Name: microsurgical obstruction of testes fusion in spodoptera litura
Uploaded: 2021-07-16T13:00:00.000+00:00
Duration: 3 min 38 s
Description: Watch this Scientific Journal Video about Microsurgical Obstruction of Testes Fusion in Spodoptera litura at JoVE.com

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).

1. Insect rearing and maintenance
<ol>
	<li>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. 
	&#8203;NOTE: Larvae rearing and maintenance techniques were published previously4,14.</li>
</ol>
2. Presurgical preparation
<ol>
	<li>Trim the aluminum foil into rectangular pieces with rounded corners (1 mm x 2 mm, Figure 2).</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>Ensure that the operators wear clean laboratory clothes, surgical masks, and sterile gloves.</li>
</ol>
3. Microsurgical placement of a barrier between the testes
NOTE: The general work-flow is as follows: Freezing &#8594; Fixing &#8594; Disinfection &#8594; Incision &#8594; Barrier Placement &#8594; Suturing&#8594; Postoperative Feeding and Inspection
<ol>
	<li>Place male larvae (L6D4) on ice for 10-30 min to keep them anesthetized during the operation.</li>
	<li>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 9th body segment (Figure 3A).</li>
	<li>Disinfect the surgical area by applying 3% iodine tincture with a cotton swab to the epidermis (9th 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.</li>
	<li>Make a 2 mm-long incision on the dorsal epidermis of the 9th 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 9th body segment or at the joint between the 9th and 10th 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&#176; towards the epidermis before evenly and continuously cutting through the epidermis (Figure 4B).</li>
	<li>Use surgical tweezers to insert a piece of aluminum foil between the testes (Figure 5).</li>
	<li>At the end of the surgery, close the incision to avoid infection, and allow the larvae to recover from the surgery.
	<ol>
		<li>Close the epidermis with a running suture (Figure 6).</li>
		<li>Use a needle holder and surgical tweezers to tie a surgical square knot, requiring two opposing mirror-image simple knots (Figure 6D,E).</li>
		<li>Use scissors to cut the excess suture from the loop tails, leaving a 2 mm thread behind.</li>
	</ol>
	</li>
	<li>After suturing, gently lay the larva in the rearing box and maintain them in a clean environmental simulation chamber. Continue observing the larvae. 
	&#8203;NOTE: The wound stops leaking hemolymph, and the larvae gradually recover after the surgery. The worms continue to complete their metamorphosis.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62524/62524fig02.jpg" /> 
Figure 2: Physical barrier prepared using aluminum foil (1 mm x 2 mm). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62524/62524fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62524/62524fig03.jpg" /> 
Figure 3: Before incision. (A)&#160;Fixing the larva. (B)&#160;Disinfection of the epidermis of the surgical area. (C)Performing surgery under the microscope. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62524/62524fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62524/62524fig04.jpg" /> 
Figure 4: Incision. (A) Slit the larvae vertically with the blade.&#160;(B)&#160;Turn the blade 45&#176; toward the epidermis before cutting through. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62524/62524fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62524/62524fig05.jpg" /> 
Figure 5: Inserting the physical barrier (aluminum foil) between the testes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62524/62524fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62524/62524fig06.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62524/62524fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have nothing to disclose.

