Cantharidin as a nuptial gift from males to females in false blister beetles

Appendix 3-2 Distribution of the cantharidin-producing beetles (Meloidae and Oedemeridae) on the mainland Tokyo, Izu Peninsula, and 11 islands, based on

4. Cantharidin as a nuptial gift from males to females in false blister beetles

4-1. Introduction

Nuptial gifts are food or items that are transferred between mates in several groups of animals, generally from males to females. Nuptial gifts are used by recipients for reproduction and to maintain physical condition (Boggs 1995; Vahed 1998; Gwynne 2008). Lewis et al. (2014) redefined nuptial gifts as materials (beyond the obligatory gametes) provided by a donor to a recipient during courtship or copulation in order to improve donor fitness, and divided them into four types: exogenous oral gifts, endogenous oral gifts, endogenous genital gifts, and endogenous transdermal gifts.

Exogenous oral gifts are materials, such as nuptial prey (e.g., Thornhill 1976; Cumming 1994) or plant seeds (e.g., Albo & Costa 2010), collected by donors. Such gifts may improve mating success, copulation duration, and the quantity of sperm transferred by donors. Endogenous oral gifts comprise orally ingestible gifts derived from the donor’s physiological systems, such as spermatophores that attach externally to the female genitalia (often called the spermatophylax) (Gwynne 1984), haemolymph, or other body parts (Fedorka & Mousseau 2004). Endogenous genital gifts are also manufactured by the donor (particularly by the donor’s reproductive glands) and absorbed within the recipient’s genital tract; they consist of spermatophores containing nutrients (e.g., Rooney & Lewis 1999) or non-nutritive substances, such as immunostimulants or antibiotics (Poiani 2006), water (Arnqvist et al. 2005), ions or minerals (Engebretson &

Mason 1980), or defensive compounds (Eisner & Meinwald 1995). In some insects, leeches, squid, polychaetes, turbellarians, and acochlidan sea slugs, males traumatically inject their ejaculates and accessory gland fluids into their mates (Lange et al. 2013);

such fluids are considered endogenous transdermal gifts.

Males of some insect species that use defense chemicals against predators store defensive compounds as nuptial gifts with their spermatophores; for example, pyrrolizidine alkaloids (Dussourd et al. 1988; Eisner & Meinwald 1995) and cyanogenic glycosides (Cardoso & Gilbert 2007). These chemicals are transferred from

males to females through copulation, and allow females to protect their eggs from predation. Cantharidin, a toxic terpenoid compound, may also serve as a nuptial gift in some beetles in the same context as above. This defensive compound is produced by true blister beetles (Family Meloidae) and false blister beetles (Oedemeridae) (Carrel &

Eisner 1974; Carrel et al. 1986), and stored in their haemolymph and various other tissues at the larval and adult stages (Dixon et al. 1963; Carrel et al. 1986, 1993; Frenzel

& Dettner 1994; Holz et al. 1994). In true blister beetles, only males continue to synthesise cantharidin after adult eclosion, and newly synthesised cantharidin is moved to their reproductive accessory glands, then transported to the epididymis and vas deferens, and finally deposited and accumulated in the testes (Nikbakhtzadeh et al.

2007). Adult females synthesise cantharidin only during the larval period (Sierra et al.

1975; Carrel et al. 1993). Thus, adult females require supplemental cantharidin from male-derived spermatophores to defend their eggs successfully. After mating, cantharidin accumulates in the female spermatophoral receptacle (bursa copulatrix) and is allocated to eggs (Carrel et al. 1993; Nikbakhtzadeh et al. 2007, 2012).

In contrast, false blister beetle adult females, as well as adult males, can synthesise cantharidin (Carrel et al. 1986; Frenzel & Dettner 1994). In most cases, field-caught females contain more cantharidin than males (Frenzel & Dettner 1994; Abtahi et al.

2012). Holz et al. (1994) reported that no cantharidin, or only a very small amount of cantharidin, is transferred from males to females at mating, and thus that its contribution as a nuptial gift may be negligible in Oedemera femorata. Thus, it is still unclear whether male false blister beetles use cantharidin as a nuptial gift.

In our preliminary observations of male and female false blister beetle internal reproductive organs, we found that some species have conspicuous sclerotized spines within the female bursa copulatrix (bursal spines), while other species have no such spines. These bursal spines have not previously been described in these beetles. The females of most moths and butterflies (Lepidoptera) also have spines in the bursa, and

spermatophores. First, we mapped the evolutionary patterns of bursal spines using phylogenetic trees inferred from mitochondrial 16S and nuclear 28S ribosomal DNA sequences from 11 genera and 39 species collected in Japan. Second, we observed the size and transfer process of spermatophores in the laboratory, and compared them with the characteristics of female bursal spines. Finally, using a new bioassay system with the small beetle Mecynotarsus tenuipes from the family Anthicidae, we compared the quantity of cantharidin contained in eggs laid by spiny and spineless species. M.

tenuipes was attracted to a droplet of cantharidin/acetone solution to feed upon, and the number of individuals attracted increased with cantharidin concentration. Our working hypothesis is that females from species with bursal spines can consume larger spermatophores and provide more cantharidin to their eggs than females from species without spines. Spermatophores thereby serve as endogenous genital gifts according to the definition of Lewis et al. (2014).

