Introduction

Two genital openings, one for mating and one for oviposition, are reported for Ditrysia (Lepidoptera) females, which includes 98% of the world butterfly and moth species (Kristensen & Skalski 1999; Capinera 2008). Yponomeutoidea, with approximately 230 genera and 1800 described species, includes microlepidoptera, such as those in the Bedelliidae family, with one genus and 16 species (Van Nieukerken 2011).

The introduced microlepidopteran Bedellia somnulentella Zeller, 1847 (Lepidoptera: Bedelliidae) was recorded in Brazil defoliating the sweet potato plant Ipomoea batatas (Convolvulaceae) (Santos et al. 2018). The life cycle of B. somnulentella is approximately 30 days, with five and seven for the pupal and adult stages, respectively, without apparent sexual dimorphism (Santos et al. 2021).

Sexual dimorphism in Lepidoptera is based on life history, behavioral, and morphological traits (Allen et al. 2011), including color pattern, body shape and size, antennae, wings, and adult abdomen morphology. Sex determination in immature stages of moths (e.g., pupae) is necessary for studies such as behavioral tests with pheromones and ecotoxicological ones using virgin insects (Posada et al. 2011). The objective was to identify morphological characters of sexual dimorphism in B. somnulentella pupae and adults.

Material and methods

Bedellia somnulentella was reared in the Cellular Biology laboratory of the “Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM)” in Diamantina, Minas Gerais state, Brazil, in wooden cages (33 × 33 × 33 cm), with screened sides and a glass lid at room temperature. Images of B. somnulentella adults were obtained with a CANON EOS Rebel 850D camera.

Bedellia somnulentella pupae were collected from mass rearing. The body length of two groups of 15 individuals was measured using the Image J2 software (Rueden et al. 2017) and obtained 3,0 and 3,5 mm long for those in both groups. These pupae were individualized in Petri dishes (60 × 15 mm), and the sex of the resulting adults was confirmed and used in other analyses.

A pair of female and male hindwings were removed and bleached in 10% potassium hydroxide overnight for 12 h in the cold. The frenulum of these wings was removed and photographed using a Leica DM 500 microscope with an attached digital camera. Pupae and abdomens of B. somnulentella adults were dehydrated in an increasing series of ethanol (70%, 80%, 90%, 95%, 99%) at 2-min intervals per concentration and mounted on aluminium supports with tape adhered to the surface, kept in a desiccator with silica for 6 h and analyzed using a Hitachi model TM 3000 microscope (Hitachi High-Technologies Corporation, Tokyo, Japan) of the UFVJM Integrated Research Laboratory.

The weight (mg) of B. somnulentella pupae was obtained on a precision scale (Shimadzu Corporation, Kyoto, Japan), and these data were subjected to the Student t-test at 5% significance using the R Software (R Core Team 2021).

Results

External structures, such as fissures or pores, were not detected on the abdominal segments (Fig. 1C) or the seventh to ninth tergites of males (Fig. 1B) and females (Fig. 1D) pupae of B. somnulentella as sex dimorphism.

Fig. 1
figure 1

The final part of the abdomen of the male pupa in ventral (A) and dorsal (B) views and the female in ventral (C) and dorsal (D) views of Bedellia somnulentella (Lepidoptera: Bedelliidae)

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Female pupae (0.99 ± 15 mg) of B. somnulentella were heavier than male ones (0.68 ± 0.15 mg), which may indicate sex dimorphism.

The yellow pattern of the head, thorax, and abdomen and filiform-type appendages and antennae (Fig. 2A) is similar for females and males of B. somnulentella.

Fig. 2
figure 2

Adult (A) and frenulum of the hind wing of male (B) and female (C) Bedellia somnulentella (Lepidoptera: Bedelliidae)

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One frenulum was observed on the hindwings of males (Fig. 2B) and two on those of females B. somnulentella (Fig. 2C).

Short cerci surround the last sternite (Fig. 3B) of the abdomen of B. somnulentella females, which is modified into an ovipositor (Fig. 3A). A bipartite valve (Fig. 3C) and long cerci were observed in the last segment of the abdomen of males (Fig. 3D), making it possible to differentiate the sexes of B. somnulentella in ventral view.