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 Rhodnius prolixus of Hemiptera, Lymantria dispar of Lepidoptera)15,16. The process of decapitation involves removing the head with a scalpel, disinfection with antibiotics, and paraffin wax sealing of the wound after operation17. After extraction and transplantation of the prothoracic glands of Bombyx mori, the wound was sealed with paraffin wax18. However, the inevitable consequences of these conventional treatments are infection and a high mortality rate, which makes it difficult to analyze the physiological state during the late stages of development of the insect. 
Therefore, this protocol was designed to ensure a minimally invasive surgery done under the microscope to minimize the wound. Moreover, compared to carbon dioxide anesthetization, freezing anesthetization is more feasible and convenient. Aluminum foil, used as the obstructer, was cut into a size of 1 mm x 2 mm, an area equivalent to the space between the testes. Following microsurgery, the suture threads fall off with the early epidermis during molting, allowing metamorphosis and development to proceed normally. The reproduction results suggest that successful microsurgery did not significantly influence insect development. When the testes did not fuse, the numbers of total, eupyrene, and apyrene sperm bundles were significantly lower than those in the Ctl group. These results indicate that male reproductive capability is affected by testes fusion. Assessment of sperm cell quality and vitality have different indexes and methods in various animals, including sperm acrosomal status in mammals19, sperm motility20, mitochondrial activity21, plasma membrane integrity22, and other markers23,24 . Because of insects' unique sperm cell development, future studies need to examine changes in reproduction capability (mating, incubation25).
Critical steps in this protocol require particular attention to ensure reliable results. Avoidance of injury to other tissues is important when making the incisions. Second, the selection of the barrier material must be based on its nontoxic and sterile properties and lack of sharp boundaries. Finally, the incision was closed with a running suture and a surgical square knot, followed by sealing in the surgical area to effectively prevent postoperative infection. Operations on the insect's internal structure such as transplantation, extraction, and application of drugs can still be performed, followed by sealing in the surgical area.
High success rates require proficient skills, and this technique has some disadvantages. First, it is not efficient, as the operations are done one by one, due to which individual variation is inevitable. Preliminary studies showed that when using medical venous transfusion tubes, rubber diaphragms, absorbent beads, and dental materials to separate the testes, the outcomes were not as successful as expected. Moreover, the technique is not successful when the obstructer is adrift. Possible reasons for a decreased surgical success rate include the barrier slipping off when the insects move, shrink, molt, and re-organize organs during the prepupal phase. Alternatively, the aluminum foil can be inserted too close to the lubricous gut, causing the barrier to float away. Therefore, a suitable material should be further optimized. 
Despite the limitations of microsurgery, it provides a method to obtain preliminary results about biological phenomena before establishing a transgenic model system. Sweeney and Waterson analyzed rid development in chick embryos by inserting tantalum foil blocks26, while Wilde and Logan used aluminum foil as an impermeable barrier to study the role of retinoic acid signaling in the induction and subsequent initiation of fore- and hindlimbs27. In invertebrate Spodoptera litura, this microsurgery successfully enables normal worm growth and development, providing a way to study physiological phenomena.

Fusion is a common phenomenon in tissue or organ development. Examples include dorsal closure and thorax closure in Drosophila1&#160;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, including China4,5,6. 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 sperm9,10. The 6th day of the pupa is the period during which the testes have abundant eupyrene and apyrene sperm bundles.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62524/62524fig01.jpg" /> 
Figure 1: Schematic diagram of the testicular structure of Lepidoptera insects11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62524/62524fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Testicular fusion occurs in most insects of the Lepidoptera order11,12, 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 5th instar (L5D1) to day 4 of the 6th 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&#39; 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.

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

microsurgical obstruction of testes fusion in spodoptera litura

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.

