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Cuticular protein gene LmACP8 is involved in wing morphogenesis in the migratory locust, Locusta migratoria

ZHAO Xiao-ming, YANG Jia-peng, GOU Xin, LIU Wei-min, ZHANG Jian-zhen

2021, 20 (6): 1596-1606. DOI: 10.1016/S2095-3119(20)63248-8

Abstract （115） PDF in ScienceDirect

Cuticular proteins (CPs) are major components of the insect cuticle-associated organs such as integument and wings, although the importance of CPs for wing development and function in hemimetabolous insects remains understudied. In the present study, a wing cuticular protein LmACP8 was identified from Locusta migratoria, which belongs to the RR-2 subfamily of cuticular protein R&R consensus (CPR) chitin-binding proteins. LmACP8 was mainly expressed in the wing pads and showed high expression levels before ecdysis of third-, fourth-, and fifth-instar nymphs, with its encoded protein located in the procuticle of wing pads and adult wings. Depletion of LmACP8 by RNA interference markedly reduced the amount of its protein, which consequently caused abnormal wing morphogenesis in the transition from nymph to adult of L. migratoria. We further demonstrated that the abnormal morphogenesis was caused by severe damage of the endocuticle in the wings. LmACP8 was suppressed by 20-hydroxyecdysone (20E) in vivo, however, its expression was significantly up-regulated after knocking down the hormone receptor gene LmHR39. Thus, the LmACP8 that is negatively regulated by the LmHR39-mediated 20E signaling pathway is involved in wing development during the nymph to adult transition.

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Egg tanning improves the efficiency of CRISPR/Cas9-mediated mutant locust production by enhancing defense ability after microinjection

ZHANG Ting-ting, WEN Ting-mei, YUE Yang, YAN Qiang, DU Er-xia, FAN San-hong, Siegfried ROTH, LI Sheng, ZHANG Jian-zhen, ZHANG Xue-yao, ZHANG Min

2021, 20 (10): 2716-2726. DOI: 10.1016/S2095-3119(21)63736-X

Abstract （135） PDF in ScienceDirect

The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects. In the construction of CRISPR/Cas9-mediated dsLmRNase2^–/–mutant locusts, we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newly-laid eggs that were less tanned. However, the heritable efficiency of the dsLmRNase2 deletion to the next generation G₁ progeny was similar between adults derived from the tanned or less tanned engineered eggs. Further, the similar effective mutant ratios in the normally developed eggs and G₀ adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9. Moreover, we found that the syncytial division period, which was longer than the time for tanning, conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs. We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection. Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure. In summary, tanned eggs possess stronger defense responses and higher efficiency of genome editing, providing an improved model for developing Cas9-mediated gene editing procedures in locusts.

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Characteristics and roles of cytochrome b₅ in cytochrome P450-mediated oxidative reactions in Locusta migratoria

LIU Jiao, ZHANG Xue-yao, WU Hai-hua, MA Wen, ZHU Wen-ya, Kun-Yan ZHU, MA En-bo, ZHANG Jian-zhen

2020, 19 (6): 1512-1521. DOI: 10.1016/S2095-3119(19)62827-3

Abstract （115） PDF in ScienceDirect

Cytochrome b₅ (Cyt-b₅) is a small heme protein and known to be involved in a wide range of biochemical transformations, including cytochrome P450 monooxygenase (CYP)-mediated metabolism of endogenous and exogenous compounds. Studies on Cyt-b₅ are more concentrated in mammals, but are relatively rare in insects. The characteristics and function of Cyt-b₅ from Locusta migratoria have not been described yet. We sequenced the full-length cDNA sequence of Cyt-b₅ from L. migratoria (LmCyt-b₅) by reverse transcription-PCR (RT-PCR) based on locust transcriptome database. The phylogenetic analysis showed that LmCyt-b₅ was closely related to the Cyt-b₅ from Blattodea. LmCyt-b₅was highly expressed in ovary, Malpighian tubules, midgut, gastric caeca, and fat bodies. Silencing of LmCyt-b₅ had no effect on the susceptibility of L. migratoria to four different insecticides. Suppression of LmCyt-b₅ or silencing of both LmCyt-b₅ and LmCPR did not significantly change the total CYP activity toward the substrate 7-ethoxycoumarin (7-EC). However, coexpression of LmCYP6FD1 with LmCPR and LmCyt-b₅together in Sf9 cells by using Bac-to-Bac baculovirus expression system significantly increased the catalytic activity of LmCYP6FD1 toward 7-EC as compared with the coexpression of LmCYP6FD1 with cytochrome P450 reductase (LmCPR) or LmCyt-b₅ separately. These results suggest that LmCyt-b₅ plays an important role in the catalytic reaction of LmCYP6FD1 toward 7-EC in our in vitro experiments. Further study is needed to clarify the role of LmCyt-b₅ in CYP-mediated catalytic reactions in L. migratoria.

