Unusual Occurrence of Syncytial Epithelia in the Male Accessory Glands of Shore Bugs (Leptopodomorpha in Hemiptera)

Abstract

(1) Background: The insect male accessory gland (MAG) produces seminal fluid components crucial for male reproduction, analogous to the mammalian prostate. While some insect MAGs exhibit binucleate epithelial cells for luminal volume plasticity, the diversity of cellular arrangements and their functional implications across insects remain largely unknown. (2) Methods: We investigated the cellular architecture of MAG epithelia in various shore bug species (infraorder Leptopodomorpha, Hemiptera) and their mechanisms of multinucleation and potential MAG volume regulation. (3) Results: The MAG epithelia of shore bugs comprise a small number of large, plastic syncytial cells with varying nuclear numbers. We hypothesize that these syncytia facilitate effective MAG volume expansion post-eclosion. Uniquely, MAG shrinkage involves the localized contraction of limited muscle fibers, unlike the systematic contraction of circular muscles in most other insects. We further describe sequential cell fusion during the nymphal stage as the mechanism of multinucleation. (4) Conclusions: The unique syncytial organization of Leptopodomorpha MAG epithelia represents an evolutionary divergence from typical binucleate or mononucleate structures in other insects; it is likely that this enables distinct mechanisms for reproductive fluid storage and evacuation. This study highlights the evolutionary diversity of male reproductive organ morphology and function within insects.

1. Introduction

Exocrine glands, which secrete various substances externally, often exhibit binucleate cells when a large quantity of material needs to be supplied. Examples include mammary gland cells in mice Mus musculus Linnaeus, 1758 (Muridae: Rodentia), salivary gland cells in the assassin bug Rhodnius prolixus Stål, 1859 (Hemiptera: Reduviidae), and male accessory gland (MAG) cells in the fruit fly Drosophila melanogaster Meigen, 1830 (Diptera: Drosophilidae) [1,2,3]. While an increased number of nuclei might seem to suggest greater transcriptional activity and thus higher substance production, it is often observed that cell mass increases proportionally with nuclear number and does not necessarily lead to enhanced substance production [4]. Furthermore, if the goal were simply increased transcription, DNA amplification alone would suffice, implying that binucleation incurs the cost of dividing the nucleus. Therefore, an alternative hypothesis proposes that binucleation enhances cell pliability. This idea is not unreasonable, considering that various muscle cells are commonly multinucleated. Indeed, in D. melanogaster males, the morphological plasticity of the male accessory gland (MAG) between contraction and distension was predicted to be 4% lower in polyploid mononucleate cells compared to binucleate epithelial cells, and 23% lower in paired diploid mononucleate cells with the same ploidy, without significant alteration in gene expression levels [4]. Furthermore, in MAGs temporarily overexpressing the fizzy-related gene during development, where most binucleate cells are replaced by these mononucleate cells, the contraction rate drops to approximately 70% [5]. This strongly suggests that binucleation contributes more to increasing the storage and release capacity of secretions rather than their synthesis.

The insect MAG is considered to be an exocrine gland analogous to the mammalian prostate, responsible for producing and secreting seminal fluid components other than sperm [6,7]. Its roles are diverse, including sperm activation, nutritional provisioning to females, female behavioral control, and producing physiologically active substances to gain an advantage in sperm competition [8,9,10,11,12,13,14,15]. For example, in D. melanogaster, a mutation in the gene for Sex Peptide, a 36-amino acid exocrine peptide synthesized in the MAG, almost completely abolishes its ability to suppress female re-mating 48 h post-copulation, a behavior typically inhibited when mated with wild-type males [16]. Peptides with similar functions have been identified in other insects. Head peptide-1, a 10-amino acid peptide from the MAG of the dengue mosquito Aedes aegypti Linnaeus, 1762 (Diptera, Culicidae), can prevent the paternity of subsequently mated males by up to 38% [17,18]. Similarly, Maccessin, a 56-amino acid peptide recently discovered in the brown planthopper Nilaparvata lugens Stål, 1854 (Hemiptera, Delphacidae), significantly reduces female re-mating, while its knockdown significantly increases it [19]. Generally, it is believed that delivering more of these substances to the female increases male reproductive success [3,20,21], and the MAG is presumed to have undergone various functional evolutions to achieve this. Hemiptera, alongside Drosophila mentioned above, is a distinctive taxonomic group that frequently exhibits binucleation or multinucleation in MAGs [22,23]. However, since both groups have undergone homoplasious evolution, their binucleation may not serve the same purpose. In Hemiptera, elongated binucleate cells can form structures suited for a slender lumen or a distended storage organ in the MAG by altering the nuclear position arrangement [22]. Changes in nuclear arrangement to form larger MAGs from the small, confined space of the nymphal stage have also been identified [23]. Regarding multinucleation, systematic observations are lacking, although some old information is available on Dysdercus fasciatus Signoret, 1861 (Pyrrhocoridae) [24].