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 differences in the appearance of the pupae that did and did not undergo surgery. After eclosion, adult females successfully mated with the adult males previously operated on, resulting in fertilized eggs and hatching larvae (Figure 7).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62524/62524fig07.jpg" /> 
Figure 7: Spodoptera litura Development after microsurgery. (A) Male larva at L6D4.&#160;(B) L6D4 larva immediately after surgery. (C) Pre-pupa (L6D6). (D) P0, the red arrow indicates the location of the surgery; the yellow arrow shows the discarded epidermis with suture thread. (E) Mating adults. (F) Eggs and hatched larvae from a female adult mating with a male that underwent surgery. Scale bars = 1 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62524/62524fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The larvae went through the pupal stage and eclosed after the microsurgical placement of aluminum foil between the testes. Detailed results following this operation have been published previously11. Although the barrier stopped the testes from fusing in some larvae, most larvae underwent testicular fusion during larval to pupal metamorphosis.
In this research, individuals were grouped by three treatments: Experimental (Exp), Sham-operation (Ctl-sham), and no operation (Ctl). Individuals of the Exp group underwent microsurgery to insert a physical barrier, and their testes remained separated during the pupal and adult stages. Individuals of the Ctl-sham group underwent the same microsurgery; however, their testes were not blocked and fused for unknown reasons. The Ctl group contained the larvae that grew naturally without surgery; their two testes fused normally during the prepupal stage.
The microsurgery group contained two subgroups: larvae that underwent microsurgery to place a barrier between the two testes (Group A) and those that underwent microsurgery to remove one testis (Group B: left testis removed in Group B-1; right testis removed in Group B-2). Table 1 shows the numbers of operation larvae, larval mortality rates, numbers of pupae, percentage of pupation, numbers of adults, percentages of adult emergence, percentages of successful mating, and percentages of successful operations in different groups. Group A includes larvae that underwent microsurgery to insert a barrier between the testes. The success of this procedure could only be determined after dissection, which is when they were further divided into the Exp and Ctl-sham groups.
As shown in Figure 8, the larval mortality rate was slightly higher in the surgical group, whereas the percentages of pupation, adult emergence, and successful mating were slightly lower in the surgical group than the control group. However, none of the differences were significantly different, indicating that the microsurgery did not markedly influence the growth and development of Spodoptera litura larvae.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Group</td>
			<td>Number of Larvae</td>
			<td>Larval Mortality Rate/%</td>
			<td>Number of Pupae</td>
			<td colspan="2">Percentage of Pupation/%</td>
			<td>Number of Adult</td>
			<td colspan="2">Percentage of Adult Emergence/%</td>
			<td colspan="2">Percentage of Successful Mating/%</td>
			<td colspan="2">Percentage of Successful Operation/%</td>
		</tr>
		<tr>
			<td colspan="2">Microsurgery group A-1*</td>
			<td>79</td>
			<td>35.4</td>
			<td>39</td>
			<td colspan="2">76.5</td>
			<td>N</td>
			<td colspan="2">N</td>
			<td colspan="2">N</td>
			<td colspan="2">10.3</td>
		</tr>
		<tr>
			<td colspan="2">Microsurgery group A-2*</td>
			<td>117</td>
			<td>12.8</td>
			<td>102</td>
			<td colspan="2">100</td>
			<td>N</td>
			<td colspan="2">N</td>
			<td colspan="2">N</td>
			<td colspan="2">11.8</td>
		</tr>
		<tr>
			<td colspan="2">Microsurgery group A-3*</td>
			<td>73</td>
			<td>13.7</td>
			<td>57</td>
			<td colspan="2">90.5</td>
			<td>N</td>
			<td colspan="2">N</td>
			<td colspan="2">N</td>
			<td colspan="2">10.5</td>
		</tr>
		<tr>
			<td colspan="2">Microsurgery group A-4</td>
			<td>101</td>
			<td>4</td>
