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Journal of Insect Physiology 「The production and transfer of spermatophores in three Asian species of Luciola fireflies 」 

Adam South†, Teiji Sota±, Norio Abe‡, Masahide Yuma+ and Sara M. Lewis†*

†Department of Biology, Tufts University, 163 Packard Ave, Medford MA 02155, USA
‡Firefly Breeding Institute, Itabashi-Ward, Toyko, Japan
±Department of Zoology, Kyoto University, Kyoto, Japan
+Department of Environmental Solution Technology, Ryukoku Univeristy, Otsu, Japan

*Address for Editorial Correspondence
Dr. Sara M. Lewis
Department of Biology
Tufts University
Medford, Massachusetts 02155 USA
Email: sara.lewis@tufts.edu
Phone: 617.627.3548
Fax: 617.627.3805


During mating, many male insects transfer sperm packaged within a spermatophore that is produced by reproductive accessory glands. While spermatophores have been documented in some North American fireflies (Coleoptera: Lampyridae), little is known concerning either production or transfer of spermatophores in the aquatic Luciola fireflies widespread throughout Asia. We investigated this process in Japanese Luciola lateralis and L. cruciata by feeding males rhodamine B, a fluorescent dye known to stain spermatophore precursors. We then mated males with virgin females, and dissected pairs at various timepoints after mating. In both of these Luciola species, spermatophores were produced by three pairs of male accessory glands and were transferred to females during the second stage of copulation. Male spermatophores were highly fluorescent, and were covered by a thin outer sheath; a narrow tube leading from an internal sperm-containing sac fit precisely into the female spermathecal duct, presumably for sperm delivery. Both L. lateralis and L. cruciata females have a spherical spermatheca as well as a highly extensible gland where spermatophore breakdown commences by 24 h postmating. Similar reproductive anatomy was observed for both sexes in Luciola ficta from Taiwan. These results suggest that nuptial gifts may play an important role in many firefly mating systems.
Keywords: firefly; male accessory glands; nuptial gifts; spermatheca; spermatophore digestion

Before, during or after copulation, males in diverse insect taxa provide females with nuptial gifts in the form of male body parts, food items, or spermatophores produced by male accessory glands (Mann, 1984, Boggs, 1990, 1995, Vahed, 1998). Such nuptial gifts have the potential to increase a female’s lifetime fecundity, and may thus select for polyandrous mating systems and postcopulatory female choice (Gwynne, 2008, Vahed, 2007). In certain insects, however, male seminal products have been shown to reduce female longevity (Chapman & Davies, 2004), suggesting they may have evolved through sexual conflict. In either case, it is clear that male nuptial gifts represent an important feature of insect mating systems, with far-reaching implications for the evolution of reproductive anatomy, physiology, and behavior in both sexes.
Nuptial gifts are likely to be of particular economic importance in the nutrient budgets of any insects that have non-feeding adults, since all reproductive activities are based on resources obtained by larval feeding. One such group is North American Photinus fireflies (family Lampyridae), in which males transfer spermatophores that are known to increase female lifetime egg production (reviewed by Lewis et al., 2004). However, little is known about nuptial gifts in Luciola firelies, a genus widespread in Asia and characterized by aquatic larvae that feed primarily on freshwater snails (Ohba, 1984, 2004). Hayashi & Suzuki (2003) postulated spermatophore transfer in the Japanese Genji firefly, L. cruciata, based on the morphology of male accessory glands, and observed multiple spermatophores in the reproductive tract of a Lucidina natsuminae female. The only other spermatophore description for any Asian firefly was by Fu and Ballantyne (2006), who noted a spermatophore found within the reproductive tract of a field-collected female of the Chinese firefly, Luciola leii.
In this study we describe in detail male and female reproductive anatomy for three Luciola firefly species: Luciola lateralis, L. cruciata, and L. ficta. In L. lateralis and L. cruciata we investigated the process of spermatophore production and transfer by feeding males rhodamine B, a fluorescent dye known to stain spermatophore precursors. We then allowed males to mate with virgin females, and dissected mated pairs at various timepoints after mating. Here we report the first description of spermatophore production and transfer for any Asian firefly species.