In this study, through comparative observations of MAGs from various taxa, we discovered that a unique form of multinucleated MAGs is widely established in species belonging to the infraorder Leptopodomorpha, including Saldula and Teloleuca. We also observed that these MAGs increase in size from the nymphal stage to the mature adult stage, in addition to the peculiar morphology of their outer muscle cells and their mechanism of syncytium formation. We then examined the characteristics of these MAGs in comparison to those of other species. The organization of Hemipteran MAGs is not uniform, making this an interesting and unique morphological study.

2. Materials and Methods

2.1. Shore Bug Species Used in This Study

For comparative studies between evolutionary lineages, it was necessary to use live materials for protein staining, as shown below. For this purpose, the following five species, which are easy to obtain in Japan and can be readily used for observation, were chosen:

• Saldula pallipes Fabricius, 1794 (Hemiptera, Saldidae) (Figure 1A): Takao, Tokyo, Japan, 22 May 2018, by TAY (ID: yA4, aTK10, and aTK14). Suwa, Nagano, Japan, 8 September 2018, and 12 May 2020, by JY/KT (ID: aSW03).
• Teloleuca kusnezowi Lindberg, 1934 (Hemiptera, Saldidae) (Figure 2B): Hamamasu, Hokkaido, Japan, 10 June 2018; 24 June 2021; and 21 June 2022, by TAY/AK/RH (for nymphs) (ID: aH54, aH56, aH85, and aH2051). Hamamasu, Hokkaido, Japan, 14 July 2020, by RN/KT (for adults) (ID: tX0).
• Macrosaldula violacea Cobben, 1985 (Hemiptera, Saldidae) (Figure 2C): Sapporo, Hokkaido, Japan, 16 July 2020, by RNR/KT (ID: tH05 and aH2012).
• Salduncula decempunctata Miyamoto, 1963 (Hemiptera, Saldidae) (Figure 2D): Ishigaki, Okinawa, Japan, 23 June 2019, by JY (ID: y1–2, y1–3, and aIG11).
• Corallocoris satoi Miyamoto, 1963 (Hemiptera, Omaniidae) (Figure 2E): Nago, Okinawa, Japan, 30 June 2019, by MO/NI/KT (ID: t1.10 and aOK2501).

2.2. Husbandry of T. kusnezowi Nymphs and Adults

The collected T. kusnezowi nymphs were reared at 25 °C on 1% agar medium S-6 (Ina Food Industry Co., Ltd., Nagano, Japan). This medium contained 5% of the isotonic beverage Pocari Sweat (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) as a mineral source and 0.1% methyl parahydroxybenzoate (Sigma-Aldrich Co. LLC, St Louis, MO, USA) as an antifungal agent. Commercially available frozen chironomid larvae (Kyorin Co., Ltd., Hyogo, Japan) were fed to the T. kusnezowi nymphs and adults several times per week.

