Description of the larva and pupa of Apion brevicorne Gerstaecker, 1854 (Coleoptera: Brentidae: Apioninae) with biological information

Apion brevicorne Gerstaecker, 1854 (Coleoptera: Brentidae: Apioninae) is an ecologically and economically important weevil that feeds on seeds and tissues of trees in the genus Copaifera L. (Fabaceae). Although the genus Apion comprises 16 species restricted to the Paleartic region, the Neotropical species A. brevicorne is still considered as incertae sedis due to the absence of a systematic study about it. The first descriptions and illustrations of Apion brevicorne are provided here. Diagnostic characters of larva and pupa are included and compared with 13 species from other biogeographic regions. Details of immature Apioninae species associated with host plants from the Neotropical region are described for the first time. Key-Words. Apionini; Cerrado; Copaifera malmei; South America; Trichapiina.

In the Neotropical region, all 400 species (190 in South America) belong in the Apionitae with 22 genera (19 in South American) and three ISSN On-Line: 1807-0205 ISSN Printed: 0031-1049 ISNI: 0000-0004-0384-1825 tribes. The Apionini includes 10 genera (seven from South America) and the monotypic South American Chilapiini and Noterapiini. Ten genera remain incertae sedis within the Apionitae (Kissinger, 1968(Kissinger, , 2002(Kissinger, , 2003(Kissinger, , 2005a(Kissinger, , 2005bWibmer & O'Brien, 1986;Alonso-Zarazaga, 2004;De Sousa et al., 2019). A recent, updated, classification of the Apioninae in Brazil includes 10 genera, two new species, and a new record, for a current total of 89 species (De Sousa & Ribeiro-Costa, 2018;De Sousa, et al., 2019). Fifty-three species remain incertae sedis, including Apion brevicorne Gerstaecker, 1854 (see De Sousa et al., 2019). We assume that this species most likely does not belong in Apion, which is monophyletic and includes 16 species restricted to the Palearctic region (Alonso-Zarazaga, 1990). However, in the absence of a systematic study for the species, we are not sure which genus to assign it to. Thus, for the purpose of the immatures descriptions, this species is kept being regarded as Apion brevicorne.
Because Apion brevicorne is an important fruit and seed predator of Copaifera spp., here we describe and illustrate in detail the external morphology of their last larval instar and their pupa. This is the first description of immature stages in the Neotropical Apioninae, and this information is fundamental for future studies of the biology, ecology, taxonomy and systematics of weevils in the Neotropical region, and for providing foundations for a future re-assessment of the Neotropical species currently classified in Apion.

MATERIAL AND METHODS
The larvae and pupa were collected individually by hand, from April through September 2018, from seed pods of Copaifera malmei Harms, in the Cerrado (Brazilian savanna) at the Universidade Federal de Rondonópolis (UFR, 16°46′S, 54°58′W). Seed pods were dissected to find larvae and pupae in the Water Analysis and Applied Ecology Laboratory (LAHEA/UFR). About 50 larvae and 50 pupae were given in hot water and then set in 70% alcohol and deposited in the Entomological Reference Collection of the Department of Biology (DBFR) for morphological study.
Larvae, their mouthparts, and pupae were drawn using a Zeiss Stemi 200-C stereomicroscope (50×) and Nikon Eclipse E200 microscope coupled to a camera lucida (40×). Larvae were decapitated, and the head was cleared in a 10% potassium hydroxide (KOH) solution and then rinsed in water. The head capsule and all mouthparts were first examined using a Zeiss Stemi 200-C stereomicroscope, then mounted on slides or placed in depression slides containing glycerin (May, 1994), and subsequently examined under optical Nikon Eclipse E200 microscope coupled to a camera lucida. Illustrations were made by a line drawing CorelDRAW X5 program. Specimens were later examined using stereo-and compound light microscopes and scanning electron microscopy (SEM). For the latter, specimens were first dehydrated in a series of progressively increasing concentrations of ethanol (70-100%) and then critical-point dried. Specimens were then mounted on stubs, sputter coated with 35 nm of gold and examined using a JEOL JSM-6390LV scanning electron microscope. The material was photographed at the Optical and Scanning Microscopy Image Laboratory at the Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
Morphological characters were measured by using a BEL Photonics stereomicroscope with Optical Microscope Imaging Software. Larval measurements (reported in mm) included body length (with head, as distance between apical margin of head capsule and dorsal margin of the tenth abdominal segment) in lateral view (N = 20 individuals); body width (at metathorax, N = 10); width of head capsule in front view (N = 10). Pupal (10 individuals) measurements included length (lateral view) and width (dorsal view). The number of setae and bilateral structures were counted on one side of the body. Terminology, abbreviations and chaetotaxy of both larvae and pupae followed Alonso-Zarazaga & Wanat (2014) and Oberprieler et al. (2014). Suprageneric classification follows Bouchard et al. (2011).
General: Adecticous and exarate; body setae on tubercles, setae greatly reduced in number and size.