Materials and Methods

L. cruciata and L. lateralis adults used in this study were from Japan: the former is endemic to Japan, while latter is widely distributed in Japan, Korea, and eastern Siberia (Ohba 1984). These two species were reared at the Firefly Breeding Institute in Itabashi Ward, Tokyo; a few L. cruciata males were field-collected in the Shiga Mountains north of Kyoto. L. ficta adults were field-collected in Taiwan. To examine male and female reproductive anatomy, adults of each species were frozen at -20°C in 70% EtOH until dissection in 1x phosphate buffered saline (PBS).
Mating experiments were conducted with virgin L. cruciata and L. lateralis to determine if spermatophores were transferred and to characterize the time course of this process. Adults of L. cruciata and L. lateralis were kept in same sex groups, then separated into individual containers with moistened paper towel. Males were fed 40% sucrose solution with rhodamine B, a thiol-reactive fluorescent dye that forms covalent bonds to proteins. This product is known to stain spermatophores (Sparks & Cheatham, 1973, van der Reijden et al., 1997), allowing us to visualize portions of the male reproductive tract responsible for producing spermatophore precursors and to track the location of male spermatophores within the female reproductive tract at various timepoints post-mating. Following rhodamine B exposure for 5-24 hours, males were paired with females and monitored until mating occurred. Firefly copulation consists of two distinct stages (Lewis & Wang, 1991); in stage 1 males mount females dorsally, while stage 2 begins after the male swivels around to face in the opposite direction in abdomen-to-abdomen position. Mating pairs were monitored, and copulations were terminated by freezing at various timepoints after the beginning of stage 1 or 2 (ranging from 0 min to 24 h), after which pairs were stored in 70% EtOH until dissection in 1x PBS. Reproductive tracts removed from males and females were observed with a Nikon SMZ1500 stereomicroscope equipped with an X-Cite 120 fluorescence illuminator, and photographed with an Insight 4 Mega-pixel Color Mosaic camera (Diagnostic Instruments, Michigan). For L. lateralis and L. cruciata, 11 males and 9 females of each species were dissected, while 4 of each sex of L. ficta were used. Wet weights were determined by removing specimens from alcohol and allowing them to air-dry. Three spermatophores were dissected out of female reproductive tracts, placed on a pre-weighed plastic platter and kept in a dessicator at room temperature for 24 hours. The male’s body was placed on a pre-weighed aluminum foil platter and allowed to dry at 40 ºC for 72 hours. All structures were weighed on a MT 400 AT261 microbalance.
To confirm sperm presence, structures within the female reproductive tract, male seminal vesicles and spermatophores were separated on slides, stained with propidium iodide, a fluorescent DNA stain, and examined at 400x on an Olympus BX40 fluorescence compound microscope. This procedure allowed us to determine the location of sperm within the female reproductive tract at various times post-mating.
In the rhodamine B mating experiments, only 2 of 5 L. cruciata pairs successfully mated, and these copulations did not progress beyond stage 1. For L. lateralis, 13 of 20 pairs successfully mated, and 6 of these progressed to stage 2. Because we had a greater number of L. lateralis specimens, and because this species appeared more conducive to mating in captivity, below we use L. lateralis to describe female and male reproductive anatomy, as well as the process of spermatophore transfer, and then present comparisons of L. cruciata and L. ficta.


Female reproductive system:

In the reproductive tract of L. lateralis females (Fig. 1A and B), the ovaries contained ovarioles with oocytes in many developmental stages, with mature oocytes occupying the lateral oviducts. These lateral oviducts converged into a common oviduct, which emerged posteriorly from the dorsal midpoint of the bursa copulatrix (BC). The BC was about 1.5 mm long and 0.5 mm wide, with relatively thick walls on the lateral sides of which two needle-shaped, sclerotized plates were embedded. There was an additional small, sclerotized plate embedded in the dorsal wall of the BC posterior to the entrance of the common oviduct (Fig. 1B; Fu & Ballantyne 2006 termed the analogous structure in L. leii the median oviduct plate). Toward the posterior end of the BC two sclerotized ovipositors were attached, and the lumen narrowed slightly as the bursal wall thickened.
Located at the anterior end of the BC were two structures: a small, spherical spermatheca and a much larger spermatophore-digesting gland (SDG: Fig. 1B). The spermatheca was connected via a short duct to the dorsal side of the BC anterior to the entrance of the common oviduct (Fig. 1A and B). Anterior to the spermatheca was a semi-spherical, thin walled SDG, which eventually contained the spermatophore as it was being digested. Typically this structure was distended and fluid-filled, and in mated females also contained heterogenous granules from the male spermatophore; unmated females lacked these granules. Regardless of female mating status, each SDG also contained a small white mass of unknown origin. Posterior to the common oviduct, a small tubular gland of unknown function entered the dorsal side of the BC.
In L. cruciata and L. ficta, female reproductive anatomy was quite similar to that of L. lateralis (Table 1). In both species, the bursa copulatrix terminated in a hemispherical SDG, with a single spherical spermatheca connected via a short duct. Females in both L. cruciata and L. ficta had a median sclerotized plate located in the bursal wall posterior to the common oviduct, but this structure was greatly reduced in L. cruciata females. Females in both species lacked the needle-like sclerotized plates that were embedded in the bursal wall in L. lateralis females.

Male reproductive system

In L. lateralis males, the paired testes were located at the anterior end of the abdomen (Fig. 2A and B) and each testis was thickly covered by fat body and connective tissue The tubular vasa deferentia led to the seminal vesicles, which appeared as ovoid enlargements at the proximal ends of these ducts. Sperm within the seminal vesicles were packaged into sperm bundles, each consisting of 50-80 sperm. The seminal vesicles emptied into the ejaculatory duct at its junction with the accessory glands.
The most conspicuous structures in the reproductive tract of L. lateralis males were three pairs of bilaterally symmetrical accessory glands (Fig. 2A and B) that entered the ejaculatory duct at a common point. The most central were the paired curled glands, which were tapering, slightly twisted glands arranged longitudinally in the abdominal cavity; these glands were approximately 2.2 mm long and measured 0.3 mm at their widest point. In males that had injested rhodamine B dye, these curled glands were the most fluorescent portion of the reproductive system (Fig. 2B), and dissections of virgin males revealed spermatophore precursors located within these glands. Near the curled glands were two additional pairs of tubular accessory glands. The thin-walled long glands were approximately 5.3 mm long and narrow, widening distally to about 0.35 mm and ending in a bulbous mass of white, spongy tissue; these glands contained heterogeneous granules similar to those found in spermatophores that had been transferred to females (see below). The short accessory glands (about 0.82 mm long and 0.23 mm wide) were also thin-walled, and contained a slightly opaque fluid. In some specimens, both the long and short glands showed slight fluorescence, suggesting that they may also produce proteins.
In L. ficta and L. cruciata males, reproductive structures were quite similar to those of L. lateralis (Table 1). Male accessory glands were virtually identical in all three species, with only slight variations in the long accessory glands; the distal swelling in the long glands of L. ficta and L. cruciata males was not as pronounced as in L. lateralis.

Spermatophore structure and transfer

In several L. lateralis pairs where copulation was interrupted during stage 1, spermatophore precursors were visible emerging from the curl gland and merging in the ejaculatory duct to begin spermatophore formation. Out of the 4 pairs interrupted during Stage 1, none had transferred the spermatophore. Instead, spermatophore transfer appears to take place during stage 2 of copulation (Fig. 3A and B).
Several intact spermatophores were dissected from the reproductive tracts of L. lateralis females that had mated with rhodamine B dyed males (Fig. 2C). Three spermatophores were weighed, and they constituted approximately 1.5% of total dry male body weight. The spermatophore consisted of an outer membranous sheath surrounding a spongy matrix which contained granules resembling the contents of the curled and long accessory glands. In turn, this matrix surrounded an inner sac containing sperm bundles. This inner sperm sac emptied through a lightly sclerotized tube that emerged from the spermatophore at a 45 degree angle. Overall, the male spermatophore in L. lateralis resembles a mitten (Fig. 2C), with the tube emerging from the sperm sac representing the thumb, and the inner sperm sac located in the mitten’s palm.
This spermatophore structure appears to function in delivering sperm directly into the female’s spermatheca. For all mated L. lateralis females that were killed between 1 and 9 h following the initiation of copulation stage 2 (n=4), the male spermatophore was always positioned partially in the bursa copulatrix and partially in the SDG, with the spermatophore thumb extending up into the spermathecal duct (Fig. C and D). However, in a single L. lateralis female frozen 24 h following the initiation of copulation stage 2, the male spermatophore was located entirely within the female’s SDG, which was extremely distended. At this timepoint the male spermatophore was still intact, but the membranous outer sheath had begun to disintegrate.
In 2 out of 4 field-collected L. ficta females, spermatophores resembling those of L. lateralis were dissected from their reproductive tracts. Because none of the L. cruciata pairs progressed beyond stage 2 of copulation, we were unable to directly observe spermatophore transfer in this species. However, dissection of a L. cruciata male frozen during stage 1 revealed a spermatophore forming in the ejaculatory duct (Fig. 2D), as we had also observed in L. lateralis.