2.3. Dissection, Optical Microscopy, and Fixation of MAGs

Whole reproductive organs were dissected from the abdomens of adult bugs or nymphs in standard phosphate-buffered saline (PBS). Dissections were performed using fine forceps (Dumont #5, Fine Science Tools, Foster City, CA, USA) and Vannas spring scissors (#15002-08, Fine Science Tools) under a binocular microscope (Leica Microsystems, Wetzlar, Germany). The excised organs were photographed using a VHX-2000 digital microscope (Keyence, Tokyo, Japan). Subsequently, the organs were fixed in ultra-pure formaldehyde (16%, Polysciences, Warrington, PA, USA) diluted 4-fold (final 4%) with PBS (pH 7.4) for 20–60 min with intermittent mixing at room temperature.

2.4. Immunofluorescence of MAG

After fixation, the organs were thoroughly washed in PBT (PBS containing 0.1% Triton X-100; #12967, Nacalai Tesque, Japan). The following reagents were used for tissue staining: DAPI (0.1 mg/mL, 4′,6-diamidino-2-phenylindole, #D8417, Sigma-Aldrich, St. Louis, MO, USA) for nuclear staining. Rhodamine-labeled phalloidin (#R415, Thermo Fisher Scientific Inc., Waltham, MA, USA) was used at a 1:500 dilution for F-actin staining. A 1:200 dilution of rabbit anti-p-H3 antibody (#05-806, Sigma-Aldrich) was used to stain phospho-histone H3 on chromosomes in the mitotic phase. Additionally, a 1:200 dilution of Alexa Fluor 647-labeled anti-rabbit IgG (#111-605-003, Jackson ImmunoResearch, West Grove, PA, USA) was used. Staining procedures followed previously described methods [25].

2.5. Laser Confocal Microscopy

Laser confocal microscopic images of the optical tissue sections were acquired using FV1000, FV3000 (Olympus, Tokyo, Japan), or Digital-Eclipse C1Si (Nikon, Tokyo, Japan) systems. For overall views of MAGs, multiple image pieces were stitched together using FV31S-SW version 2.6.1.243 software (Evident Scientific, Tokyo, Japan).

2.6. Transmission Electron Microscopy (TEM)

Fixation: Samples were fixed overnight at 4 °C in a solution containing 2% paraformaldehyde (PFA) and 2% glutaraldehyde (GA) in 0.1 M cacodylate buffer (pH 7.4). Following primary fixation, samples were washed three times for 30 min each in 0.1 M cacodylate buffer, and then postfixed in 2% osmium tetroxide (OsO₄) in 0.1 M cacodylate buffer at 4 °C for 2 h. Dehydration: Samples were dehydrated through a graded ethanol series: 50% and 70% ethanol for 30 min each at 4 °C, followed by 90% ethanol for 30 min at room temperature (RT). Dehydration was completed with three 30 min changes in 100% ethanol at RT. Infiltration: Infiltration was performed by immersing samples twice in propylene oxide (PO) for 30 min each. Samples were then transferred to a 70:30 mixture of PO and Quetol-812 resin (Nisshin EM Co., Tokyo, Japan) for 1 h, after which the tubes were uncapped to allow PO to volatilize overnight. Embedding and Polymerization: Samples were then transferred to fresh 100% resin and polymerized at 60 °C for 48 h. Ultra-thin Sectioning and Staining: Ultra-thin sections (70 nm) were cut from the polymerized resin blocks using a diamond knife on an ultramicrotome (Ultracut UCT; Leica, Vienna, Austria) and mounted on copper grids. Sections were stained with 2% uranyl acetate at RT for 15 min, washed with distilled water, and then counterstained with Lead stain solution (Sigma-Aldrich Co., Tokyo, Japan) at RT for 3 min. Observation and Imaging: Grids were observed using a transmission electron microscope (JEM-1400Plus; JEOL Ltd., Tokyo, Japan) operated at an acceleration voltage of 100 kV. Digital images (3296 × 2472 pixels) were captured with a CCD camera (EM-14830RUBY2; JEOL Ltd., Tokyo, Japan).

