Sclerotised spines in the female bursa associated with male’s spermatophore production in cantharidin-producing false blister beetles
Graphical abstract
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 and 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 and 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 and Lewis, 1999) or non-nutritive substances, such as immunostimulants or antibiotics (Poiani, 2006), water (Arnqvist et al., 2005), ions or minerals (Engebretson and Mason, 1980), or defensive compounds (Eisner and 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 defence chemicals against predators store defensive compounds as nuptial gifts with their spermatophores; for example, pyrrolizidine alkaloids (Dussourd et al., 1988, Eisner and Meinwald, 1995) and cyanogenic glycosides (Cardoso and 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 and 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, Carrel et al., 1993, Frenzel and 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, Nikbakhtzadeh et al., 2012).
In contrast, false blister beetle adult females, as well as adult males, can synthesise cantharidin (Carrel et al., 1986, Frenzel and Dettner, 1994). In most cases, field-caught females contain more cantharidin than males (Frenzel and 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 sclerotised 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 these spines are thought to break and digest spermatophore envelopes (Cordero and Baixeras, 2015). In this study, therefore, we examined the possible functions of false blister beetle bursal spines in relation to cantharidin donation from males to females via 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 relative 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 digest 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).
Section snippets
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 min. 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,
Female bursal spines and male spermatophores
We found sclerotised spines in the inner walls of the female bursa copulatrix (bursal spines) in 16 of 39 species examined, 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. 3a); many short spines in four bands in N. konoi, N. osawai, and N. luteipennis (Fig. 3b); many short, uniformly arranged spines in N. deformis, N. spinicoxis, and Chrysanthia geniculata (Fig.
Discussion
The family Oedemeridae consists of three subfamilies, Oedemerinae, Calopodinae, and Polypriinae in the world, of which we treat only Oedemerinae in this study. This subfamily is further subdivided into five tribes; Asclerini, Ditylini, Nacerdini, Oedemerini, and Stenostomini (Švihla, 2008). Although our phylogenetic trees constructed using both mitochondrial 16S and nuclear 28S partial sequences are based on Japanese materials, these well-defined four tribes are included; the only exception is
Acknowledgments
We thank Yuki Murakami and Ryota Matsuyama for their kind assistance in the field sampling and Sachiko Sugawara, Haruki Suenaga, Yuki Murakami, Kaede Nishino, Kaho Horiuchi, Shoichi Matsumoto, Namiki Kikuchi, Hisanori Okamiya, and Yurika Torii for providing valuable specimens. This study was partly supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (Research Project Number: 14J05882).
References (50)
- et al.
Nuptial gift giving behaviour and male mating effort in the Neotropical spider Paratrechalea ornata (Trechaleidae)
Anim. Behav.
(2010) - et al.
Towards the phylogeny of the Curculionoidea (Coleoptera): reconstructions from mitochondrial and nuclear ribosomal DNA sequences
Zool. Anz.
(2009) - et al.
Molecular phylogenies of fig wasps: partial cocladogenesis of pollinators and parasites
Mol. Phylogenet. Evol.
(2001) - et al.
Cantharidin biosynthesis and function in meloid beetles
- et al.
Intraspecific transfer of cantharidin within selected members of the family Meloidae (Insecta: Coleoptera)
J. Insect Physiol.
(2007) Phylogenetic relationships of the carabid subfamily Harpalinae (Coleoptera) based on molecular sequence data
Mol. Phylogenet. Evol.
(2002)- et al.
Quantitative characterization of cantharidin in the false blister beetle, Oedemera podagrariae ventralis, of the southern slopes of Mount Elborz
Iran. J. Insect Sci.
(2012) - et al.
Using RNA sequencing to characterize female reproductive genes between Z and E Strains of European Corn Borer moth (Ostrinia nubilalis)
BMC Genomics
(2014) - et al.
Mating rate and fitness in female bean weevils
Behav. Ecol.
(2005) Male nuptial gifts: phenotypic consequences and evolutionary implications
A male gift to its partner? Cyanogenic glycosides in the spermatophore of longwing butterflies (Heliconius)
Naturwissenschaften
Cantharidin: potent feeding deterrent to insects
Science
Identification of cantharidin in false blister beetles (Coleoptera. Meloidae) from Florida
J. Chem. Ecol.
Cantharidin production in a blister beetle
Experientia
Sexual selection within the female genitalia in Lepidoptera
Elateridae Leach, 1815
Sexual selection and the evolution of dance fly mating systems (Diptera: Empididae; Empidinae)
Can. Entomol.
Isolation of cantharidin from Melöe proscarabeus
Can. Pharm. J.
Biparental defensive endowment of eggs with acquired plant alkaloid in the moth Utetheisa ornatrix
Proc. Natl. Acad. Sci. U.S.A.
The chemistry of sexual selection
Proc. Natl. Acad. Sci. U.S.A.
Chemical basis of courtship in a beetle (Neopyrochroa flabellata): cantharidin as “nuptial gift”
Proc. Natl. Acad. Sci. U.S.A.
Transfer of 65Zn at mating in Heliothis virescens
Environ. Entomol.
Female mating bias results in conflicting sex-specific offspring fitness
Nature
Quantification of cantharidin in canthariphilous Ceratopogonidae (Diptera), Anthomyiidae (Diptera) and cantharidin producing Oedemeridae (Coleoptera)
J. Chem. Ecol.
Courtship feeding increases female reproductive success in bushcrickets
Nature
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