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. 2021 Jul 2;22(13):7157.
doi: 10.3390/ijms22137157.

Molecular Characterization of TRPA Subfamily Genes and Function in Temperature Preference in Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

Xiao-Di Wang  1 , Ze-Kai Lin  1 , Shun-Xia Ji  1 , Si-Yan Bi  1 , Wan-Xue Liu  1 , Gui-Fen Zhang  1 , Fang-Hao Wan  1   2 , Zhi-Chuang Lü  1
Affiliations

Molecular Characterization of TRPA Subfamily Genes and Function in Temperature Preference in Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

Xiao-Di Wang et al. Int J Mol Sci. .

Abstract

To reveal the mechanism of temperature preference in Tuta absoluta, one of the top 20 plant pests in the world, we cloned and identified TaTRPA1, TaPain, and TaPyx genes by RACE and bioinformatic analysis, and clarified their expression profiles during different development stages using real-time PCR, and revealed their function in preference temperature by RNAi. The full-length cDNA of TaPain was 3136 bp, with a 2865-bp open reading frame encoding a 259.89-kDa protein; and the partial length cDNA of TaPyx was 2326-bp, with a 2025-bp open reading frame encoding a 193.16-kDa protein. In addition, the expression of TaTRPA1 and TaPyx was significantly lower in larvae than other stages, and it was significantly higher in pupae and newly emerging males for TaPain. After feeding target double-stranded RNA (dsRNA), the preferred temperature decreased 2 °C more than the control group. In conclusion, the results firstly indicated the molecular characterization of TRPA subfamily genes and their key role in temperature perception in T. absoluta, and the study will help us to understand the temperature-sensing mechanism in the pest, and will provide some basis for study of other Lepidoptera insects' temperature preference. Moreover, it is of great significance in enriching the research progress of "thermos TRP".

