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Materials

References

Biology

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

Published: July 16th, 2021

DOI:

10.3791/62524

1South China Normal University-Shipai Campus: South China Normal University, 2Southwest University
* These authors contributed equally

Aluminum foil was microsurgically inserted between the testes of Spodoptera litura to obstruct the fusion of testis. The procedure includes freezing, fixing, disinfection, incision, placing the barrier, suturing, postoperative feeding, and inspection. This approach provides a method to interfere with tissue formation.

Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease Cas9, a physical barrier was microsurgically inserted between the testes of Spodoptera litura to study the impact of this microsurgery on its growth and reproduction. After inserting aluminum foil between the testes, insect molting during metamorphosis proceeded normally. Insect growth and development were not remarkably altered; however, the number of sperm bundles changed if testes fusion was stopped by the microsurgery. These findings imply that blocking testicular fusion can influence male reproduction capability. The method can be further applied to interrupt communication between organs to study the function of specific signaling pathways. Compared to conventional surgery, microsurgery only requires freezing anesthetization, which is preferable to carbon dioxide anesthetization. Microsurgery also minimizes the surgery site area and facilitates wound healing. However, the selection of materials with specific functions needs further investigation. Avoiding tissue injury is crucial when making incisions during the operation.

Fusion is a common phenomenon in tissue or organ development. Examples include dorsal closure and thorax closure in Drosophila1 and palate morphogenesis, neural tube morphogenesis, and heart morphogenesis in mice and chicken2. CRISPR and RNAi have been applied to investigate the roles of genes in the process of fusion2,3,4.

Spodoptera litura (S. litura, Lepidoptera: Noctuidae) is a detrimental polyphagous pest that is widely distributed in tropical and subtropical areas of Asia,....

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1. Insect rearing and maintenance

  1. Culture the Spodoptera litura larvae in environmental simulation chambers with an artificial diet until they reach day 4 of the 6th instar (L6D4). Select male larvae when the worms enter the first day of the 6th instar (L6D0) based on the inverse triangle-shaped structure on the eighth abdomen14.
    ​NOTE: Larvae rearing and maintenance techniques were published previously4,

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The effects of microsurgery on Spodoptera litura growth and development
The microsurgery left a 2 mm-long wound on the dorsal larval epidermis that eventually stopped leaking hemolymph and healed. The larvae went through prepupal and pupal stages and eclosed, indicating that the microsurgery had no major impact on growth and development. When the larvae molted into pupae, the suture threads were discarded along with the epidermis. There were no obvious dif.......

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After microsurgically obstructing testes fusion in Spodoptera litura, the number of sperm bundles decreased, which supported the hypothesis that this fusion is beneficial to the reproductive capability. Surgical manipulation has been used to study the physiological development of insects since the early 20th century. To determine whether the cranial nerve is regulated by insect metamorphosis, some researchers performed procedures such as ligation and decapitation on different insects (including Rh.......

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This work were supported by National Natural Science Foundation of China (Nos.:31772519, 31720103916; ) and an open grant from the State Key Laboratory of Silkworm Genome Biology, South West University (No.: sklsgb2013003).

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Name Company Catalog Number Comments
75% Rubbing alcohol Qingdao Hainuo Nuowei Disinfection Technology Co., Ltd Q/370285HNW 001-2019
Colored Push Pins Deli Group Co.,LTD 0042
Corneal Scissors Suzhou Xiehe Medical Device Co., Ltd MR-S221A Curved and blunt tip
Glad Aluminum Foil Clorox China(Guangzhou) Limited 831457 10 cm*2.5 cm*0.6
Medical Cotton Swabs (Sterile) Winner Medical Co., Ltd. 601-022921-01
Medical Iodine Cotton Swab Winner Medical Co., Ltd. 608-000247
Needle holder Shanghai Medical Instruments (Group) Ltd., Corp. J32030 14 cm fine needle
Sterile surgical blade Shanghai Pudong Jinhuan Medical Supplies Co., LTD #11
Suigical Blade Holder Shanghai Pudong Jinhuan Medical Supplies Co., LTD K6-10 Straight 3#
Suture thread with needle Ningbo Medical Stitch Needle Co., Ltd needle: 3/8 Circle, 2.5*8 ; Thread: Nylon, 6/0, 25 cm
Tying Forceps Suzhou Xiehe Medical Device Co., Ltd MR-F201T-3 Straight-pointed; long handle; 0.12 mm-wide-head

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