This study adds considerably to our knowledge of reproductive anatomy and spermatophore transfer in Luciola fireflies, a genus widespread throughout Asia. Hayashi and Suzuki (2003) dissected males of 20 species of Japanese fireflies, and reported pre-spermatophores from 12 of these, including L. cruciata, based on male internal anatomy. Fu and Ballantyne (2006) described a spermatophore in a field-collected female of L. leii. Our study provides a comprehensive description of male and female reproductive anatomy in L. lateralis and L. cruciata, as well as describing the time-course of spermatophore transfer in L. lateralis. We also observed the early stages of spermatophore formation in a mating pair of L. cruciata, and documented the presence of spermatophores in field-collected females of L. ficta.
Males of these three Asian Luciola species show similarities to several North American Photinus fireflies for which spermatophore structure, transfer, and fate was described by van der Reijden et al. (1997). In both genera, sperm coming from the testes are packaged into sperm bundles, and males have multiple pairs of reproductive accessory glands (three in Luciola spp., four in Photinus spp.) that are involved in spermatophore production. The main body of the spermatophore appears to be produced by the largest accessory glands (curled glands in Luciola, spiral glands in Photinus), as these contain spermatophore precursors that are very similar in shape to portions of the final spermatophore. In addition, the strong rhodamine B fluorescence observed in these glands suggests they produce spermatophore components with a high protein content. Our findings may also provide insight into Luciola mating systems. In a comparison of 4 Photinus species, species known to exhibit polyandrous mating systems were characterized by males with multiple accessory glands (Demary & Lewis, 2007). Our results suggest that female multiple mating may also occur in the three Luciola species studied here.
Female reproductive anatomy in these three Luciola species is also similar to that described for North American lamyprids by van der Reijden et al. (1997) and Rooney and Lewis (2000). Females have a single, spherical spermatheca, although in Luciola spermathecal size is much reduced compared to Photinus and Ellychnia (P. greeni females have two spermathecae; Demary, 2005). In addition, females in both genera possess specialized structures within their reproductive tract where male spermatophores eventually break down. Fu and Ballantyne (2006) describe female reproductive anatomy for Luciola leii, which is similar to that described here.
This study reveals a novel method of delivering sperm from the spermatophore into the female spermatheca in L. lateralis. In Photinus and Ellychnia fireflies, sperm bundles are released from the anterior end of the spermatophore while it is positioned within the female spermatheca, which can expand greatly to receive the spermatophore (van der Reijden et al., 1997). However, in Luciola females the spermatheca is smaller, and is connected to the bursa copulatrix by a narrow duct. When the spermatophore is transferred to the female reproductive tract, it is positioned so that its thumb enters the spermathecal duct, allowing sperm rings to be transferred from the sperm sac directly into the spermatheca.
Our results indicate that diverse substances produced by multiple male accessory glands are incorporated into spermatophores of these Luciola fireflies. Additional work is needed to identify these compounds and to determine whether these substances might influence female oviposition, re-mating rates, or sperm competition, as documented for seminal products of other insects (see Wolfner, 2002, Chapman & Davies, 2004 for review).
In Photinus fireflies, male spermatophores provide a net benefit to females by increasing their lifetime fecundity (reviewed by Lewis et al., 2004). In Photinus ignitus, 62% of radiolabelled proteins derived from male spermatophores were found in females’ mature oocytes at 2 days after mating (Rooney & Lewis, 1999), and triply-mated females showed a 73 % increase in their lifetime fecundity compared to singly mated females (Rooney & Lewis, 2002). Luciola fireflies do not eat as adults (Hayashi & Suzuki, 2003, Fu & Ballantyne, 2006), suggesting that spermatophores may represent an important nutritional supplement for Luciola spp. females. Hayashi and Suzuki (2003) postulated that spermatophore production in fireflies may be linked with a high degree of sexual dimorphism and female flightlessness, with spermatophores being absent in species that have larviform, apterous (wingless), or brachypterous (short-winged) adult females. Lewis and Cratsley (2008) extended this into a model linking nuptial gifts to female mobility and sexual size dimorphism based on the potential for male nutrient contributions to increase female fecundity. In this study, we found spermatophore production in three Luciola species where females are capable of flight, although sexual size dimorphism (measured as ratios of average female:male wet mass) ranged from 1.4 to 2.6 for L. lateralis and L. cruciata, respectively. Future studies examining whether Luciola spermatophores increase females’ lifetime fecundity will provide a more complete understanding of the relationship between sexual dimorphism and nuptial gift evolution.