			<td>97</td>
			<td colspan="2">96</td>
			<td>29</td>
			<td colspan="2">29.9</td>
			<td colspan="2">N</td>
			<td colspan="2">26.9</td>
		</tr>
		<tr>
			<td colspan="2">Microsurgery group A-5</td>
			<td>176</td>
			<td>20.1</td>
			<td>140</td>
			<td colspan="2">79.5</td>
			<td>28</td>
			<td colspan="2">20</td>
			<td colspan="2">44.4</td>
			<td colspan="2">25</td>
		</tr>
		<tr>
			<td colspan="2">Microsurgery group A-6</td>
			<td>434</td>
			<td>12.4</td>
			<td>376</td>
			<td colspan="2">98.9</td>
			<td>209</td>
			<td colspan="2">55.6</td>
			<td colspan="2">26.8</td>
			<td colspan="2">14.3</td>
		</tr>
		<tr>
			<td colspan="2">Microsurgery group A-7</td>
			<td>260</td>
			<td>10.8</td>
			<td>135</td>
			<td colspan="2">58.2</td>
			<td>66</td>
			<td colspan="2">48.9</td>
			<td colspan="2">47</td>
			<td colspan="2">48.4</td>
		</tr>
		<tr>
			<td colspan="2">Microsurgery group A-8</td>
			<td>49</td>
			<td>24.5</td>
			<td>37</td>
			<td colspan="2">100</td>
			<td>21</td>
			<td colspan="2">56.8</td>
			<td colspan="2">81</td>
			<td colspan="2">58.8</td>
		</tr>
		<tr>
			<td colspan="2">Microsurgery group B-1</td>
			<td>117</td>
			<td>29.1</td>
			<td>71</td>
			<td colspan="2">85.5</td>
			<td>30</td>
			<td colspan="2">42.3</td>
			<td colspan="2">23.3</td>
			<td colspan="2">N</td>
		</tr>
		<tr>
			<td colspan="2">Microsurgery group B-2</td>
			<td>188</td>
			<td>6.9</td>
			<td>172</td>
			<td colspan="2">98.3</td>
			<td>115</td>
			<td colspan="2">66.9</td>
			<td colspan="2">35.7</td>
			<td colspan="2">N</td>
		</tr>
		<tr>
			<td colspan="2">Average of Microsurgery Group (mean&#177; SD)</td>
			<td>159</td>
			<td>17&#177;10.1</td>
			<td>123</td>
			<td colspan="2">88.3&#177;13.7</td>
			<td>71</td>
			<td colspan="2">45.8&#177;16.3</td>
			<td colspan="2">43&#177;20.8</td>
			<td colspan="2">25.8&#177;18.5</td>
		</tr>
		<tr>
			<td colspan="2">Control Group 1*</td>
			<td>40</td>
			<td>17</td>
			<td>37</td>
			<td colspan="2">100</td>
			<td>N</td>
			<td colspan="2">N</td>
			<td colspan="2">N</td>
			<td colspan="2">N</td>
		</tr>
		<tr>
			<td colspan="2">Control Group 2</td>
			<td>300</td>
			<td>0</td>
			<td>281</td>
			<td colspan="2">93.7</td>
			<td>184</td>
			<td colspan="2">65.5</td>
			<td colspan="2">N</td>
			<td colspan="2">N</td>
		</tr>
		<tr>
			<td colspan="2">Control Group 3</td>
			<td>354</td>
			<td>11</td>
			<td>305</td>
			<td colspan="2">96.8</td>
			<td>127</td>
			<td colspan="2">41.6</td>
			<td colspan="2">N</td>
			<td colspan="2">N</td>
		</tr>
		<tr>
			<td colspan="2">Control Group 4</td>
			<td>679</td>
			<td>2.7</td>
			<td>638</td>
			<td colspan="2">96.5</td>
			<td>534</td>
			<td colspan="2">83.7</td>
			<td colspan="2">41.2</td>
			<td colspan="2">N</td>
		</tr>
		<tr>
			<td colspan="2">Control Group 5</td>
			<td>448</td>
			<td>4.2</td>
			<td>399</td>
			<td colspan="2">93</td>
			<td>232</td>
			<td colspan="2">58.1</td>
			<td colspan="2">60</td>
			<td colspan="2">N</td>
		</tr>
		<tr>
			<td colspan="2">Control Group 6</td>
			<td>490</td>
			<td>7.1</td>
			<td>448</td>
			<td colspan="2">98.5</td>
			<td>355</td>
			<td colspan="2">79.2</td>
			<td colspan="2">48</td>
			<td colspan="2">N</td>
		</tr>
		<tr>
			<td colspan="2">Average of Control Group (mean&#177; SD)</td>
			<td>385</td>
			<td>5.4&#177;6.2</td>
			<td>351</td>
			<td colspan="2">96.4&#177;2.7</td>
			<td>286</td>
			<td colspan="2">65.6&#177;15.1</td>
			<td colspan="2">50&#177;9.5</td>
			<td colspan="2">N</td>
		</tr>
	</tbody>
</table>
Table 1: The effects of microsurgery on Spodoptera litura development. Microsurgery groups B-1 and B-2 underwent microsurgery to remove unilateral testis (left in Microsurgery Group B-1 and right in Microsurgery Group B-2). Note: Microsurgery groups A-1 to A-8 underwent microsurgery to insert a barrier between the testes; Microsurgery groups B-1 and B-2 underwent microsurgery to remove unilateral testis (left in Microsurgery Group B-1 and right in Microsurgery Group B-2); the rates and percentages are given as Mean &#177; SD. Asterisks indicate that the individuals in the group were dissected at the pupal stage, and there were no statistics on the number of adults, percentage of adult emergence, or percentage of successful mating; N indicates no data.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62524/62524fig08.jpg" /> 