4-2. Materials and methods

4-2-1. Morphological measurements and mating experiments

A total of 554 false blister beetle individuals, consisting of 11 genera and 39 species, were collected across Japan from March to June, 2012 to 2015, and reared at 25 ± 1°C with a 14 h light and 10 h dark cycle. Within 3 days after collection, they were cooled at -20°C for 10 minutes. After their maximum elytral lengths were measured to the nearest 0.1 mm using a binocular eyepiece (Leica MZFL3), internal reproductive organs were dissected in insect saline consisting of 0.9 g NaCl, 0.02 g CaCl2, 0.02 g KCl, and 0.02 g NaHCO3 in 100 mL water. A Nikon H550S stereomicroscope with a Nikon DS-L3 camera system was used to examine bursal spines, count the number of spines, and measure the length of spines. If the female carried a spermatophore, the major and minor axes of the spermatophore were measured under a Nikon H550S stereomicroscope with a Nikon DS-L3 camera system, and its volume was calculated as a spheroid.

Mating was observed in plastic vials (27 mm in diameter, 110 mm in depth) that contained a male and female. If mating occurred, copulation duration was recorded.

Females were removed and dissected about 30 min after copulation to determine the presence or absence of the spermatophore. If present, its volume was calculated as mentioned above.

4-2-2. Molecular phylogenetic analysis

139 individuals from 11 genera and 39 species of false blister beetles were used for molecular phylogenetic analysis (Appendix 4-1). These beetles were collected across Japan from January to November, 2007 to 2015, and comprised all four tribes of the subfamily Oedemerinae distributed in Japan: Asclerini, Ditylini, Nacerdini, and Oedemerini. Collected beetles were preserved in 99.5% ethanol or as dried specimens.

Meloe coarctatus of the family Meloidae was used as an outgroup species (Appendix 4-1).

Total genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). For all 140 samples, fragments of mitochondrial 16S rRNA and nuclear 28S rRNA genes were amplified using Ex Taq® (TaKaRa, Tokyo, Japan) with three primer sets: 5ʹ-GGGAGGAAAAGAAACTAAC-3ʹ (Ober 2002) and 5ʹ-CACGTACTCTTGAACTCTCTCT-3ʹ (Snäll et al. 2007) for the 28S D1 region, 5ʹ-AGAGAGAGTTCAAGAGTACGTG-3ʹ (Snäll et al. 2007) and 5ʹ-TTGGTCCGTGTTTCAAGACGGG-3ʹ (Snäll et al. 2007) for the 28S D2 region, and 5ʹ-CGCCTGTTTAWCAAAAACAT-3ʹ (Hundsdoerfer et al. 2009) and 5ʹ-CTCCGGTYTGAACTCAGATCAAGT-3ʹ (Hundsdoerfer et al. 2009) for the 16S region. The PCR reaction mix (total volume 10 μL) contained 1.0 μL 10× Ex Taq Buffer, 0.8 μL 25 mM dNTP mix, 0.5 μL each of the forward and reverse primers (10 pM), 0.05 μL Taq polymerase, 6.15 μL distilled deionized water, and 1.0 μL template DNA. PCR

extension at 72°C (Lopez-Vaamonde et al. 2001); and for the 16S rRNA gene, an initial 2-min denaturing step at 94°C, 35 cycles of 30 s at 94°C, 30 s at 53°C, and 80 s at 72°C, with a final 5-min extension at 72°C. PCR products were purified with illustra™

ExoProStar™ 1-Step (GE Healthcare, Buckinghamshire, UK) and sequenced using BigDye® Terminator ver. 3.1 (Applied Biosystems, Foster City, CA, USA) on an ABI 3130xl Genetic Analyzer (Applied Biosystems). Direct sequencing data were aligned to 448 bp16S and 805 bp 28S rRNA sequences using MEGA5 (Tamura et al. 2011).

Phylogenetic analyses were performed using MEGA5 with the neighbor-joining (NJ) method based on p-distance and maximum likelihood (ML) estimation based on Kimura’s 2-parameter model with gamma-distributed rates (28S), the Tamura-Nei model with gamma-distributed rates and invariant sites (16S), and the General Time Reversible model with gamma-distributed rates and invariant sites (28S+16S) (Tamura et al. 2011). The best-fit nucleotide substitution model was estimated based on the Bayesian Information Criterion (BIC: Schwarz 1978) using MEGA5 (Tamura et al.

2011).

4-2-3. Bioassay of egg cantharidin

To assess the relative quantities of cantharidin in the eggs of false blister beetles, we developed a new bioassay system using adult males of the small (ca. 2 mm in body length) beetle Mecynotarsus tenuipes (Anthicidae), which is strongly attracted to cantharidin (Hemp & Dettner 2001). This beetle was collected at Iyo, Ehime Prefecture, Western Japan, from May to August, 2015, with cantharidin traps. Traps were made from plastic cups (95 mm in internal diameter, 50 mm in depth) with two square openings (10

× 10 mm²) in the covers. 0.5 mL volumes of a 10⁻²M solution of cantharidin in acetone were applied to filter paper disks (55 mm in diameter, #2, Whatman, Little Chalfont, UK), which were then wrapped in a paper towel and placed on the cup bottom.

Wrapping discs with a paper towel prevented the direct exposure of trapped insects to cantharidin. Field-caught M. tenuipes were placed in plastic boxes (165 × 225 × 25 mm³ in volume), the bottoms of which were covered with the sand substrate obtained from the

trapping area (ca. 5 mm in depth), and kept at 25 ± 1°C with a 14 h light and10 h dark cycle. A piece of wet cat food (Pro Plan; Nestlé, Ibaraki, Japan) was provided daily.