2.7. Nucleotide Sequencing of mtCOI and Phylogenetic Analysis

To support the taxonomic identification of naturally collected bugs, DNA samples were prepared from a few legs or a part of the carcass of each bug using a NucleoSpin DNA Insect DNA extraction kit (Macherey-Nagel, Düren, Germany). To obtain representative barcode sequences, mitochondrial cytochrome oxidase subunit I (mtCOI) gene fragments were amplified by polymerase chain reaction (PCR) using LCO1490 and HCO2198 primers [26,27]. The PCR conditions were as follows: denaturation at 94 °C for 30 s, annealing at 45–55 °C for 90 s, extension at 72 °C for 60 s, and 40 cycles. The thermostable DNA polymerase used here was EmeraldAmp PCR Master Mix (Takara Bio Inc., Kusatsu, Japan). If this did not work, we performed another PCR using the primers tRWF1 and LepR1 [28,29] under the following conditions: denaturation at 98 °C for 10 s, annealing at 48 °C for 30 s, extension at 72 °C for 30 s, and 30 cycles. The thermostable DNA polymerase used here was ExTaq (Takara Bio Inc., Kusatsu, Japan). Nucleotide sequences of the PCR products were analyzed by Eurofins Genomics Japan (Tokyo, Japan).

Using the obtained nucleotide sequences of four Saldid species, we constructed a multiple alignment across the 606 nucleotide segment using the default parameters of ClustalW in the program MEGA-X [30]. Based on this alignment, a phylogenetic tree was conducted using the Maximum Likelihood method. The bootstrap tests were performed with 1000 replicates.

3. Results

3.1. Discovery of Syncytial Epithelium in the MAG of Saldula pallipes

While observing the diversity of MAG morphology across various insect species, we focused on specimens from the infraorder Leptopodomorpha. In mature male Saldula pallipes (Figure 1A), a pair of spindle-shaped MAGs, smaller than the testes, originates near the confluence with the seminal vesicle (Figure 1B). Confocal microscopic observation of this epithelium revealed a remarkable morphology: numerous nuclei were present within irregularly shaped regions enclosed by cell membranes, visualized by F-actin detection (Figure 1C,C’). These multiple nuclei appeared to be arranged in a horizontal plane, seemingly maximizing the volume of the MAG cavity (Figure 1C’’,C’’’). Conversely, the typical circular muscles which usually contract the MAG during copulation were absent in this species’ MAGs. Instead, several irregularly shaped longitudinal muscles were attached to the MAG surface, with their fibers not contacting all cells (Figure 1C’’’’,G).

Concerned that not all cell membranes might be visible under F-actin staining, as some cell membranes in various tissues had sparse F-actin lining, we examined cross-sections of the epithelium using TEM (Figure 1D,D’ and Supplementary Figure S1). We studied a total of 13 cells within a single MAG, showing endoplasmic reticulum membranes between the numerous nuclei but no cell membranes whatsoever, strongly suggesting that these cells are multinucleate. The irregular number of nuclei per cell, ranging from 9 to 41, suggested that these were not coenocytes formed by endomitosis or nuclear division, but rather syncytia formed by cell fusion, as subsequently demonstrated. Detailed observations of individual cells revealed instances where nuclear spacing and chromatin condensation were synchronized among the multiple nuclei within a single cell but differed between adjacent cells (Figure 1E). Additionally, in cells undergoing apoptosis, all nuclei within the multinucleated cell exhibited synchronized nuclear fragmentation (Figure 1F). These observations are consistent with the interpretation that multiple nuclei share a common cytoplasm.

3.2. Diversity of MAG Epithelial Multinucleation Across Various Taxa in Leptopodomorpha

We investigated the evolutionary diversity of this unique syncytial epithelium. Figure 2A illustrates the phylogenetic relationships of five genera based on our mtCOI sequence analysis, although it is not entirely consistent with the published results [31,32]. We compared the tissue and cellular morphologies among representative species of these genera. Teloleuca shared many characteristics with Saldula, including the overall external appearance of the internal reproductive organs, the multinucleated state of the MAG epithelia, and the arrangement of muscle fibers, differing only slightly in the aspect ratio of MAG morphology (approximately 2 in Teloleuca and 3 in Saldula) (Figure 2B’–B’’’’). T. kusnezowi, the species used from this genus, was larger in individual and organ size (Figure 2B,B’) than S. pallipes and the following species, and its MAG contained an overwhelmingly greater number of cells, making it suitable for subsequent developmental studies due to the ease of observing cell division and fusion events.