Figure 8: The influence of microsurgery on Spodoptera litura growth and development of (n &#8805; 6). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62524/62524fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The influence of microsurgery on the number of sperm bundles of Spodoptera litura 
Microsurgery was performed to insert a physical barrier to stop the testes fusion or remove unilateral testis in Spodoptera litura. Eupyrene and apyrene sperm bundles were counted to calculate the percentage of eupyrene sperm bundles on the sixth day of the pupal stage. The individuals were grouped by treatment, as described above. The numbers of sperm bundles (eupyrene sperm bundles, apyrene sperm bundles, and total) were significantly lower in the Exp group than in the Ctl-sham and Ctl groups. The mean number of eupyrene sperm bundles from two separated testes in the Exp group was 2082 &#177; 599. In the Ctl-sham and Ctl groups with fused testes, the number of eupyrene sperm bundles ranged from 4652 to 6200.
The number of apyrene sperm bundles in the Exp group was 1602 &#177; 703, while it ranged from 3299 to 4632 in the Ctl-Sham and Ctl groups. The total of sperm bundles in the Exp group was 3684 &#177; 985; it ranged from 9284 to 10832 in the Ctl-Sham and Ctl groups. Thus, the percentages of eupyrene sperm bundles ranged from 50% to 60%, with no significant differences among all three groups. Figure 9 shows that when fusion is prevented, the amount of eupyrene and apyrene sperm bundles decreased, whereas the percentage of eupyrene sperm bundles was unchanged.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62524/62524fig09.jpg" /> 
Figure 9: The numbers of sperm bundles and percentages of eupyrene sperm bundles in different groups. (A) The number of sperm bundles in the Exp group was significantly lower than in the Ctl-sham and Ctl groups. (B) The percentages of eupyrene sperm bundles were not significantly different among the three groups. Asterisk indicates a significant difference when compared with Ctl. P &#60; 0.05, Mean &#177; SD (n &#8805;5). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62524/62524fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
After removing a unilateral testis of the larvae, the numbers of eupyrene and apyrene sperm bundles were counted to calculate the percentage of eupyrene sperm bundles on the sixth day of the pupal stage. The number of eupyrene and apyrene sperm bundles ranged from 1286 to 1638 and 720 to 850, respectively, which means the total number ranged from 2006 to 2488, corresponding to a eupyrene sperm bundle percentage of 63% to 65%. Figure 10 shows that the number of sperm bundles decreased significantly after unilateral testis removal (reduced by 60% to 70%), without much influence on the percentage of eupyrene sperm bundles.
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/62524/62524fig10.jpg" /> 
Figure 10: The numbers of sperm bundles and percentages of eupyrene sperm bundles after removing unilateral testis. (A) The numbers of sperm bundles in pupae that underwent unilateral testis removal were significantly different among the three groups (left and right testis removed in Microsurgery Group B-1 and Microsurgery Group B-2, respectively) (B) The percentage of eupyrene sperm bundles in pupae that underwent unilateral testis removal was not significantly different compared with Ctl. The asterisk indicates a significant difference compared with Ctl. P &#60; 0.05, Mean &#177; SD (n &#8805;8). Control group = no surgery, the testes fused naturally during the prepupal stage; Ctl-Sham group = operation unsuccessful and testes fused after microsurgery; Exp. Group = microsurgery performed to insert a physical barrier between the two testes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62524/62524fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Microsurgical Obstruction of Testes Fusion in Spodoptera litura at JoVE.com