We assessed the attractancy of M. tenuipes to cantharidin. Bioassays were conducted in petri dishes 93 mm in diameter and 9 mm in depth. Four equally sized circles of white paper (12 mm in diameter, each 23 mm distant) were placed under the petri dish (Fig. 4-1). A 6 µL droplet of cantharidin solution was placed on each of two diagonally adjacent circles, and 6 µL control droplets were placed on the other two circles. 20 male M. tenuipes individuals were then released into the petri dish. After a 1 min acclimation to the petri dish, individuals within each circle were counted at 0, 5, 10, 15, 20, 25, and 30 min, and averaged for the two test and two control circles. M. tenuipes individuals were never used for more than one assay. The results for assays using five concentrations (10^-2, 10^-3, 10^-4, 10^-5, and 10^-6 M) of cantharidin/acetone solution showed that M. tenuipes beetles were clearly attracted to cantharidin, and that their response was stronger for cantharidin concentrations between 10^-3 and 10^-6 M. Beetles were not attracted to the control circles (Fig. 4-2). Their attraction to cantharidin was highest at the 10^-3 M concentration. Because attractancy decreased gradually with exposure time (Fig. 4-2), we used the number of M. tenuipes beetles within each circle just after 1 min of acclimation as the response variable for the relative cantharidin concentrations.

Egg cantharidin was compared between congeneric false blister beetles with conspicuous bursal spines (Nacerdes caudata and N. katoi) and no spines (N.

waterhousei). Mature females of these species were collected at four sites in Yamagata, Saitama, Tokyo, and Kanagawa Prefectures, Central to Northern Japan, from June to August, 2015. Females were kept individually in plastic cups (43 mm in diameter, 27 mm in depth) at 25 ± 1°C with a 14 h light and10 h dark cycle. A wet paper towel was placed into the cups to provide a substrate for egg deposition. Beetles were provided with pollen powder (Bee Pollen; Swanson Hearth Products, ND, USA) as a food source.

When egg masses were deposited, the number of eggs was counted and the major and

acetone using a pestle. Another 100 µL of acetone was used to wash the material on the pestle into the tube, which was then centrifuged at 5,000 rpm for 5 min. The supernatant was removed, added to a new 0.5 mL tube, and preserved at -80°C until further use. In the bioassay, the number of M. tenuipes individuals per circle was multiplied by 33.3 (200 µL /6 µL) and then divided by the total egg volume used for extraction (egg number × mean egg volume) to compare the attractancy among species.

Egg cantharidin was also assessed for three species of insects that have never been reported to produce cantharidin; two dragonfly species, Anax parthenope (Odonata:

Aeshnidae) and Orthetrum albistylum (Libellulidae); and one true bug Riptortus pedestris (Hemiptera: Alydidae). For these species, 500, 100, and 9 eggs were removed from the deposited eggs, respectively, and treated as described above.

4-3. Results

4-3-1. Female bursal spines and male spermatophores

We found sclerotized spines in the inner walls of the female bursa copulatrix (bursal spines) in 16 of 39 species examined (Fig. 4-3), but not in the other 23 species. The spines differed in shape and size, and were divided into four types: a few long spines in Nacerdes katoi, N. hilleri, N. umenoi, and N.caudata (Fig. 4-3a); many short spines in four bands in N. konoi, N. osawai, and N. luteipennis (Fig. 4-3b); many short, uniformly arranged spines in N. deformis, N. spinicoxis, and Chrysanthia geniculata (Fig. 4-3c, d);

and a few rose thorn-like spines in Dryopomera kurosai, Oedemera venosa, O.

manicata, O. sexualis, O. lucidicollis, and O. testaceithorax (Fig. 4-3e, f).

We were able to dissect a total of 13 species of females mated in the laboratory or appearing to have recently mated in the field. Spermatophores were observed in 9 species, Nacerdes katoi, N. hilleri, N. umenoi, N.caudata, N. konoi, N. luteipennis, Chrysanthia geniculata, Oedemera manicata, and O. lucidicollis. In four species, only the sperm mass was observed: N wadai, N. waterhousei, O. subrobusta, and O. robusta (Fig. 4-4). In Nacerdes caudata, spermatophores in various states were observed in

female bursae. Just after mating, a large spermatophore was placed in the bursa (Fig.

4-4a). For field-caught females, we usually found no spermatophore or only a spermatophore residue (Fig. 4-4b) when they were unmated or had oviposited (Fig.

4-4c). The spermatophore thus rapidly disappeared after copulation from the storage area in the apical part of the bursa, where spines are densely distributed. Sperm were stored in another tubular organ, the spermatheca.

4-3-2. Female bursal spine distribution on phylogenetic trees

Two groups at the tribe level, Nacerdini+Ditylini and Oedemerini, were identified with relatively high bootstrap probabilities in both NJ and ML trees, although the basal parts of the phylogenetic trees had poor statistical support and topologies differed slightly between NJ (Appendix 4-2) and ML trees (Fig. 4-5). The tribe Asclerini was paraphyletic, and its branching patterns varied depending on the method used for tree construction. Monophyly of each genus was well-supported in both NJ and ML trees, excluding Eobia, which consisted of two subgenera (Eobia + Pareobia), and Oedemera including Dryopomera (Fig. 4-5).