In Macrosaldula violacea (Figure 2C), the MAG was more elongated (aspect ratio of approx. 4), and it had simple testicular follicles (two pairs per each side), showing differences from the previous two species (Figure 2C’). The MAG of M. violacea was composed of a syncytial epithelium with nuclei arranged in a planar fashion, similar to the aforementioned species (Figure 2C’’,C’’’), but the number of nuclei per cell was not as high as in the previous two species. While the outer muscle fibers were not as sparse as in the previous two species, the circular muscles were still poor, and thin longitudinal muscles were present (Figure 2C’’’’). Salduncula decempunctata (Figure 2D), similarly to Macrosaldula, had two pairs of testicular follicles per each side and small MAGs (approx. 250 μm) with fewer cells. However, various features were fundamentally consistent with the preceding three species (Figure 2D’–D’’’’).

Among the specimens examined, an intertidal dwarf bug Corallocoris satoi (Figure 2E) was the only species that did not belong to Saldidae. In this species, the seminal vesicle was located directly beneath the two pairs of testicular follicles per each side, and a long vas deferens reached the ejaculatory duct, where it merged with the MAG, which had a narrow, tubular base (Figure 2E’). This morphology was distinct from the Saldidae species mentioned above. The MAG epithelia consisted of a population of binucleate cells, but, uniquely, trinucleate cells were frequently interspersed (approximately 30%), showing a multinucleated state not observed in other species (Figure 2E’’,E’’’). The outer muscles resembled the standard circular muscles found in other insects, but their arrangement and spacing were not as orderly as in other insects, exhibiting some similarities with the Saldidae species discussed above (Figure 2E’’’’).

3.3. Expansion of MAG Luminal Volume During Adult Maturation

In Aphelocheirus vittatus, a benthic water bug species within Heteroptera, the MAG rapidly increases in size after adult eclosion due to feeding, similar to many other insects. During this process, the binucleate cell population forming the MAG increases the luminal volume by changing the angle of the binucleate epithelial surface from vertical to horizontal [23]. Therefore, we investigated whether the arrangement of nuclei within the MAG syncytia of Saldidae changes during maturation.

Our results showed that in both Saldula (Figure 3A,A’) and Teloleuca (Figure 3B,B’), the multiple nuclei contained within a single syncytium of a teneral adult were not arranged in a horizontal plane like those in a mature adult. Instead, they were highly stacked towards the lumen of the MAG. This stacking reached a maximum of about five nuclear layers in Saldula and about seven nuclear layers in Teloleuca. On the other hand, the outer muscle fibers, which were sparsely present in mature adults, were only found in considerable numbers on one side of the dorsoventral surface in teneral adults (Figure 3B’’-1,B’’-2). However, their thickness was extremely poor, differing in appearance from the standard circular muscles of other insects.

We predict that if the highly stacked nuclei spread into a horizontal plane with maturation due to feeding, the cross-sectional area of the cell, and consequently the volume of the MAG lumen, would significantly increase (Figure 3C). Indeed, measuring the diameter of the cavity in Figure 1C’’, Figure 2B’’’ and Figure 3A’,B’ showed an 8.1-fold increase in Saldula (266 μm/33 μm) and a 9.1-fold increase in Teloleuca (578 μm/63 μm) between teneral and mature adults. For comparison, the same measurement in Aphelocheirus (mentioned above) showed a 4.3-fold increase (144 μm/34 μm) (Figure 3D). Therefore, multinucleation is predicted to be an effective method for expanding cavity volume during growth.

3.4. Mechanism of Multinucleation in Teloleuca MAG Epithelia

To investigate how multinucleation is achieved in this syncytial epithelium, we dissected final-instar nymphs of T. kusnezowi, which have the highest total nuclear count (Figure 4A,B). We confirmed that these nymphs were the same species as the adults based on their mtCOI nucleotide sequences.