We thank H. Hirata for technical and navigational assistance, and Jen Zon-Ho for providing L. ficta specimens. This research was supported by Tufts University and by U.S. National Science Foundation grant 10B-0543738 to S.M.L.


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Table 1. Reproductive characters for three species of Luciola fireflies based on the following sample sizes: L. lateralis and L. cruciata = 11 males, 9 females; L. ficta = 4 males, 4 females. Wet masses reported as means (± 1 SE).

Species Number of male accessory glands Spermatophore present? SDG Present? Number of SPT Male mass (mg) Female mass (mg)
L. lateralis Three Yes Yes One 25.15 (± 0.983) 34.17 (± 2.43)
L. cruciata Three Yes Yes One 65.95 (± 7.51) 171.25 (± 10.98)
L. ficta Three Yes Yes One 18.5 (± 0.912) 32.63 (±7.24)

Figure Legends
Figure 1. Female reproductive anatomy in Luciola fireflies. A) Entire reproductive system of L. cruciata female: Ovaries (OV) contain both mature and immature oocytes, and lead into the lateral oviducts (LO). The common oviduct (CO) enters the bursa copulatrix (BC) immediately adjacent to the spermatheca (SPT), the site of sperm storage and the spermataphore digesting gland (SDG), the site of eventual spermatophore digestion. B) Close-up of hemispherical spermataphore digesting gland (SDG), common oviduct (CO) and smaller, spherical spermatheca (SPT) of L. lateralis female. Median sclerotized plate indicated by arrowhead.
Figure 2. Male reproductive anatomy and spermatophore structure in Luciola fireflies. A) Reproductive system of L. lateralis male in visible light. B) Same specimen under fluorescence illumination showing rhodamine B staining. Labelled structures: a- long accessory glands, b- curled glands, c- short accessory glands, d-testes, e-ejaculatory duct. C) Intact spermatophore dissected from the reproductive tract of a L. lateralis female. D) Reproductive system of a male L. cruciata interrupted during Stage 1 mating under fluorescence illumination showing rhodamine B staining of the pre-spermatophore emerging from the curled glands into the ejaculatory duct.
Figure 3. Process of spermatophore transfer in Luciola lateralis fireflies. A) Reproductive tracts of male (lower right) and female (upper left) in copula, with spermatophore in the process of being transferred into the female bursa copulatrix (taken in visible light). B) Same specimen under fluorescence illumination showing rhodamine B staining of male reproductive tract and male spermatophore inside female. C) Close-up of female reproductive tract taken in visible light, showing male spermatophore (pink, mitten-shaped structure) located partly within the female’s spermatophore digesting gland (SDG). Sperm delivery tube (indicated by white arrow) leading from the internal sperm sac is positioned within the female’s spermathecal duct. Needle-shaped sclerotized plate is visible embedded in bursal wall. D) Same specimen under fluorescence illumination showing rhodamine B stained male spermatophore with sperm delivery tube (white arrow).



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