Spineless bursa appears to be an ancestral state, and the acquisition of bursal spines seemed to occur multiple times (Fig. 4-6). Four types of spine development were specific to phylogenetic lineages: a small number (< 1,000) of thick and long (70-100 μm) spines distributed uniformly in the bursa of Nacerdes katoi, N. hilleri, N. umenoi, and N.caudata; a medium number (1,000 to 3,000) of thick but short (< 10 μm) spines arranged in four longitudinal bands in N. konoi, N. osawai, and N. luteipennis; a large number (2,000 to 9,000) of fine and short (7-13 μm) spines distributed uniformly in N.

deformis, N. spinicoxis, and Chrysanthia geniculata; and a very small number (< 400) of robust (40-80 μm), rose thorn-like spines in Dryopomera kurosai, Oedemera venosa, O. manicata, O. sexualis, O. lucidicollis, and O. testaceithorax (Fig. 4-6).

sperm masses lacked spines (Fig. 4-6).

4-3-3. Bioassay of egg cantharidin

Nacerdes caudata and N. katoi had long spines in the female bursa, but N.

waterhousei lacked bursal spines, despite being a very closely related species (Fig. 4-7).

Spermatophores were found in the female bursa in the former two species, but only sperm masses were found in the latter species (Fig. 4-7). Other internal reproductive organs seemed to be similar in shape and size among species. In this experiment, therefore, the data from the former two species were pooled for comparison of eggs laid by the spiny and spineless females. The canthariphilous beetle Mecynotarsus tenuipes was not attracted to egg-extracted droplets from dragonflies (A. parthenope and O.

albistylum) or a true bug (R. pedestris), but was attracted to those from false blister beetles (Fig. 4-8). However, the attractancy of M. tenuipes was much higher per unit egg mass for N. caudata/katoi than for N. waterhousei (Fig. 4-8). The number of M.

tenuipes individuals attracted per unit egg mass was similarly high among the false blister beetles: 29.2 (N = 4, SD = 14.6) for N. caudata, 25.7 (N = 8, SD = 11.4) for N.

katoi, and 19.1 (N = 17, SD = 7.6) for N. waterhousei (ANOVA: F2, 28 = 3.4, P = 0.11).

However, the attractancy of M. tenuipes was much higher for N. caudata/katoi, which had bursal spines, than for N. waterhousei which lacked spines (Fig. 4-8). The mean elytral length of egg-laying females was 9.95 mm (N = 12, SD = 0.91) in N.

caudata/katoi and 10.11 mm (N = 17, SD = 0.92) in N. waterhousei (Student’s t-test, t₂₇

= 0.49, P = 0.63). The number of eggs per mass was 104 (N = 12, SD = 32) and 121 (N

= 17, SD = 27), respectively (t-test, t27 = 1.51, P = 0.14). The mean egg volume was 0.076 mm³ (N = 12, SD = 0.012) and 0.076 mm³ (N = 17, SD = 0.011), respectively (t27

= 0.003, P = 1.00). The mean egg volumes of the other insect groups (A. parthenope, O.

albistylum, and R. pedestris) were 0.096 (N = 1), 0.019 (N = 2, SD = 0.001), and 1.02 (N = 3, SD = 0.05), respectively.

4-4. Discussion

Some species of false blister beetles have sclerotized spines on the inner walls of the female bursa. However, even within the same genus, there are species that lack the spines.

Molecular phylogenetic trees constructed using both mitochondrial 16S and nuclear 28S partial sequences suggest that spineless bursa is the ancestral state, and that the acquisition of bursal spines occurred multiple times. These multiple acquisitions may be supported by the fact that four types of bursal spines can be recognised, each corresponding to a monophyletic phylogenetic group: a few long spines in Nacerdes katoi, N. hilleri, N. umenoi, and N.caudata; many short spines in four bands in N. konoi, N. osawai, and N. luteipennis; many short, uniformly arranged spines in N. deformis, N.

spinicoxis, and Chrysanthia geniculata; and a few rose thorn-like spines in Dryopomera kurosai, Oedemera venosa, O. manicata, O. sexualis, O. lucidicollis, and O.

testaceithorax (see Figs. 4-3, 4-6). Spines or spine-like sclerites in the female genital tract are also known in other insect groups, including most moths and butterflies (Lepidoptera) (e.g., Sánchez et al. 2011; Cordero & Baixeras 2015), some click beetles (Coleoptera, Elateridae) (Ôhira 2002; Costa et al. 2010), some marsh beetles (Coleoptera:

Scirtidae) (Yoshitomi 2005), some cardinal beetles (Coleoptera: Pyrochroidae) (Kai &

Yoshitomi 2013), some bark lice (Psocoptera: Psyllipsocidae) (Lienhard & Ferreira, 2013), and some sandflies (Diptera: Psychodidae) (Zhang & Leng 1997;

Kakarsulemankhel 2004). In moths and butterflies, the spines, known as ‘signa,’ assist in tearing open the received spermatophore in the female bursa (review by Cordero &

Baixeras 2015). However, the functions of bursal spines have never been examined in other groups of insects.

In false blister beetles, spermatophores were transferred only in species with bursal spines. In species without these spines, only sperm masses were directly ejaculated into the female. Spermatophores were much larger than sperm masses, because the former

digest the spermatophore envelope, as is the case in moths and butterflies (Cordero &

Baixeras 2015).

