Microsurgical Obstruction of Testes Fusion in Spodoptera litura

Summary

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.

Abstract

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.

Introduction

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 fusion^2,3,4.

Spodoptera litura (S. litura, Lepidoptera: Noctuidae) is a detrimental polyphagous pest that is widely distributed in tropical and subtropical areas of Asia, including China^4,5,6. The wide distribution of S. litura is partly attributed to its powerful reproductive capability, which is relevant to gonad development. Male infertility is one approach to control this pest. As shown in the schematic figure of testicular structure, the testes are enclosed by the testicular sheath, including the external sheath (peritoneal sheath) and inner basal lamina. The basal lamina extends internally to form the follicular epithelium and separates the inner area of the testis into four chambers named follicles (Figure 1).

In the follicles, spermatogonia develop into spermatozoa after mitosis and meiosis, and then the spermatozoa in the sperm sacs align in the same direction to form sperm bundles⁷. During spermatogenesis, the primary spermatocytes differentiate into eupyrene sperms or apyrene sperms. Spermatocytes in the larval phase develop into eupyrene sperm with a long tail connected to a head of an elongated nucleus; these can fertilize eggs. Conversely, spermatocytes in the mid-pupal phase develop into apyrene sperm with a discarded nucleus; these sperm assist the survival, motion, and fertilization of eupyrene sperm^9,10. The 6^th day of the pupa is the period during which the testes have abundant eupyrene and apyrene sperm bundles.

Figure 1: Schematic diagram of the testicular structure of Lepidoptera insects¹¹. Please click here to view a larger version of this figure.

Testicular fusion occurs in most insects of the Lepidoptera order^11,12, especially in those species that are agricultural pests. Testicular fusion refers to a pair of testes growing bilaterally in the larval phase, approaching and adhering to each other, eventually integrating into a single gonad¹¹. In Spodoptera litura, it happens during metamorphosis from the larval to the pupal stage. From day 1 of the 5^th instar (L5D1) to day 4 of the 6^th instar (L6D4), the pair of testes grows gradually in size, and the color turns light yellow from ivory-white. It becomes faint red as it reaches the prepupal phase (L6D5 to L6D6). Two bilateral symmetrical testes approach each other during the prepupal stage, fuse into one, and twist anticlockwise (doral view) to produce a single testis in the pupal and adult phases¹¹. This phenomenon does not occur in silkworms, which have considerable economic importance and have been domesticated for 5000 years¹³. Thus, it is assumed that the testes' fusion improves reproductive capability.

To determine the significance of Spodoptera litura testicular fusion, it is important to investigate the effects of blocking the process. In this protocol, aluminum foil was microsurgically inserted between the testes to keep them separated, and the consequent changes in the development of the insects and their testes were studied.

Protocol

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 6^th instar (L6D4). Select male larvae when the worms enter the first day of the 6^th instar (L6D0) based on the inverse triangle-shaped structure on the eighth abdomen¹⁴.
   ​NOTE: Larvae rearing and maintenance techniques were published previously^4,14.

2. Presurgical preparation

1. Trim the aluminum foil into rectangular pieces with rounded corners (1 mm x 2 mm, Figure 2).
2. Sterilize the surgery platform and related items (table surface, microscope, icebox, insect box, wax tray, pins, and thread) by spraying 75% alcohol on their surface and wiping them down.
3. Sterilize surgical tools (including the aluminum foil) with a high-pressure steam sterilizer for 30 min, and place them in a heating and drying oven at 120 °C.
4. Ensure that the operators wear clean laboratory clothes, surgical masks, and sterile gloves.

3. Microsurgical placement of a barrier between the testes

NOTE: The general work-flow is as follows: Freezing → Fixing → Disinfection → Incision → Barrier Placement → Suturing→ Postoperative Feeding and Inspection

1. Place male larvae (L6D4) on ice for 10-30 min to keep them anesthetized during the operation.
2. Place a larva on the wax tray with the dorsal side up, and then fix the head and the tail of the larva with pins and threads, showing the surgical area that is the dorsal surface on the 9^th body segment (Figure 3A).
3. Disinfect the surgical area by applying 3% iodine tincture with a cotton swab to the epidermis (9^th body segment), followed by 70% alcohol to remove the iodine (Figure 3B).
   NOTE: Focus on the larva through coarse and fine adjustment of the surgical microscope (Figure 3C). Place the wax tray on a larger culture dish filled with ice to keep the anesthesia.
4. Make a 2 mm-long incision on the dorsal epidermis of the 9^th body segment. Next, use a sterile cotton swab to remove any leaking hemolymph and fat bodies and obtain a clear view of the surgical area.
   NOTE: It is important to avoid the heart during the procedure. This can be done by making the incision slightly next to the mid-line in the 9^th body segment or at the joint between the 9^th and 10^th body segments to prevent the testes from popping out due to the larval internal pressure. While using the scalpel, make a vertical slit with the blade first (Figure 4A), and then turn it 45° towards the epidermis before evenly and continuously cutting through the epidermis (Figure 4B).
5. Use surgical tweezers to insert a piece of aluminum foil between the testes (Figure 5).
6. At the end of the surgery, close the incision to avoid infection, and allow the larvae to recover from the surgery.
   1. Close the epidermis with a running suture (Figure 6).
   2. Use a needle holder and surgical tweezers to tie a surgical square knot, requiring two opposing mirror-image simple knots (Figure 6D,E).
   3. Use scissors to cut the excess suture from the loop tails, leaving a 2 mm thread behind.
7. After suturing, gently lay the larva in the rearing box and maintain them in a clean environmental simulation chamber. Continue observing the larvae.
   ​NOTE: The wound stops leaking hemolymph, and the larvae gradually recover after the surgery. The worms continue to complete their metamorphosis.

Figure 2: Physical barrier prepared using aluminum foil (1 mm x 2 mm). Please click here to view a larger version of this figure.

Figure 3: Before incision. (A) Fixing the larva. (B) Disinfection of the epidermis of the surgical area. (C)Performing surgery under the microscope. Please click here to view a larger version of this figure.

Figure 4: Incision. (A) Slit the larvae vertically with the blade. (B) Turn the blade 45° toward the epidermis before cutting through. Please click here to view a larger version of this figure.

Figure 5: Inserting the physical barrier (aluminum foil) between the testes. Please click here to view a larger version of this figure.

Figure 6: Suturing. (A) Insert the needle. (B) Withdraw the needle. (C) Withdraw and clamp the needle. (D) Tie the first simple knot. (E) Tie the opposing mirror-imaged simple knot. (F) Cut excess suture thread. Please click here to view a larger version of this figure.

Results

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...

Discussion

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 20^th 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...

Materials

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