Keywords: Painless; Pyrexia; RNA interference; TRPA1; Tuta absoluta; temperature preference.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A). Full-length cDNA sequence of Tuta absoluta TaPain and its deduced amino acid sequence. The full-length cDNA of T. absoluta TaPain is 3136 bp, and the open reading frame (72–2936 bp) encodes a polypeptide of 954 amino acids. Eight boxes in different colors are marked with eight ankyrin repeats. (B). Partial length cDNA sequence of Tuta absoluta TaPyx and its deduced amino acid sequence. The partial length cDNA of T. absoluta TaPyx is 2326 bp, and the open reading frame 178–2202 bp encodes a polypeptide of 674 amino acids.
Figure 2
Figure 2
(A). Transmembrane structure prediction of the TaPain protein in Tuta absoluta. Six transmembrane structures were found in Tuta absoluta TaPain. The transmembrane structural positions of TM1, TM2, TM3, TM4, TM5, and TM6 were located at the amino acid positions of 535–558, 570–588, 608–626, 633–653, 673–692, and 754–775, respectively. (B). Transmembrane structure prediction of the TaPyx protein in Tuta absoluta. Six transmembrane structures were found in Tuta absoluta TaPyx. The transmembrane structural positions of TM1, TM2, TM3, TM4, TM5, and TM6 was located at the amino acid positions of 252–275, 314–332, 353–373, 379–404, 416–436, and 483–505, respectively.
Figure 2
Figure 2
(A). Transmembrane structure prediction of the TaPain protein in Tuta absoluta. Six transmembrane structures were found in Tuta absoluta TaPain. The transmembrane structural positions of TM1, TM2, TM3, TM4, TM5, and TM6 were located at the amino acid positions of 535–558, 570–588, 608–626, 633–653, 673–692, and 754–775, respectively. (B). Transmembrane structure prediction of the TaPyx protein in Tuta absoluta. Six transmembrane structures were found in Tuta absoluta TaPyx. The transmembrane structural positions of TM1, TM2, TM3, TM4, TM5, and TM6 was located at the amino acid positions of 252–275, 314–332, 353–373, 379–404, 416–436, and 483–505, respectively.
Figure 3
Figure 3
The conserved domains of Painless and Pyrexia in Tuta absoluta. The light blue to dark blue represent different transmembrane domains structure, and the light yellow to red represent different ankyrin repeats.
Figure 4
Figure 4
(A). Alignment of TRP protein from Tuta absoluta and other insects. Black represents that the amino acid sequences of seven species in multiple sequence alignment are completely identical; purple represents that six species out of seven species have the same amino acid sequences at this site; red represents that four to five species out of seven species have the same amino acid sequences at this site; and no color represents that there are differences among species. B. mori: Bombyx mori Painless (001296553.1); P. xylostella: Plutella xylostella Painless (XP_011560772.2); H. Melpomene: Heliconius Melpomene Painless (QDR50965.1); P. rapae: Pieris rapae Painless (XP_022120653.1); O. furnacalis: Ostrinia furnacalis Painless (XP_028177403.1); M. sexta: Manduca sexta Painless (XP_030023596.2). (B). Alignment of Pyrexia protein from Tuta absoluta and other insects. Black represents that the amino acid sequences of seven species in multiple sequence alignment are completely identical; purple represents that six species out of seven species have the same amino acid sequences at this site; red represents that four to five species out of seven species have the same amino acid sequences at this site; and no color represents that there are differences among species. B. mori: Bombyx mori Pyrexia (NP_001296553.1); H. armigera: Helicoverpa armigera Pyrexia (XP_021194189.1); T. ni: Trichoplusia Pyrexia ni(XP_026735810.1); O. furnacalis: Ostrinia furnacalis Pyrexia (XP_028163008.1); P. aegeria: Pararge aegeria Pyrexia (XP_039759808.1); V. tameamea: Vanessa tameamea Pyrexia (XP_026483194.1). (C). Alignment of TRPA1 protein from Tuta absoluta and other insects. Black represents that the amino acid sequences of six species in multiple sequence alignment are completely identical; purple represents that five species out of six species have the same amino acid sequences at this site; red represents that four species out of six species have the same amino acid sequences at this site; and no color represents that there are differences among species. B. mori: Bombyx mori TRPA1 (NP_001296525.1); B. mandarina: Bombyx mandarina TRPA1(XP_028033887.1); H. armigera: Helicoverpa armigera TRPA1(XP_021185779.1); G. mellonella: Galleria mellonella TRPA1(XP_031767554.1); M. sexta: Manduca sexta TRPA1(XP_037299698.1).