The outer muscles of these nymphal MAGs differed from those of mature adults; myoblasts almost completely covered only one side of the dorsoventral surfaces (Figure 4C), which can be understood as a precursor stage to the muscle distribution in teneral adults (Figure 2B’’’’-1,B’’’’-2). The epithelial cells also differed from those of mature adults. In addition to the multinucleated epithelium already present in mature adults (though with fewer nuclei), another epithelium, composed of cells each with a single diploid nucleus, covered the outer side (Figure 4D,D’,E). This was not necessarily a monolayer; in some areas (especially on the side contacting the seminal vesicle), it formed a stratified epithelium (Figure 4E’).

To identify the locations of dividing nuclei, we used an anti-phospho-Histone H3 antibody to detect nuclei in the mitotic phase. Interestingly, dividing nuclei were found to be localized and scattered within the outer diploid cell layer (Figure 4D,D’’,E,E’’). While the irregular number of nuclei per syncytium in Teloleuca (17–39) suggested that multinucleation occurred via cell fusion, nuclear division within the multinucleated cells was never observed despite multinucleation being ongoing. This suggests that multinucleation progresses through cells fusing one at a time from the outer proliferating layer. Indeed, round-shaped interphase cells often detached from the outer cell layer on the inner surface of the diploid cell layer (white arrowhead in Figure 4E’’’), which were considered to be undergoing fusion. This proliferation and fusion were also observed in the previous instar (Figure 4F,F’), but it was not possible to determine when this phenomenon began, as obtaining younger nymphs is challenging (we did not succeed in hatching eggs after overwintering.). After adult eclosion, proliferating cells were no longer found, but cell fusion continued until the external diploid cell layer disappeared (Figure 4G,G’). During this period, the MAG epithelial structure became simpler, making cell fusion easier to observe. The diploid cells undergoing fusion at interfaces are thought to lose the hexagonal shape and apicobasal polarity typically found in single-layer epithelial cells, becoming rounded from all perspectives (Figure 4G’’). Therefore, if the numerous and widely distributed round cells in the nymphal diploid epithelial cell layer (Figure 4D’) are not in the mitotic phase, they are considered to be cells undergoing fusion.

4. Discussion

4.1. Evolutionary Diversity of MAG Syncytia in Hemiptera

The syncytial epithelia of the male accessory gland (MAG) reported in this study are a common feature in Saldidae but are rare in other taxa. While the MAG epithelia of Heteroptera are typically composed of binucleate cells [22,23], the first instance of a MAG where all cells were stable multinucleate cells was discovered over 40 years ago through TEM observations of Dysdercus fasciatus [24]. This is considered the earliest record of broad multinucleation in Hemipteran MAG epithelia. However, the number of nuclei per cell in D. fasciatus was only 3–5, significantly fewer than the 9–41 nuclei in S. pallipes or 17–39 in T. kusnezowi reported in this study. Furthermore, the complex, branched morphology of MAGs in D. fasciatus differs greatly from the simple, sac-like structure of Saldidae. Thus, it is unlikely that the homoplasiously evolved multinucleation in these two groups serves the same purpose.

We also found scattered multinucleate cells within the binucleate cell populations of MAGs across a wide range of Heteropteran suborders, including Enicocephalomorpha, Cimicomorpha, and Pentatomomorpha (JY: unpublished results). However, we hypothesize that these are likely unavoidable errors that occur when MAG epithelial binucleation is achieved by cell fusion, as seen in the bed bug Cimex lectularius Linnaeus, 1758 [22]. This mechanism is distinctly different from the active multinucleation observed in Saldidae. Conversely, the widespread presence of this latent capacity for multinucleation through cell fusion across diverse taxa is likely the foundation upon which the advanced multinucleation of all MAG cells in Saldidae evolved. It is likely that the MAGs of intertidal dwarf bugs Corallocoris are intermediates in this evolutionary pathway, consisting primarily of binucleate cells but with a high frequency (approximately 30%) of trinucleate cells interspersed (Figure 2E’’).