The spermatophore sheath includes nutrients (e.g., Rooney & Lewis 1999) and non-nutritive substances, such as immunostimulants and antibiotics (Poiani 2006), water (Arnqvist et al. 2005), ions or minerals (Engebretson & Mason 1980), and defensive compounds (Eisner & Meinwald 1995). True and false blister beetles can synthesise cantharidin and use it as a defense chemical. It was confirmed in several species that cantharidin is transferred from males to females and also incorporated into eggs to deter egg predators (Sierra et al. 1975; Carrel et al. 1993; Nikbakhtzadeh et al. 2007, 2012;

Holz et al. 1994). In some butterfly species, males provide defensive chemicals, such as pyrrolizidine alkaloids (e.g., Drummond 1984) and cyanogenic glycosides (e.g., Cardoso

& Gilbert 2007), in spermatophores to females. The most likely functional consequence of the transfer of defensive cyanogenic glycosides is enhanced female and/or egg survival (Cardoso & Gilbert 2007). Males of the beetle Neopyrochroa flabellata (Coleoptera:

Pyrochroidae) also transfer cantharidin acquired from cantharidin-producing insects to females during copulation; mated females then provide the cantharidin to developing eggs (Eisner et al. 1996b). Eggs laid by females mated with cantharidin-fed males are better-defended than eggs laid by females mated with males not fed cantharidin (Eisner et al. 1996b).

Using a novel bioassay system, we demonstrated that female false blister beetles of the genus Nacerdes also incorporate cantharidin into eggs. In true blister beetles, cantharidin content in haemolymph ranges from 6 × 10^-4 to 1 × 10^-1 M (Carrel & Eisner 1974).

Although their precise cantharidin content has not been determined, false blister beetles contain enough cantharidin to cause blisters on human skin (Vaurie, 1951; McCormick &

Carrel 1987). In our bioassay system, the canthariphilous beetle M. tenuipes was most strongly attracted to 10^-3 M cantharidin. This can be used as a bio-indicator for naturally occurring concentrations of cantharidin. Higher concentrations of egg cantharidin were detected in species with bursal spines than in species without bursal spines. This suggests that the former species use sclerotized spines to break and absorb large spermatophores, which are used to enhance egg provisioning. Quick consumption of spermatophores may

also allow for additional mating opportunities, which enables females to acquire more cantharidin from other spermatophores.

The evolutionary significance of female bursal spines remains unclear; in particular, why some species possess such spines while others do not. However, we found an interesting parallel between hind-leg sexual dimorphism and bursal spine development in the tribe Oedemerini. In 6 species of this tribe, male hind legs are much larger than female hind legs; the female bursa is spiny in this species, but not spiny in the 2 species that lack hind-leg sexual dimorphism (Figs. 4-5, 4-6). Exaggerated traits in males can be the result of both sexual selection, including mate choice, and male-male competition (e.g., Emlen 2008). Males of the leaf beetle Sagra femorata have enlarged hind legs, and they grasp and remove rival males with their hind legs to guard females while mating and to defend their feeding territories (Katsuki et al. 2014). In some species of Oedemerini, sexual selection may promote hind-leg sexual dimorphism and also affect the development of bursal spines in females. However, in other false blister beetles, species with bursal spines do not exhibit such sexual dimorphism. Closer analysis of sexual selection in Oedemerini will be important to understanding the evolutionary significance of female bursal spines.

False blister beetles are globally distributed and consist of three subfamilies:

Oedemerinae, Polypriinae, and Calopodinae. In Japan, only the subfamily Oedemerinae is present; this study was therefore limited to that group. However, female bursal sclerites (not spines) have been found in Polypriinae and Calopodinae and some (e.g., Microsessinia) Asclerini within Oedemerinae (Lawrence & Ślipiński 2010). In future studies, the construction of more comprehensive phylogenetic trees, including all subfamilies, will help to resolve the evolutionary dynamics of male spermatophores and female bursal spines and sclerites.

a b

Figure 4-1 Bioassay of cantharidin using small canthariphilous beetle Mecynotarsus tenuipes (Anthicidae) in a petri dish. 6 µL of acetone extract (experimental sample) were deposited within each of two diagonally adjacent circles, and 6 µL of acetone (control) were deposited within two other circles (all circles were 12 mm in diameter). 20 M.

tenuipes males were released into the petri dish, and the number of individuals within each circle was counted after a 1 min acclimation period. Controls and acetone extracts from eggs of the bug Riptortus pedestris (a) do not attract M. tenuipes, but acetone extracts from eggs of the false blister beetle Nacerdes caudata (b) do attract them.

M ean num ber of i ndi v idual s per c ir c le

Elapsed time (min)

0 5 10 15 20 25 30

Figure 4-2 Temporal changes at 5 min intervals in the mean number of Mecynotarsus tenuipes attracted to cantharidin/acetone solution concentrations of 10^-6 (circles), 10^-5 (rails), 10^-4 (triangles), 10^-3 (squares), and 10^-2 M (rhombuses) (closed symbols and solid lines), and to acetone controls (open symbols and dotted lines).

a b

c d

e f

100 μm

100 μm 100 μm

100 μm

100 μm 100 μm

Figure 4-3 Sclerotized spines in the female bursa copulatrix of six species of false blister beetle: Nacerdes umenoi (a), N. luteipennis (b), N. deformis (c), Chrysanthia geniculata (d), Dryopomera kurosai (e), and Oedemera testaceithorax (f).

a b c

d e

spp

spm

Figure 4-4 Fate of the spermatophore (spp) in the female bursa copulatrix of the false blister beetle Nacerdes caudata (a-c) and the sperm mass (spm) in the female bursa of N.

waterhousei (d, e). The large spermatophore is formed in the bursa just after copulation

Nacerdes katoi (Fujieda-shi) Nacerdes katoi (Kimotsuki-gun)

Nacerdes katoi (Hachioji-shi) Nacerdes katoi (Chichibu-shi) Nacerdes katoi (Hinohara-mura) Nacerdes katoi (Katsuyama-shi) Nacerdes katoi (Iida-shi01) Nacerdes katoi (Iida-shi02) Nacerdes katoi (Shimoina-gun) Nacerdes katoi (Aso-shi01) Nacerdes katoi (Aso-shi02)

