Mating behavior and sexual dimorphism of the Lobiopa insularis (Coleoptera: Nitidulidae) strawberry pest

Abstract The objective of this work was to describe the sexual dimorphism and mating behavior of Lobiopa insularis for the development of pest control and management programs. The morphological characteristics for separating males and females were described with the aid of a stereomicroscope. Mating sequence was recorded over 72 hours with a digital camera. There are differences between males and females in the last abdomen segments, and males maintain a guarding position after mating. The obtained data can be used in monitoring programs to precisely identify the sex of the insects, also shedding light on the ecological features of this pest and Nitidulidae.

Pesq. agropec.bras., Brasília, v.58, e03187, 2023 DOI: 10.1590/S1678-3921.pab2023.v58.03187 In Brazil, this pest is one of the most important of strawberry crops, standing out among the species with a frugivorous habit (Dorzenoni et al., 2019).In the country, an outbreak of L. insularis larvae and adults caused injuries in strawberry fruits produced for in natura consumption and commercialization, representing a damage of 14.3% per insect/fruit and 70% on total production (Bernardi et al., 2015;Dorzenoni et al., 2019;Souza et al., 2019).In this scenario, strategies to monitor and control this pest have been implemented based on the life cycle of the species and on pesticide use (Guimarães et al., 2009;Bernardi et al., 2015;Greco et al., 2017).However, despite the economic importance of L. insularis, its morphology still needs to be further detailed and its sexual behavior described.
The objective of this work was to describe the sexual dimorphism and mating behavior of Lobiopa insularis for the development of pest control and management programs.
For the experiment, adults of L. insularis were collected from strawberry crops in the municipality of São José dos Pinhais, in the state of Paraná, Brazil.The specimens were identified according to the key proposed by Parsons (1943) and to additional information from Guimarães et al. (2009) and Lasoń & Przewoźny (2009).
For an accurate sexual differentiation (N=5), male and female genitalia were analyzed following procedures modified from Cline & Kinnee (2012).First, the beetles were killed by freezing and, then, their cuticula was softened in warm and soapy water.Afterwards, the specimens were boiled in 10% KOH water solution to clean internal body tissue and facilitate its removal.Morphological features were observed at the Electronic Microscopy Center of Universidade Federal do Paraná, under the Discovery.V8 stereomicroscope (Zeiss, Oberkochen, Germany), and imagens were captured by the VEGA-3 LMU scanning electron microscope (Tescan Group, a.s., Kohoutovice, Czech Republic).
After visually observing sexual dimorphism by the analysis of the pygidium and hypopygidium, live insects (N=40) were sexed, selected, and kept under laboratory conditions, at a temperature of 25°C±2, humidity of 70%±5, and photoperiod of 12 hours of light/12 hours of darkness.
Although, externally, the bodies of male and female L. insularis appeared quite similar, the pygidium and hypopygidium differed significantly (Figure 1).For males, the pygidium had a wider basal part, becoming narrow and triangular near its apical part, whereas the hypopygidium had an angulate apical margin.For females, the pygidium was membranous without a triangular part near the apical part, while the hypopygidium had a broad apical part.
Among Nitidulidae, a similar sex dimorphism in tergite VIII was observed in males of Aethina tumida Murray, 1867 and Carpophilus dimidiatus (Fabricius, 1792), which showed a pygidium with an angulate apical margin (Neumann et al., 2013) and a concave structure in ventrite V at the top of the pygidium (Reales et al., 2018), respectively.Before the beginning of the experiment, the diet of the insects was changed every two days, and, then, only strawberry was offered for five days before reproductive behavior was observed.For this, ten insect couples were kept in five Petri dishes (5 cm height x 10 cm internal diameter), covered with a PVC film perforated (N=30) with a needle (1 mm in diameter) to allow air circulation and avoid their exit.The behavior of each couple was recorded over 72 hours using the DSC-H400 digital camera (Sony, Tokyo, Japan), directed to the arena with the Petri dishes.The behavior sequences observed during the experiment were used for reproductive description.
Disregarding oviposition, after the behavior analysis, the three main stages established for L. insularis were: pre-copulation, copulation, and postcopulation, according to the terminology and methods of Kamiya et al. (2015) and Mustafa et al. (2015).
The main stages of reproductive behavior were observed for L. insularis (Figure 2).In the precopulation stage under a natural diet with ripe strawberries, the males approached the females from the lateral side during feeding, starting sexual arousal by performing a brief courtship by vibrating the right and left hind legs three times.A similar courtship behavior was reported for A. tumida, with a similar pattern of male mating stages and female choice (Neumann et al., 2013;Mustafa et al., 2015).In this stage, the male touches the female's pronotum with its antennas, and, if accepted, climbs on the female, then touches the female's head and pronotum with its antennas.In Tenebrionidae, a family of the same infraorder (Coleoptera: Cucujiformia) as Nitidulidae (Lawrence et al., 2011), beetles also displayed leg movements during mating behavior, but associated to a possible sperm transference (Fedina & Lewis, 2015).In the present work, the leg movements of the analyzed male L. insularis were observed before mating, which may indicate two possibilities: a behavior of sexual arousal or to check mating viability.The taxis behavior of a male touching the body of a female is a way of identifying the correct partner through the cuticular hydrocarbons present in the female's body, as observed by Kamiya et al. (2015) for Cyrtomon luridus (Curculionidae) and Mustafa et al. (2015) for A. tumida.Based on this finding, the mating interaction of L. insularis could be related to a mixture of visual and chemical cues; however, to confirm this assumption, further studies are necessary.After pre-copulation, the copulatory stage began, when the male started to tremble with the aedeagus everted and inserted in the female genitalia (Figure 2); the tremble behavior occurred from right to left for ±5 s.
The post-copulatory stage was initiated with the everted aedeagus, when males stayed over the female's body for a very short period, no longer than 1 min, which could suggest a possible mate guarding behavior.In fact, mate guarding plays an important role as it aims to protect and ensure paternity after mating as observed by Eberhard (1997) for other arthropods.
In the present study, it was also noted that the females only laid their eggs when the males were present.This reproductive behavior was also recorded by Greco et al. (2017) for L. insularis, meaning it could be another mechanism for paternity protection.Therefore, other researches are necessary to verify if the presence of more males can interfere in the reproductive behavior of courtship, mating, and oviposition since only couples were evaluated in the present study.This is the first known work to confirm and describe the sex dimorphism of L. insularis based on high-resolution images, as well as some aspects of the species mating behavior.These data could contribute to a precise sex identification for monitoring program purposes, also shedding light on the ecological features of this insect pest and Nitidulidae.

Figure 1 .
Figure 1.Images of the sexual dimorphism of Lobiopa insularis captured with a scanning electron microscope, showing: A, male pygidium with a wider basal part, becoming narrow and triangular near its apical part; B, membranous female pygidium without a triangular part near the apical part; C-D, male hypopygidium with an angulate apical margin; and E-F, female hypopygidium with a broad apical part.

Figure 2 .
Figure 2. Lobiopa insularis mating sequence scheme recorded over 72 hours using a digital camera.