Figure 4
Figure 4
(A). Alignment of TRP protein from Tuta absoluta and other insects. Black represents that the amino acid sequences of seven species in multiple sequence alignment are completely identical; purple represents that six species out of seven species have the same amino acid sequences at this site; red represents that four to five species out of seven species have the same amino acid sequences at this site; and no color represents that there are differences among species. B. mori: Bombyx mori Painless (001296553.1); P. xylostella: Plutella xylostella Painless (XP_011560772.2); H. Melpomene: Heliconius Melpomene Painless (QDR50965.1); P. rapae: Pieris rapae Painless (XP_022120653.1); O. furnacalis: Ostrinia furnacalis Painless (XP_028177403.1); M. sexta: Manduca sexta Painless (XP_030023596.2). (B). Alignment of Pyrexia protein from Tuta absoluta and other insects. Black represents that the amino acid sequences of seven species in multiple sequence alignment are completely identical; purple represents that six species out of seven species have the same amino acid sequences at this site; red represents that four to five species out of seven species have the same amino acid sequences at this site; and no color represents that there are differences among species. B. mori: Bombyx mori Pyrexia (NP_001296553.1); H. armigera: Helicoverpa armigera Pyrexia (XP_021194189.1); T. ni: Trichoplusia Pyrexia ni(XP_026735810.1); O. furnacalis: Ostrinia furnacalis Pyrexia (XP_028163008.1); P. aegeria: Pararge aegeria Pyrexia (XP_039759808.1); V. tameamea: Vanessa tameamea Pyrexia (XP_026483194.1). (C). Alignment of TRPA1 protein from Tuta absoluta and other insects. Black represents that the amino acid sequences of six species in multiple sequence alignment are completely identical; purple represents that five species out of six species have the same amino acid sequences at this site; red represents that four species out of six species have the same amino acid sequences at this site; and no color represents that there are differences among species. B. mori: Bombyx mori TRPA1 (NP_001296525.1); B. mandarina: Bombyx mandarina TRPA1(XP_028033887.1); H. armigera: Helicoverpa armigera TRPA1(XP_021185779.1); G. mellonella: Galleria mellonella TRPA1(XP_031767554.1); M. sexta: Manduca sexta TRPA1(XP_037299698.1).
Figure 4
Figure 4
(A). Alignment of TRP protein from Tuta absoluta and other insects. Black represents that the amino acid sequences of seven species in multiple sequence alignment are completely identical; purple represents that six species out of seven species have the same amino acid sequences at this site; red represents that four to five species out of seven species have the same amino acid sequences at this site; and no color represents that there are differences among species. B. mori: Bombyx mori Painless (001296553.1); P. xylostella: Plutella xylostella Painless (XP_011560772.2); H. Melpomene: Heliconius Melpomene Painless (QDR50965.1); P. rapae: Pieris rapae Painless (XP_022120653.1); O. furnacalis: Ostrinia furnacalis Painless (XP_028177403.1); M. sexta: Manduca sexta Painless (XP_030023596.2). (B). Alignment of Pyrexia protein from Tuta absoluta and other insects. Black represents that the amino acid sequences of seven species in multiple sequence alignment are completely identical; purple represents that six species out of seven species have the same amino acid sequences at this site; red represents that four to five species out of seven species have the same amino acid sequences at this site; and no color represents that there are differences among species. B. mori: Bombyx mori Pyrexia (NP_001296553.1); H. armigera: Helicoverpa armigera Pyrexia (XP_021194189.1); T. ni: Trichoplusia Pyrexia ni(XP_026735810.1); O. furnacalis: Ostrinia furnacalis Pyrexia (XP_028163008.1); P. aegeria: Pararge aegeria Pyrexia (XP_039759808.1); V. tameamea: Vanessa tameamea Pyrexia (XP_026483194.1). (C). Alignment of TRPA1 protein from Tuta absoluta and other insects. Black represents that the amino acid sequences of six species in multiple sequence alignment are completely identical; purple represents that five species out of six species have the same amino acid sequences at this site; red represents that four species out of six species have the same amino acid sequences at this site; and no color represents that there are differences among species. B. mori: Bombyx mori TRPA1 (NP_001296525.1); B. mandarina: Bombyx mandarina TRPA1(XP_028033887.1); H. armigera: Helicoverpa armigera TRPA1(XP_021185779.1); G. mellonella: Galleria mellonella TRPA1(XP_031767554.1); M. sexta: Manduca sexta TRPA1(XP_037299698.1).