Nacerdes hilleri (Yatsushiro-shi) Nacerdes hilleri (Iida-shi) Nacerdes hilleri (Higashiusuki-gun) Nacerdes umenoi (Nago-shi) Nacerdes caudata (Fujieda-shi) Nacerdes caudata (Nikko-shi) Nacerdes caudata (Higashiusuki-gun) Nacerdes caudata (Shimoina-gun) Nacerdes caudata (Katsuyama-shi) Nacerdes caudata (Aso-shi) Nacerdes caudata (Iida-shi02) Nacerdes caudata (Hinohara-mura) Nacerdes caudata (Iida-shi01) Nacerdes caudata (Uenohara-shi)

Nacerdes wadai (Matsuyama-shi) Nacerdes wadai (Nankoku-shi) Nacerdes wadai (Aso-shi) Nacerdes waterhousei (Nishitama-gun) Nacerdes waterhousei (Hinohara-mura02)

Nacerdes waterhousei (Hinohara-mura03) Nacerdes waterhousei (Chichibu-shi) Nacerdes waterhausei (Sannohe-gun) Nacerdes waterhausei (Hinohara-mura01) Nacerdes waterhousei (Hachioji-shi) Nacerdes waterhousei (Katsuyama-shi) Nacerdes waterhousei (Iida-shi) Nacerdes waterhousei (Shimoina-gun) Nacerdes waterhousei (Koshu-shi) Nacerdes waterhousei (Miyoshi-gun) Nacerdes waterhousei (Aso-shi) Nacerdes waterhousei (Kimotsuki-gun)

Nacerdes atriceps (Yufutsu-gun) Nacerdes atriceps (Matsumoto-shi)

Nacerdes atriceps (Chichibu-shi)

Anogcodes coarctata (Ashoro-gun) Nacerdes melanura (Chiyoda-ku)

Nacerdes konoi (Sado-shi) Nacerdes konoi (Iida-shi) Nacerdes konoi (Hachioji-shi) Nacerdes konoi (Koshu-shi) Nacerdes konoi (Hinohara-mura) Nacerdes konoi (Fujieda-shi)

Nacerdes osawai (Katsuyama-shi) Nacerdes osawai (Hinohara-mura) Nacerdes osawai (Iida-shi) Nacerdes luteipennis (Hachioji-shi)

Nacerdes luteipennis (Higashiusuki-gun) Nacerdes luteipennis (Towada-shi) Nacerdes luteipennis (Hinohara-mura) Nacerdes luteipennis (Sado-shi) Nacerdes luteipennis (Katsuyama-shi) Nacerdes luteipennis (Iida-shi01) Nacerdes luteipennis (Iida-shi02) Nacerdes luteipennis (Shimoina-gun) Nacerdes deformis (Hinohara-mura) Nacerdes deformis (Higashiusuki-gun) Nacerdes deformis (Hachioji-shi) Nacerdes deformis (Fujieda-shi) Nacerdes spinicoxis (Takayama-shi)

Chrysanthia geniculata (Hidaka-gun) Chrysanthia geniculata (Tsumagoi-mura) Chrysanthia geniculata (Yamanashi-shi) Ditylus laevis (Rikubetsu-cho)

Ditylus laevis (Ashoro-cho)

Oedemera subrobusta (Tohmi-shi) Oedemera robusta (Hanno-shi) Oedemera robusta (Hinohara-mura) Dryopomera kurosai (Ishigaki-jima01) Dryopomera kurosai (Ishigaki-jima03) Dryopomera kurosai (Ishigaki-jima02) Oedemera venosa (Chichibu-shi) Oedemera venosa (Naka-cho) Oedemera manicata (Hayama-cho01) Oedemera manicata (Hayama-cho02) Oedemera sexualis (Matsuyama-shi) Oedemera sexualis (Iyo-shi) Oedemera sexualis (Ishigaki-jima) Oedemera sexualis (Kunigami-gun) Oedemera sexualis (Amami-shi) Oedemera sexualis (Nago-shi)

Oedemera lucidicollis (Hanno-shi) Oedemera lucidicollis (Hinohara-mura) Oedemera lucidicollis (Yamanashi-shi) Oedemera lucidicollis (Hitachinaka-shi) Oedemera testaceithorax (Kunigami-gun) Oedemera testaceithorax (Ishigaki-jima) Asessinia vittata (Amami-shi01)

Asessinia vittata (Amami-shi02) Asessinia flavomarginata (Tosa-shi)

Asessinia flavomarginata (Amami-shi) Asessinia geniculata (Ishigaki-jima01) Asessinia geniculata (Ishigaki-jima02) Eobia (Pareobia) florilega (Sado-shi) Eobia (Pareobia) matsumurai (Chichi-jima) Eobia (Pareobia) matsumurai (Muko-jima) Indasclera brunneipennis (Hinohara-mura01) Indasclera brunneipennis (Hinohara-mura02) Indasclera japonica (Kunigami-gun01) Indasclera japonica (Kunigami-gun02)

Indasclera subrugosa (Kunigami-gun01) Indasclera subrugosa (Kunigami-gun02)

Hyperopselaphus ikedai (Kuro-shima)

Ischnomera nigrocyanea (Hanno-shi) Ischnomera nigrocyanea (Hachioji-shi) Ischnomera okushimai (Kunigami-gun) Eobia (Eobia) magna (Ishigaki-jima01)

