abstract

Lepidopteran insects are mostly monophagous or oligophagous. Female butterflies distinguish their host plants by detecting a combination of specific phytochemicals through the gustatory sensilla densely distributed on their foreleg tarsi, thereby ensuring oviposition on appropriate host plants. In this study, to gain insight into the molecular mechanism underlying host plant recognition by the gustatory sensilla, using Asian swallowtail, Papilio xuthus, we focused on a family of small soluble ligand-binding molecules, odorant-binding proteins (OBPs), and found that three OBP genes showed enriched expression in the foreleg tarsus. Multicolor fluorescence in situ hybridization analyses demonstrated the coexpression of these three OBP genes at the bases of the foreleg gustatory sensilla. Further analyses on other appendages revealed that PxutOBP3 was exclusively expressed in the tissues which could have direct contact with the leaf surface, suggesting that this OBP gene specifically plays an important role in phytochemicals perception.

Keywords: Papilio xuthus; butterfly; fluorescence in situ hybridization; gustatory sensilla; host plant selection; odorant-binding protein.

Figure 1

NJ tree of OBPs from P. xuthus and other lepidopteran species.

Figure 2

Expression levels of identified PxutOBPs. (A) Appendages examined for gene expression with a magnified view of the foreleg tarsus. Orange brushes in the dotted ellipse are the gustatory sensilla. As few gustatory sensilla are distributed in the first tarsomere (11), we collected the other four tarsomeres as “leg tarsus” samples. (B) Comparison of expression levels of OBPs between the tarsus of the forelegs and that of the other legs sampled from 0-day-old adult females performed by qRT-PCR. Three individuals were used per lot. All data are shown as the means ± SEM (n = 5, each leg). Student's t-test was conducted. OBP genes expressed significantly higher in foreleg tarsus (p < 0.05) or exhibiting prominent average fold change (the ratio of expression levels in foreleg tarsus to those in the other leg tarsus > 5) were indicated by orange-colored numbers. (C) Comparison of expression levels of OBPs among the foreleg tarsus (FLt), midleg tarsus (MLt), hindleg tarsus (HLt), antenna (An), proboscis (Pb), and ovipositor (Ovp) sampled from 0-day-old adult females performed by qRT-PCR. Two individuals were used per lot. All data are shown as the means ± SD (n = 6, each appendage). Multiple comparisons were performed using Dunnett's test. Asterisks indicate significant differences compared to expression levels of the foreleg tarsus (*, p < 0.05; **, p < 0.01).

Figure 3

Visualization of PxutOBP2, 3, and 9 expression in the foreleg tarsus. (A) Developmental time-course of the expression of PxutOBP2, 3, and 9 in the female foreleg tarsus analyzed by qRT-PCR. −3, −2, −1: 3, 2, and 1 day before eclosion, respectively; 0: the day of eclosion; 3v: 3-day-old virgin female; 3m: 3-day-old mated female. Two individuals were used per lot. All data are shown as the means ± SD (n = 5, each developmental stage). Multiple comparisons were performed using Tukey-Kramer's test. Different letters indicate significant differences (p < 0.05). (B) Spatial distribution of OBP-expressing cells in the female fifth tarsomere visualized by FISH. As shown in (A), PxutOBP3 expression was peaked on 1 day before eclosion. Therefore, −1-day-old females were used for detection of PxutOBP3 transcripts while 0-day-old females were analyzed for the visualization of PxutOBP2 and 9 expression. (C) Simultaneous detection of PxutOBP2, 3, and 9 expression in the female fifth tarsomere by triple-color FISH. −1-day-old females were used. DNP-, DIG-, and FLU-labeled riboprobes were synthesized for PxutOBP2, 3, and 9, respectively. (D) Simultaneous detection of PxutGr1 and PxutOBP9 expression in the female fifth tarsomere. 0-day-old females were analyzed. DIG- and FLU-labeled riboprobes were synthesized for PxutGr1 and PxutOBP9, respectively.

Figure 4

Coexpression of PxutOBP2, 3, and 9 in the midleg tarsus (A) and ovipositor (papilla analis) (B). −1-day-old females were analyzed. DNP-, DIG-, and FLU-labeled riboprobes were synthesized for PxutOBP2, 3, and 9, respectively. Arrows indicate signals of OBPs expression.

Figure 5

Visualization of PxutOBP2, 3, and 9 expression in the proboscis and antenna. Simultaneous detection of two different pairs of OBP genes was performed. −1-day-old females were analyzed. Arrows indicate signals of OBPs expression. (A) Longitudinal sections of the proboscis. FLU-labeled riboprobe was synthesized for PxutOBP9. For detection of PxutOBP2 or 3 transcripts, DIG-labeled riboprobes were used. (B) Longitudinal sections of the tip of antenna. FLU-labeled riboprobe was synthesized for PxutOBP2. For detection of PxutOBP3 or 9 transcripts, DIG-labeled riboprobes were used.

Figure 6

Visualization of PxutOBP2, 3, and 9 expression in the larval gustatory system. (A) Frontal view of a head of a final instar larva with a magnified view of the larval maxilla. (B,C) Simultaneous detection of two different pairs of OBP genes in the maxillary galea (MG) (B) and the maxillary palp (MP) (C). FLU-labeled riboprobe was synthesized for PxutOBP9. For detection of PxutOBP2 or 3 transcripts, DIG-labeled riboprobes were used. Arrows indicate signals of OBPs expression.