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

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.

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated ...

microsurgical-obstruction-of-testes-fusion-in-spodoptera-litura

Nanyang Technological University

Research

JoVE Journal

Biology

2.4K Views.  South China Normal University-Shipai Campus: South China Normal University. Dit protocol suggereert dat een fysieke methode kan worden gebruikt om te helpen bij het uitzoeken van biologische problemen. Deze techniek biedt een methode om materiaal in insecten te plaatsen met minimale incisie en de invloed van microchirurgie op de ontwikkeling van individuen te bestuderen. Deze methode kan van toepassing zijn op andere Lepidoptera-larven en andere delen van de insecten, waaronder het hoofd en de thorax.Het is erg belangrijk om te oefenen met het maken van incisi...

Video: Microsurgical Obstruction of Testes Fusion in Spodoptera litura

Teratoma Generation in the Testis Capsule

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers. This makes them a potentially valuable tool for the treatment of many different diseases. With the advent of induced pluripotent stem cells (iPSCs) and continuing research with human embryonic stem cells (hESCs) there is a need for assays that can demonstrate that a particular cell line is pluripotent. Germline transmission has been the gold standard for demonstrating the pluripotence of mouse embryonic stem cell (mESC) lines1,2,3. Using this assay, researchers can show that a mESC line can make all cell types in the embryo including germ cells4. With the generation of human ESC lines5,6, the appropriate assay to prove pluripotence of these cells was unclear since human ESCs cannot be tested for germline transmission. As a surrogate, the teratoma assay is currently used to demonstrate the pluripotency of human pluripotent stem cells (hPSCs)7,8,9. Though this assay has recently come under scrutiny and new technologies are being actively explored, the teratoma assay is the current gold standard7. In this assay, the cells in question are injected into an immune compromised mouse. If the cells are pluripotent, a teratoma will eventually develop and sections of the tumor will show tissues from all 3 germ layers10. In the teratoma assay, hPSCs can be injected into different areas of the mouse. The most common injection sites include the testis capsule, the kidney capsule, the liver; or into the leg either subcutaneously or intramuscularly11. Here we describe a robust protocol for the generation of teratomas from hPSCs using the testis capsule as the site for tumor growth.

Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases. The utility of these cells lies in the fact that they can differentiate into any cell type in the body. Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs.

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

Medicine

Micromanipulation of Chromosomes in Insect Spermatocytes

The micromanipulation of chromosomes has been an essential method for illuminating the mechanism for chromosome congression, the spindle checkpoint, and anaphase chromosome movements, and has been key to understanding what controls chromosome movements during a cell division. A skilled biologist can use a micromanipulator to detach chromosomes from the spindle, to reposition chromosomes within the cell, and to apply forces to chromosomes using a small glass needle with a very fine tip. While perturbations can be made to chromosomes using other methods such as optical trapping and other uses of a laser, to date, no other method allows the repositioning of cellular components on the scale of tens to hundreds of microns with little to no damage to the cell.
The selection and preparation of appropriate cells for the micromanipulation of chromosomes, specifically describing the preparation of grasshopper and cricket spermatocyte primary cultures for the use in live-cell imaging and micromanipulation, are described here. In addition, we show the construction of a needle to be used for moving chromosomes within the cell, and the use of a joystick-controlled piezoelectric micromanipulator with a glass needle attached to it to reposition chromosomes within dividing cells. A sample result shows the use of a micromanipulator to detach a chromosome from a spindle in a primary spermatocyte and to reposition that chromosome within the cell.

In this protocol, we describe the selection and preparation of appropriate cells for micromanipulation and the use of a piezoelectric micromanipulator to reposition chromosomes within those cells.

The micromanipulation of chromosomes has been an essential method for illuminating the mechanism for chromosome congression, the spindle checkpoint, and ...

Developmental Biology

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Murine Round Spermatid Injection Procedure to Study Embryonic Development

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Fluorescence In Situ Hybridization to Study Mosquito Spermatogenesis

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

Author Spotlight: Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito  

Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production.

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...

Immunology and Infection

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Vessel-Sparing Microsurgical Longitudinal Intussusception Vasoepididymostomy to Treat Epididymal Obstructive Azoospermia

The epididymis is a common site of obstruction in obstructive azoospermia (OA). Vasoepididymostomy has become an important method for the treatment of epididymal OA since 2000. There are two challenges in classic microscopic vasoepididymostomy. First, anastomosis of the vas deferens and epididymis is performed with double-needle sutures. However, there is a lack of good-quality and cost-effective double-needle sutures in China, which leads to increased difficulty and poor success rates of anastomosis. Second, the separation of the vas deferens does not retain vasculature, although the vas deferens vasculature plays an important role in the blood supply to the vas deferens, epididymis, and testis. This affects the blood supply to the anastomotic area and epididymis.
Therefore, this team has made innovative improvements to address these problems. Good-quality, cost-effective, single-needle sutures, which are easy to purchase in China and other countries, were used in microsurgical longitudinal intussusception vasoepididymostomy. This can optimize the operation procedure and shorten the operation time while ensuring the success rate of the anastomosis. The surgical method of preserving the vas deferens vessels was innovatively proposed because the etiology of epididymal OA is mostly inflammatory in China. The protection of the blood supply to the vas deferens and epididymis is maximized using microsurgical forceps to separate and protect the vasculature. Patency reached 81.7% in the postoperative follow-up, indicating a better surgical treatment effect.

The present protocol describes a vessel-sparing, longitudinal intussusception vasoepididymostomy using readily available single-needle sutures in China, a safe and effective procedure, which may improve patients' patency and natural pregnancy rates.

The epididymis is a common site of obstruction in obstructive azoospermia (OA). Vasoepididymostomy has become an important method for the treatment of ...

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

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