Fig. 3
figure 3

Dorsal view of the abdomen (A), last female abdominal segment in ventral (B), and dorsal view of the abdomen (C) and last abdominal segment (D) of Bedellia somnulentella (Lepidoptera: Bedelliidae) males. Caption – ov: ovipositor; val: valve

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Discussion

Anal pore, absent in the last three abdominal segments of B. somnulentella pupae of different sizes collected on I. batatas plants, makes sexing difficult at this stage. This differs from reports of this structure in other Ditrysia species, such as in pupae of the microlepidopteran Tonza citrorrhoa Meyrick, 1905 (Lepidoptera: Tonzidae) in the ninth abdominal segment with a distinct morphology between females and males, allowing sexing this insect (Kobayashi et al. 2018). Anal pores, with a variable shape between immature females and males, were reported in pupae of the microlepidopteran Tuta absoluta Meyrick, 1907 (Lepidoptera: Gelechiidae), with an anal fissure in the eighth abdominal segment of the former and the tenth in the latter, as a dimorphism in this stage (Genc 2016). A longitudinal ridge was reported on the eighth abdominal segment of the butterfly Nymphalis polychloros Linnaeus, 1758 female pupae (Lepidoptera: Nymphalidae) (Genc 2015). Genital opening in the middle part of the ninth abdominal segment in a short slit surrounded by a pair of semicircular ridges differentiates females and male pupae of Mythimna separata Walker, 1865 (Lepidoptera: Noctuidae) in ventral view (Lin et al. 2020). Still, this cleft was not detected in B. somnulentella pupae.

The greater weight of female than male pupae of B. somnulentella is a standard in butterflies and moths, as reported for male pupae of N. polychloros (Genc, 2015), Syssphinx molina Crame, 1780 (Lepidoptera: Saturniidae) (Batista et al. 2013), T. absoluta (Genc, 2016), and Lycoriella ingenua Dufour, 1839 (Diptera: Sciaridae) (Lewandowski et al. 2004). Heavier female pupae indicate sex dimorphism in B. somnulentella.

Similar color of the head, thorax, and wings and filiform antennae in males and females and the abdomen of males B. somnulentella thinner than that of their females is similar to that reported for Conopomorpha cramerella Snellen, 1904 (Lepidoptera: Gracillariidae) (Posada et al. 2011). The sharper body of B. somnulentella males agrees with that reported for T. citrorrhoa (Kobayashi et al. 2015), and color markings on the forewings of Spodoptera frugiperda J.E. Smith, 1797 (Lepidoptera: Noctuidae) (Navasero and Navasero 2020) are sex dimorphism.

A pair of frenulum in females and only one in males on the hind wings of B. somnulentella is similar to that reported for Mythimna separata Walker, 1865 (Lepidoptera: Noctuidae) (Lin et al. 2020), being an adequate parameter to sex B. somnulentella adults.

The external male genitalia of B. somnulentella, surrounded by a long cercus in the last segment and short in its females, are similar to that reported for T. citrorrhoa (Kobayashi et al. 2015). Circuses and shape of the genitalia of B. somnulentella and the bipartite male genitalia in the eighth male abdominal segment of Leucoptera coffeella Guérin & Méneville, 1842 (Lepidoptera: Lyonetiidae) (Posada et al. 2011; Motta et al. 2021) are parameters of sexual dimorphism. A pair of elongated limbs with cerci covering the external genitalia of females in the eighth segment and the last with a tubular shape and ovipositor surrounded by apical bristles in B. somnulentella females agree with that reported for L. coffeella (Motta et al. 2021).

Conclusion

External structures of sex dimorphism were not detected in the last abdominal segments of B. somnulentella pupae, but females at this stage are heavier, being a parameter of sex dimorphism. The color pattern and general body structures of males and females B. somnulentella are identical.

Sexual dimorphism in B. somnulentella is characterized by discrete anatomical differences. Females are heavier and have a pair of frenula per wing, whereas only one frenulum was observed on each wing of males. Additionally, elongated and smaller cerci involve the last abdominal segment of bipartite genitalia in males. In contrast, an ovipositor was observed in females. These distinct morphological features differentiate between male and female B. somnulentella.