Research Article |
Corresponding author: Gilson R.P. Moreira ( gilson.moreira@ufrgs.br ) Academic editor: Erik J. van Nieukerken
© 2019 Gilson R.P. Moreira, Oleg G. Gorbunov, Júlia Fochezato, Gislene L. Gonçalves.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Moreira GRP, Gorbunov OG, Fochezato J, Gonçalves GL (2019) A peculiar new species of gall-inducing, clearwing moth (Lepidoptera, Sesiidae) associated with Cayaponia in the Atlantic Forest. ZooKeys 866: 39-63. https://doi.org/10.3897/zookeys.866.34202
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Larvae of most clearwing moths (Lepidoptera, Sesiidae) are endophagous borers of many angiosperms, including their fruits, stems, and roots. Their localized feeding may lead to swellings on those plant parts, but whether the structures produced should be considered true galls is still controversial. In this study we describe a peculiar sesiid moth, Neosphecia cecidogena sp. nov. whose larvae induce unusual, external galls on Cayponia pilosa (Vell.) Cogn. (Cucurbitaceae) in the Atlantic Forest of southernmost Brazil. The adults, egg, larva, pupa and the gall are described and illustrated based on light and scanning electron microscopy. Galls are cylindrical and unilocular; they are induced individually on axillary buds of the C. pilosa stem. Unlike larvae of other sesiids, those of N. cecidogena sp. nov. lack abdominal pseudopodia, and show reduced stemata and chaetotaxy. Pupation occurs inside the gall, after having overwintered in the last larval instar. A maximum likelihood tree constructed based on mitochondrial DNA (COI) sequences showed that N. cecidogena sp. nov. is monophyletic and has an average distance of 13% to species of Melittia. The genera Neosphecia Le Cerf, 1916 stat. rev., Premelittia Le Cerf, 1916 stat rev., and Melittina Le Cerf, 1917 stat. rev. are restored from synonyms of Melittia Hübner, 1819 [“1816”].
Cucurbitaceae, insect galls, Melittina, Neosphecia cecidogena, Neotropical region, Premelittia, sesiid moths, taxonomy
The Sesiidae is a worldwide family with ca 160 valid genera and 1,452 species. Of these, 268 species are found in the Neotropical region (
In contrast to the adults, however, their immature stages are poorly known, especially in the tropical regions where the host plants for only a small proportion species have been documented (
This study concerns a new species of clearwing moth that induces conspicuous, external galls on the stem of a vine, Cayaponia pilosa (Vell.) Cogn. (Cucurbitaceae) in the Atlantic Forest of southernmost Brazil. A preliminary comparison of genitalia structures suggested that it belongs to a genus closely related to Melittia Hübner, 1819 [“1816”], but does not conform to any species within it. We describe and illustrate the gall, the immature stages and adults of this moth under both light and scanning electron microscopy, and provide information on its natural history. By conducting a phylogenetic analysis of mitochondrial DNA (COI) sequences using representative members of Melittini (Melittia), we provide further support for the proposition of the new taxon, as well as its genus validation.
After careful examination of the habitus we came to the conclusion that this species is congeneric with Neosphecia combusta Le Cerf, 1916, which is the type species of the monotypic genus Neosphecia Le Cerf, 1916. This genus was formally synonymized with Melittia by
All specimens used in this study were either dissected or reared from galls in small plastic vials, which were maintained under controlled conditions (14 h light/10 h dark; 25 ± 2 °C) in the Laboratório de Morfologia e Comportamento de Insetos (LMCI), Departamento de Zoologia, Universidade Federal do Rio Grande do Sul (
For descriptions of genital morphology, larvae were cleared in a 10% potassium hydroxide (KOH) solution, stained with either Eosin or Chlorazol black E and slide-mounted in either glycerin jelly or Canada balsam. Last instar larvae were prepared similarly for description of chaetotaxy. Observations were performed with the aid of a Leica M125 stereomicroscope. Structures selected to be drawn were previously photographed with a Sony Cyber-shot DSC-H10 digital camera attached to the stereomicroscope. Vectorized line drawings were then made with the software Corel Photo-Paint X7, using the corresponding digitalized images as a guide. Additional specimens were used for scanning electron microscope analyses. They were dehydrated in a Bal-tec CPD030 critical-point dryer, mounted with double-sided tape on metal stubs, coated with gold in a Bal-tec SCD050 sputter coater and examined and photographed in a JEOL JSM6060 scanning electron microscope at the Centro de Microscopia Eletrônica (CME) of
DNA was extracted from four larvae (specimens LMCI 263-33A, B, C, and D) of the new taxon using the PureLink Genomic DNA extraction kit (Invitrogen). Extracted DNA was resuspended in 50 mL Tris: EDTA (10 mm Tris-HCl, 1 mm EDTA, pH 5 8.0). Mitochondrial DNA PCR was conducted using primers LCO1490 and HCO2198 (
DNA barcode sequences of Sesiidae specimens used to infer the relationship of Neosphecia cecidogena (BOLD dataset DS-NEOSESII) within the tribe Melittiini.