4.2. Possible Function of Syncytial MAGs

The high degree of multinucleation in Saldidae MAG epithelia is undoubtedly an efficient mechanism for the development from a contracted MAG in teneral adults to a distended MAG in mature adults. However, if this were the optimal method for MAG substance storage and expulsion, it would have evolved in many insects. The fact that it has not suggests that this specialized method comes with certain constraints. It is likely that one such constraint is the degeneration of the outer muscles associated with this syncytial epithelium. Due to the significant size difference between immature and mature states (Figure 3C), the circular muscles adhering to the epithelium undergo degeneration beyond their elastic limit, resulting in only a few irregular longitudinal muscles remaining in mature adults (Figure 1C’’’’ and Figure 2B’’’’–D’’’’). This implies that these few muscle fibers would struggle to achieve the high degree of MAG contraction normally brought about by the ubiquitous circular muscles found in most insects, which are essential for expelling as much content as possible during copulation. Indeed, the muscle fibers do not reach all cells.

Therefore, it is possible that this specialized MAG is a method only usable by insects that do not require a large volume of seminal fluid per copulation. While insects often exist with accessory glands larger than their testes, the MAGs of various Saldidae species are less than half the size of their testes (Figure 1B and Figure 2B’–D’) and do not appear to occupy a large volume within the abdominal cavity. On the other hand, we have observed that male Teloleuca can copulate three times in succession immediately after a previous copulation. This suggests that this type of MAG may have the characteristic or constraint of being adapted for continuous copulation involving small amounts of seminal fluid.

4.3. Mechanism of Syncytium Formation

One could hypothesize that syncytia formation occurs through a process similar to binucleation in the bed bug Cimex, where numerous mononucleate cells proliferate, arrest their cell cycle, and then synchronously fuse to achieve multinucleation (Figure 5F–J, [22]). However, in such a scenario, the number of nuclei that can be accommodated within the limited volume of the MAG anlage would be restricted, making it difficult to develop a large MAG.

Teloleuca adopts a unique mechanism (Figure 5K–N) where the outer surrounding epithelium, composed of diploid cells, proliferates and then sends the newly formed cells one by one into the inner multinucleated cell, thereby stacking nuclei inwards. This allows nuclei to be packed into a small volume and is considered a more rational developmental strategy for enhancing MAG expansion efficiency. While MAG development does not occur in penultimate-instar Cimex nymphs [22], in Teloleuca, MAG development is presumed to begin earlier to allow a longer period of nuclear delivery to the syncytia (Figure 4F,F’). The observed cell fusion process is unusual as it differs from that of Cimex, in which the septa between adjacent cells disappear [22]. However, similar phenomena where cells are engulfed intact are also known, such as the entosis (a type of cell cannibalism) of uterine epithelial cells by syncytiotrophoblast cells seen during implantation in mammals [33].

4.4. Development and Characteristics of Outer Muscles

Another interesting observation is the nature of the myoblast population in the nymphal stage. While restricted to only one of the two dorsoventral sides of the MAG, it spreads out to cover the outer surface (Figure 4C,C’). After adult eclosion, it temporarily exhibits an appearance similar to circular muscles (Figure 3B’’-1) before subsequently degenerating (Figure 2B’’’’-1,2B’’’’-2). The elimination of a structure that was initially formed is often observed in the development of vestigial organs. This muscle degeneration appears to occur randomly, with the number and shape of the remaining muscle fibers varying completely among individual adults. However, a common feature is that most circular muscles do not remain, while many longitudinal or poorly oriented muscle fibers persist, with their termini becoming apparent (Figure 2B’’’’-1,2B’’’’-2).

Consequently, during copulation, the MAG is expected to be pulled in the proximodistal direction, causing the cells to deform as if compressed in that direction (Figure 2B’’-2) and thereby reducing the luminal volume. The degree of shape change flexibility in these multinucleated cells is astonishing; such irregularly shaped cells are not found in the MAG epithelia of other taxonomic groups. Conversely, if these syncytia maintained the hexagonal morphology common to typical epithelia, the overall plasticity of the MAG would be poor, making it difficult for a few muscle fibers to contract the entire MAG. Therefore, it is highly probable that these few muscle fibers and flexible syncytia only function rationally when they are present together.