Eobia (Eobia) magna (Ishigaki-jima02) Eobia (Eobia) chinensis (Shikine-jima) Eobia (Eobia) chinensis (Amami-shi) Eobia (Eobia) chinensis (Hachijo-jima) Eobia (Eobia) fuscipennis (To-shima) Eobia (Eobia) fuscipennis (Hachijo-jima)

Eobia (Eobia) fuscipennis (Shikine-jima) Eobia (Eobia) fuscipennis (Amami-shi) Eobia (Eobia) chinensis (Tosa-shi) Eobia (Eobia) fuscipennis (Miyake-jima) Eobia (Eobia) chinensis (Sado-shi) Eobia (Eobia) cinereipennis (Muko-jima02)

Eobia (Eobia) cinereipennis (Chichi-jima02) Eobia (Eobia) cinereipennis (Haha-jima02)

Eobia (Eobia) cinereipennis (Amami-shi) Eobia (Eobia) cinereipennis (Sado-shi) Eobia (Eobia) cinereipennis (Haha-jima01) Eobia (Eobia) cinereipennis (Muko-jima01) Eobia (Eobia) cinereipennis (Muko-jima01) Eobia (Eobia) cinereipennis (To-shima) Eobia (Eobia) cinereipennis (Miyake-jima)

Eobia (Eobia) cinereipennis (Ishigaki-jima)

Meloe coarctatus (Oamishirasato-shi) 100 100

100 100

100

83 69 100

100

66 64 62 95 69 60 96

95 90 100 72 99

99 100

100

97 44

100 91

89 95

66 100

99 25

100

31 11

12 36

99 100

9169 50 99

99 82 88

92 99

65 23 82

9978

83 97

81 96 82 87

99 71 5369 99

0.1

Nacerdes katoi (Fujieda-shi) Nacerdes katoi (Kimotsuki-gun)

Nacerdes wadai (Matsuyama-shi) Nacerdes wadai (Nankoku-shi) Nacerdes wadai (Aso-shi) Nacerdes waterhousei (Nishitama-gun) Nacerdes waterhousei (Hinohara-mura02)

Nacerdes atriceps (Chichibu-shi)

Anogcodes coarctata (Ashoro-gun) Nacerdes melanura (Chiyoda-ku)

Nacerdes konoi (Sado-shi) Nacerdes konoi (Iida-shi) Nacerdes konoi (Hachioji-shi) Nacerdes konoi (Koshu-shi) Nacerdes konoi (Hinohara-mura) Nacerdes konoi (Fujieda-shi)

Nacerdes osawai (Katsuyama-shi) Nacerdes osawai (Hinohara-mura) Nacerdes osawai (Iida-shi) Nacerdes luteipennis (Hachioji-shi)

Chrysanthia geniculata (Hidaka-gun) Chrysanthia geniculata (Tsumagoi-mura) Chrysanthia geniculata (Yamanashi-shi) Ditylus laevis (Rikubetsu-cho)

Ditylus laevis (Ashoro-cho)

Asessinia vittata (Amami-shi02) Asessinia flavomarginata (Tosa-shi)

Indasclera subrugosa (Kunigami-gun01) Indasclera subrugosa (Kunigami-gun02)

Hyperopselaphus ikedai (Kuro-shima)

Ischnomera nigrocyanea (Hanno-shi) Ischnomera nigrocyanea (Hachioji-shi) Ischnomera okushimai (Kunigami-gun) Eobia (Eobia) magna (Ishigaki-jima01)

Eobia (Eobia) cinereipennis (Chichi-jima02) Eobia (Eobia) cinereipennis (Haha-jima02)

Eobia (Eobia) cinereipennis (Ishigaki-jima)

Meloe coarctatus (Oamishirasato-shi) 100 100

100 100

100

83 69 100

100

66 64 62 95 69 60 96

95 90 100 72 99

99 100

100

97 44

100 91

89 95

66 100

99 25

100

31 11

12 36

99 100

9169 50 99

99 82 88

92 99

65 23 82

9978

83 97

81 96 82 87

99 71 5369 99

0.1

N. katoi

Nacerdini

~ ~

N. hilleri N. umenoi

N. caudata

N. wadai

N. waterhousei

N. atriceps A. coarctata

N. melanura N. konoi

N. osawai

N. luteipennis

N. deformis N. spinicoxis

C. geniculata D. laevis

O. subrobusta O. robusta D. kurosai O. venosa O. manicata O. sexualis

O. lucidicollis O. testaceithorax

A. vittata A. flavomarginata A. geniculata

E. (P.) florilega E. (P.) matsumurai

I. burunneipennis I. japonica

I. subrugosa

H. ikedai I. nigrocyanea / I. okushimai

E. (E.) magna

E. (E.) chinensis / E. (E.) fuscipennis

E. (E.) cinereipennis

Meloe coarctata

Ditylini

Oedemerini

Asclerini

Outgroup

Figure 4-5 A phylogenetic tree of false blister beetles (Oedemeridae: Oedemerinae) of Japan inferred from 448-bp of mitochondrial 16S rRNA and 805-bp of nuclear 28S rRNA sequences by the ML method. The scale bar indicates the genetic distance and numerals near branches are bootstrap probabilities with 1,000 replications. Four tribes and one outgroup are shown with different colored bars.