Rostrum in ventral view
Head: Frons with 1 seta (os), similar in size as drs, behind eyes. Antennae basally near prosternum and apically extend to propleura, oblique to protibia.
Sexual dimorphism: Female with small round convexity on each side of sternum on ninth abdominal segment (Figs. 16, 22).

Biological information (Figs. 23-28): Apion brevicorne
is associated with C. malmei. Adults may feed on nectar in flowers at any time, day or night. Copulation and oviposition occur on fruits at any stage of development. The oviposition is endophytic and eggs are easily found only in fruits at an early stage of development. As in most weevils, the damage caused by A. brevicorne to the seed is mostly by larvae that develop within the seed, where they feed on developing endocarp. Pupae were always found within seeds (without a pupal chamber) along with feces that indicate that the larvae consumed the entire endocarp, and so development is completed within the seed. The number of adults in each seed is quite variable. Seed pods that contain larvae, pupae or adults show no external evidence of presence. This suggests that for adults to disperse, they depend on the dry dehiscence of the fruit. Dead adults and pupae can be found within dry seed pods that did not dehisce.

DISCUSSION
All of the 13 species of Apioninae whose immature stages have been described are in seven of the subtribes of the Apionini (Williams, 1968;May, 1994;Sanz Benito & Gurrea Sanz, 1999;Nikulina, 2016;Wang et al., 2013;Letowski et al., 2015). We found that morphology of Apion brevicorne larvae and pupae described herein are very different from other species in these subtribes (especially the reduced chaetotaxy of the mala, thorax and abdomen) and different from other generalized endophagous Apioninae (May, 1993(May, , 1994Wang et al., 2013;Alonso-Zarazaga & Wanat, 2014) and other Curculionoidea (Oberprieler et al., 2014) (Tables 1 and 2). Establishing homologies for the reduced number of setae for the Apioninae is complicated because of the various usages of nomenclature for the Curculionoidea (Anderson, 1947, Marvaldi, 1999Wang et al., 2013). Differences in terminology and abbreviations among publications can lead to errors or inconsistencies in morphology, phylogeny and interpretation (Lira et al., 2017).
Larval characteristics that vary among the Apioninae and which can be used to identify genera and species of Apionini are mainly chaetotaxy of the head and mouthparts. Variability is common interspecifically as well as intrageneric and thus may be used to diagnose species (Sanz Benito & Gurrea Sanz, 1999;Wang et al., 2013;Alonso-Zarazaga & Wanat, 2014).
The indistinct frontal line and endocarina we describe for Apion brevicorne is the first observation of these characters in Apioninae. The frontal line may be absent in some groups of Curculionoidea (Oberprieler et al., 2014) but is always present in Apioninae (Alonso-Zarazaga & Wanat, 2014). This may be very useful in future phylogenetic analyses of Apioninae. The hypopharyngeal bracon, a bar that connects the two hypopharyngeal margins and which supports the hypopharynx, separates larvae of the Curculionoideae from those of the Bruchidae and Chrysomelidae (Lawrence, 1991;Oberprieler et al., 2014). The hypopharyngeal bracon is present and distinct in the primitive Australo-Pacific Neocyba sp. (Rhadinocybitae) (May, 1993) and absent in A. brevicorne. Its absence in Apioninae was first noted in P. botanicum (Wang et al., 2013), perhaps because it was uncommon to refer to ventral characters of the epicranium when describing apionine larvae (Bennett, 1992;Emden, 1938;Gosik et al., 2010;May, 1994;Sanz Benito & Gurrea Sanz, 1999). More apionine species need to be studied to determine the importance of the hypopharyngeal bracon as a diagnostic character (Wang et al., 2013).
In the female pupae, the gonotheca of the ninth abdominal sternum is divided into two parts. This pattern of sexual dimorphism seems to be common in pupae in a variety of Curculionoidea (Burke, 1968;May, 1994;Sarro et al., 2004;De Sousa et al., 2004), but has never been noted in the Apioninae (May, 1994;Sanz Benito & Gurrea Sanz, 1999;Wang et al., 2013).
Morphological and taxonomic studies of immature stages of Apioninae are still uncommon, yet such studies are fundamental for species identification, as well as for understanding their taxonomy and systematics. Apioninae biology is also poorly known in the Neotropics. Now, with our results, we were able to identify pests of unidentified genera of Neotropical Apioninae (Apion sp.) that are found on three species of Copaifera: C. sabulicola J. Costa & L.P. Queiroz, Copaifera luetzelburgii Harms and Copaifera depilis Dwyer (Santos et al., 2015). We confirmed that samples of these thitherto unknown species also pertain to A. brevicorne . Thus, A. brevicorne is oligophagous in contrast with other Brazilian Apioninae in their association with several Copaifera spp. Pests of Copaifera spp. may cause reduced productivity, with important detrimental effects on plant populations (Santos et al., 2015). Studies should continue using integrative taxonomy to determine the number of species of Copaifera that may host A. brevicorne, whether each host species is associated with additional taxonomic units of the weevil, and the taxonomic position of the species as a possible new genus within the Apionini.