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A Photosensitivity Insecticide, 5-Aminolevulinic Acid, Exerts EffectiveToxicity to Oxya chinensis (Orthoptera: Acridoidea)

YANG Mei-ling; YIN Kun; GUO Ya-ping; MA En-bo and ZHANG Jian-zhen

2011, 10 (7): 1056-1063. DOI: 10.1016/S1671-2927(11)60094-1

Abstract （1872） PDF in ScienceDirect

5-Aminolevulinic acid (ALA), a major photosensitivity insecticide, has attracted increasing attention as a new type of highly efficient, environmental friendly pesticide to be used to control the pest. To examine whether or not ALA acts effectively to grasshopper, Oxya chinensis and elucidate the detoxification mechanism of ALA, the susceptibility to ALA was assessed in O. chinensis and two major metabolic detoxification enzymes including glutathione S-transferases (GSTs) and general esterases (ESTs)-specific activities were compared in different development stages and different body sections
of O. chinensis treated by ALA and the control. The results showed that the ALA exhibited obvious toxicity to the grasshopper in different development stages. In the low-dose treatment (0.0597 mmol L-1), the mortalities of O. chinensis reached a significant level (55.5% in the 1st instar nymphs, 61.4% in the 2nd instar nymphs, 71.4% in the 3rd instar nymphs, and 64.4% in the 4th instar nymphs. But, there was no dose-dependent toxic effect. Thereby, we proposed that ALA has the potential for acting as photosensitivity insecticide for controlling O. chinensis. GSTs activity assays using CDNB and DCNB as substrates indicated that the thorax and abdomen of the different instar nymphs treated by ALA showed 1.52-5.56 fold significantly increased GSTs activities compared with the control. However, for the ESTs-specific activity assay, there was no significant difference between O. chinensis treated by ALA and the control within different instar nymphs, when α-NA, α-NB and β-NA were used as substrates. Therefore, GSTs-mediated metabolic detoxification
as evidenced by significantly increased GSTs activities might contribute to protect against oxidative damage and oxidative
stress by ALA in O. chinensis.

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Expression and Characterization of a Sigma-Class Glutathione S-transferase of the Oriental Migratory Locust, Locusta migratoria manilensis (Meyen)

JIA Miao, QIN Guo-hua, LIU Ting, ZHANG Jian-zhen, ZHANG Xue-yao, ZHU Kun-yan, GUO Yaping, MA En-bo

2011, 10 (10): 1570-1576. DOI: 10.1016/S1671-2927(11)60153-3

Abstract （1822） PDF in ScienceDirect

A cDNA encoding a sigma-class glutathione S-transferase of the locust, Locusta migratoria manilensis (LmGSTs1), was cloned by reverse transcriptase-polymerase chain reaction. The 830 bp-long cDNA encoded a 615 bp open reading frame (204 amino acid polypeptide), which exhibited the structural motif and domain organization characteristic of GST sigmaclass. It revealed 59, 57, 57, and 56% identities to sigma-class GSTs from Blattella germanica, Gryllotalpa orientalis, Nasonia vitripennis, and Pediculus humanus corporis, respectively. A recombinant protein (LmGSTs1) was functionally expressed in Escherichia coli cells in a soluble form and purified to homogeneity. LmGSTs1 was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GSTs, as well as with p-nitro-benzyl chloride. Its optimal activity was observed at pH 8.0 and at 30°C. Incubation for 30 min at temperatures below 50°C scarcely affected the activity. The I50 of reactive blue (RB) was 18.5 μmol L-1. In the presence of 0.05 mmol L-1 ethacrynic acid (ECA), LmGSTs1 showed (81±3)% of the original activities.

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