Number Length (μm)

―

― 0

1 ―

―

― 6.23 (0.58)

― 8 0 6.69 (0.81) 16

6.68 (0.51) 5

―

― 6.35 (0.21)

― 2 0 7.12 (0.62)

―

― 6.57 (0.06)

― 3 0

―

― 9.47 (0.21)

― 3 0 12.00 (1.15) 3

―

― 4.75 (0.07)

― 2 0 5.65 (0.07) 2

8.46 (0.22) 5

―

― 5.55 (0.07)

― 2 0 4.80

―

― 7.45 (0.64)

― 2 0 1

―

― 6.30 (0.71)

― 2 0 6.60 1

―

― 6.70

― 1 0 7.90 (1.13) 2

8.60 (0.14) 2

―

― 4.90

― 1 0 5.70

―

― 0 0 1

―

― 8.10

― 1 0 8.10 (0.85) 2

―

― 0 0 7.35 (0.07) 2

―

― 7.88 (0.94)

― 6 0 9.73 (1.02) 10

―

― 0 0 6.35 (0.07) 2

5.60 (0.50) 18

―

― 6.44 (0.85) 10 77.57 (11.67) 161 (24) 6.38 (0.98)

― 0.17 (0.07) [N = 3]

7 5.09 (0.39) 45.12 (1.64)

119 (22) 6

―

― 5.66 (0.41) 9 45.83 (3.90) 183 (42) 5.66 (0.52) 9

144 [N = 1]

0.38 [N = 1]

6.00 (0.36) 3 47.28 (4.41) 227 (11) 6.35 (0.07) 2

9.55 (0.07) 2

―

― 8.10

1 43.83 (4.11) 182 (47) 9.65 (1.18)

―

― 9.00 (0.42) 2 70.22 (2.54) 353 (39) 7

598 (76) [N = 3]

Sperm mass only 4.79 (0.31)

― 18 0 5.26 (0.37) 8

448 [N = 1]

Sperm mass only 5.15 (0.26)

― 4 0 5.88 (0.25) 4

―

― 12.36 (1.08)

― 2 0 10.90 1

10.30 1

1293 [N = 1]

0.22 (0.12) [N = 10]

4.45 (0.46) 17 7.87 (0.85) 9927 (1607) 4.82 (0.48)

―

― 0 13.79 2368 10

—

— 8.23 (0.70) 3 8.90 (0.61) 2101 (617) 8

―

― 9.76 (0.70) 7 12.60 (1.07) 4221 (1193) 10.54 (0.58) 12

8.55 (0.88) 10

268 [N = 1]

0.35 (0.04) [N = 2]

7.35 (0.59) 11 8.78 (0.47) 1719 (321) 7.90 (0.37)

522 (415) [N = 5]

0.68 (0.33) [N = 5]

8.07 (0.36) 9 9.53 (0.72) 3476 (569) 8.80 (0.37) 13

―

0 ―

―

― 5.10

― 1

―

― 6.80

― 1 0 8.70 1

―

― 7.83 (0.21)

― 3 0 8.40 (1.54) 4

961 (84)

10.05 (1.58) 2 10.25 (0.11) 0.76 [N = 1]

232 (35) 10.47 (1.35) 11

7 (1) [N = 4]

Sperm mass only 9.60 (0.54)

― 28 0 10.47 (0.85) 39

― Sperm mass only 11.38

― 1 0 11.50 (0.57) 2

44 9.95 (0.92) 257 (34) 85.15 (5.58) 31 8.95 (0.80) 0.19 [N = 1] 441 [N = 1]

1149 [N = 1]

0.71 (0.45) [N = 9]

9.12 (1.04) 32 96.42 (6.94) 284 (64) 9.92 (1.13) 36

―

― 0.23 (0.12) [N = 2]

8.75 (0.92) 2 85.59 (4.86)

6 72.86 (4.71)

Female Male

N Elytral length (mm)

Bursal spine

N Elytral length (mm)

Volume of spermatophores (mm³)

Copulation duration (sec.)

Figure 4-6 Mean values (SD) of male and female reproductive traits (elytral length, bursal spine number and size, spermatophore size, and copulation duration) of false blister beetles mapped on the phylogenetic tree. The species in red have bursal spines, whilst those in blue lack the spines (in A. coarctata, the presence or

(a) (b) (c) (d)

N. c au d at a N. k at o i N. w at er ho u s e i

100 μm

ova

bc cov

ovp

spp

spm

Figure 4-7 Habitus of adult females (a), internal reproductive organs of unmated females (b), bursal spines (c), and internal reproductive organs just after mating with the spermatophore or sperm mass (d) in the three closely species Nacerdes caudata, N. katoi, and N. waterhousei. Abbreviations: bc, bursa copulatrix; cov, common oviduct; ova, ovary; ovp, ovipositor; sp, spermatheca; spm, sperm mass; spp, spermatophore.

Acetone extracts Acetone controls

*

N = 17

N = 12

N = 3 N = 2

N = 2

N = 3

N = 17 N = 12 N = 1 N = 1

A . pa rt he n o pe O. a lb is ty lu m R . p e de s tr is N. w at er ho u s e i (w it h o ut b u rs a l s p in es ) N. c au d at a / k at o i (w it h b ur s al s pi n e s )

M ean num ber of i ndi v idual s attr ac ted per egg v ol um e (i ndi v idual s /m m

)

***

Figure 4-8 Attractancy (mean ± SD) of the canthariphilous beetle Mecynotarsus tenuipes to acetone controls (open bars) and acetone extracts (closed bars) of eggs laid by two

Appendix 4-1 Materials used for DNA extraction

ドキュメント内 Doctoral Thesis Cantharidin world in nature: structure and function of a concealed arthropod assemblage with interactions via cantharidin (ページ 39-59)