Sclerotised spines in the female bursa associated with male’s spermatophore production in cantharidin-producing false blister beetles

Highlights

• Cantharidin is a defence chemical produced by only two beetle families.

• In Oedemeridae, some species have sclerotised spines in the female bursa copulatrix.

• Transferred spermatophores were gradually broken and digested in the spiny bursa.

• Females with the spiny bursa may be able to provide more cantharidin to their eggs.

Abstract

Cantharidin is a defence chemical synthesised in only two beetle families Meloidae and Oedemeridae. In Meloidae, cantharidin is used as a defence chemical in eggs. However, in Oedemeridae the function of cantharidin remains unclear. Based on morphological comparison of female internal reproductive organs in 39 species of Oedemeridae, we found that some species have sclerotised spines in the bursa copulatrix (bursal spines), while others have no such spines. Molecular phylogenetic trees inferred from mitochondrial 16S and nuclear 28S rRNA gene sequences suggested multiple evolutionary origins of bursal spines from an ancestor without spines. In the species which lacked spines, males transferred small amounts of ejaculates to females; however, in species with spines, males transferred large spermatophores. Deposited spermatophores gradually disappeared in the bursa, probably owing to absorption. To compare the amounts of cantharidin in eggs laid by species with and without bursal spines, we constructed a new bioassay system using the small beetle Mecynotarsus tenuipes from the family Anthicidae. M. tenuipes individuals were attracted to droplets of cantharidin/acetone solution, and the level of attraction increased with cantharidin concentration. This bioassay demonstrated that the eggs of Nacerdes caudata and N. katoi, both of which species have conspicuous bursal spines, contain more cantharidin than the eggs of N. waterhousei, which lacks spines. In the former species, males transfer large spermatophores to the female, and spermatophores are eventually broken down and digested within the female’s spiny bursa. Thus, females with bursal spines may be able to provide more cantharidin to their eggs.

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).