Figure 5
Figure 5
A phylogenetic tree based on the known amino acid sequences of TRPA genes. The phylogenetic tree was generated via Maximum Likelihood method based on the poisson correction mode, and this tree was used to determine the relationships between different insects. Helicoverpa armigera Pyrexia (XP_021194189.1); Trichoplusia ni Pyrexia (XP_026735810.1); Galleria mellonella Pyrexia (XP_026761448.2); Ostrinia furnacalis Pyrexia (XP_028163008.1); Pararge aegeria Pyrexia(XP_039759808.1); Plutella xylostella Pyrexia(XP_037962898.1); Bombyx mori Pyrexia (NP_001296536.1); Manduca sexta Pyrexia (XP_030021352.2); Agrilus planipennis Pyrexia (XP_018334781.1); Nicrophorus vespilloides Pyrexia (XP_017775114.1); Leptinotarsa decemlineata Pyrexia (XP_023020123.1); Agasicles hygrophila Pyrexia (QLH02045.1); Diabrotica virgifera virgifera Pyrexia (XP_028131629.1); Melanaphis sacchari Pyrexia (XP_025205608.1); Acyrthosiphon pisum Pyrexia (XP_016657902.1); Monomorium pharaonic Pyrexia (XP_012535072.2); Nomia melanderi Pyrexia (XP_031825641.1); Apis dorsata Pyrexia (XP_006623313.1); Apis cerana Pyrexia (XP_028522925.1); Thrips palmi TRPA1 (XP_034255303.1); Frankliniella occidentalis TRPA1 (XP_026285236.1); Myzus persicae TRPA1 (XP_022167295.1); Acyrthosiphon pisum TRPA1(XP_029342925.1); Aphis gossypii TRPA1 (XP_027848279.1); Aphis craccivora TRPA1 (KAF0770012.1); Rhopalosiphum maidis TRPA1 (XP_026809343.1); Melanaphis sacchari TRPA1 (XP_025200861.1); Heliconius melpomene TRPA1(QDR50963.1); Danaus plexippus TRPA1 (QDQ16924.1); Galleria mellonella TRPA1 (XP_031767554.1); Helicoverpa armigera TRPA1 (XP_021185779.1); Manduca sexta TRPA1 (QDR51038.1); Ostrinia furnacalis TRPA1 (XP_028170510.1); Papilio xuthus TRPA1 (KPJ00566.1); Papilio machaon TRPA1 (KPJ09099.1); Bombyx mori TRPA1 (NP_001296525.1); Bombyx mandarina TRPA1(XP_028033887.1); Apis florea Painless (XP_031772455.1); Apis cerana Painless (XP_028520736.1); Apis mellifera Painless(XP_006562517.1); Apis dorsata Painless (XP_031369976.1); Bombus impatiens Painless (XP_024223159.1); Nasonia vitripennis Painless (XP_031783731.1); Nylanderia fulva Painless (XP_029174885.1); Harpegnathos saltator Painless (XP_025162136.1); Solenopsis invicta Painless (XP_039303762.1); Monomorium pharaonis Painless (XP_036145416.1); Anopheles arabiensis Painless (XP_040153163.1); Aedes aegypti Painless (XP_001652261.2); Culex quinquefasciatus Painless (XP_001849122.2); Bactrocera oleae Painless (XP_036223549.1); Rhagoletis pomonella Painless(XP_036331109.1); Bactrocera tryoni Painless (XP_039969281.1); Drosophila melanogaster Painless(NP_611979.1); Anoplophora glabripennis Painless(XP_018573376.1); Tribolium castaneum Painless (NP_001164308.1); Agasicles hygrophila Painless (QLH02046.1); Myzus persicae Painless (XP_022174286.1); Acyrthosiphon pisum Painless (XP_029346868.1); Rhopalosiphum maidis Painless (XP_026806747.1); Melanaphis sacchari Painless (XP_025190493.1); Aphis craccivora Painless (KAF0773934.1); Aphis gossypii Painless (XP_027846115.1); Danaus plexippus Painless (QDQ16926.1); Bombyx mori Painless (NP_001296553.1); Bombyx mandarina Painless (XP_028039162.1); Manduca sexta Painless (XP_030023596.2); Pieris rapae Painless (XP_022120653.1); Papilio xuthus Painless (KPJ01914.1); Plutella xylostella Painless (XP_011560772.2); Ostrinia furnacalis Painless (XP_028177403.1); Trichoplusia ni Painless(XP_026747274.1); Helicoverpa armigera Painless (XP_021191852.1).
Figure 6
Figure 6
Relative expression levels of TaTRPA1 (A), TaPain (B), TaPyx (C) in eggs, first to fourth instars, early to late pupae, newly emerged to mature females and males. Data represent means ± SEM. Bars with different lowercase letters are significantly different at p < 0.05.
Figure 7
Figure 7
Effects of dsRNA treatments on mRNA expression in the Tuta absoluta. Data are presented as means ± SEM. Data were compared by analysis of variance (ANOVA) followed by Tukey’s post hoc test (* p < 0.05).
Figure 8
Figure 8
Temperature preference responses of Tuta absoluta larvae. The data are presented as mean ± SEM.
Figure 9
Figure 9
Temperature preference responses after dsRNA feeding in Tuta absoluta larvae. The data are presented as mean ± SEM.
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