Group | Genus | Species | Specimen voucher | Accession number | |
GenBank | BOLD | ||||
Ingroup | Melittia | brabanti Le Cerf, 1917 | NS-RR051 | JN304556 | LNOUE514-11 |
calabaza Duckworth & Eichlin, 1973 | USNM ENT 00831286 | MH592747 | LNAUS302-12 | ||
cucurbitae (Harris, 1828) | USNM ENT 00831289 | MH592773 | LNAUS305-12 | ||
grandis (Strecker, 1881) | USNM ENT 00831295 | MH592813 | LNAUS310-12 | ||
gloriosa Edwards, 1880 | USNM ENT 00831292 | MH592712 | LNAUS308-12 | ||
snowii Edwards, 1882 | CSU-CPG-LEP001016 | GU685631 | ABLCU016-09 | ||
Neosphecia | cecidogena sp. nov. | LMCI 263-33A | MK210242 | MISA037-18 | |
LMCI 263-33B | MK210243 | MISA038-18 | |||
LMCI 263-33C | MK210244 | MISA039-18 | |||
LMCI 263-33D | MK210241 | MISA040-18 | |||
Outgroup | Carmenta | bassiformis (Walker, 1856) | USNM ENT 00831360 | MF124173 | LNAUS376-12 |
Synanthedon | exitiosa (Say, 1823) | 06-JKA-0240 | MH592851 | LSEU240-06 |
The final dataset included 12 specimens: four individuals originally sequenced and six species of Melittia, the only representative of Melittini available, chosen based on morphologically established relationships with the new taxon. In addition, two species of Synanthedonini were used as outgroup to Melittini according to relationships proposed based on previous molecular phylogenies (e.g.
The specific status of the new taxon was tested through a COI tree relying on monophyly. Tree reconstruction was based on the maximum likelihood (ML) algorithm performed in PHYML v. 3.0 (
Abbreviations of the institutions from which specimens were examined are:
MCTP Museu de Ciências e Tecnologia da Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
LMCI Laboratório de Morfologia e Comportamento de Insetos, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
Sequencing of COI resulted in an average amplicon size of 633 bp. The aligned data matrix including all genera resulted in 668 characters. Of these, 189 (28%) were phylogenetically informative. Maximum likelihood analysis recovered an optimal ML tree = 5897 with nucleotide frequencies of A = 29.1%, C = 16.5%, G = 14.8%, and T = 39%. The four LMCI 263-33 specimens grouped in the COI tree as a single lineage, which was placed close to the genus Melittia (Fig.
Maximum likelihood consensus tree of Neosphecia cecidogena (LMCI 263-33) and related Melittiini taxa. The monophyly and relationships were inferred based on 668 base pairs of cytochrome oxidase subunit gene I (COI) sequences (using six species of Melittia, one from Synanthedon and Carmenta from databases; the later genera of the related subfamily Synanthedonini were surveyed as outgroups). Numbers above branches indicate statistical support (bootstrap values).
Genetic distance between Neosphecia cecidogena and other members of Melittini based on a 668 base pair sequence of the cytochrome oxidase I (COI) gene using the Kimura 2-parameter model. Comparisons with the outgroup Synanthedonini (Synanthedon and Carmenta) are indicated as well. Intra-group distances are shown in brackets.
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
1. Neosphecia cecidogena | 0.00 | ||||||||
2. Melittia snowii | 0.14 | – | |||||||
3. Melittia brabanti | 0.15 | 0.11 | – | ||||||
4. Melittia calabaza | 0.13 | 0.08 | 0.11 | – | |||||
5. Melittia gloriosa | 0.13 | 0.08 | 0.08 | 0.05 | – | ||||
6. Melittia grandis | 0.12 | 0.09 | 0.09 | 0.06 | 0.06 | – | |||
7. Melittia cucurbitae | 0.14 | 0.09 | 0.10 | 0.02 | 0.05 | 0.06 | – | ||
8. Synanthedon exitiosa | 0.18 | 0.16 | 0.16 | 0.18 | 0.15 | 0.16 | 0.18 | – | |
9. Carmenta bassiformis | 0.22 | 0.21 | 0.19 | 0.19 | 0.19 | 0.18 | 0.20 | 0.12 | – |
“Genre Melittina gen. nov.” Le Cerf, 1917: 239. Type species: Melittina nigra Le Cerf, 1917, by original designation.
(after
Despite the fact that Le Cerf placed Melittina in the subfamily Aegeriinae sensu Le Cerf, 1917 [= Sesiini + Osminiini + Paranthrenini + Synanthedonini], by the venation of the hindwing this genus belongs to the tribe Melittiini. Superficially Melittina resembles the Afrotropical genus Agriomelissa Meyrick, 1931 (type species: Agriomelissa gypsospora Meyrick, 1931), but it can be differed by the practically undeveloped hair-like tuft of the hind leg (vs hair-like tuft of the hind leg well-developed in Agriomelissa) and undeveloped anal lobe of the hindwing (vs well-developed in the genus compared). Melittina can be easily distinguished from Premelittia and Neosphecia by the well-developed proboscis (proboscis undeveloped in both these compared genera) and shape of the antenna (antenna strongly clavate, short, slightly shorter than a half of forewing in Melittina, vs antenna fusiform, slightly longer than middle of forewing in Premelittia and Neosphecia). From all other genera in Melittiini, including species of New World so-called “Melittia” (
Presently this genus contains only a single species: Melittina nigra Le Cerf, 1916, stat. rev.
The genus Melittina was described by
“Premelittia rufescens ♀ nov. sp.” —
(after
Without any doubt, by the venation of the hindwing this genus belongs to the tribe Melittiini. By the undeveloped proboscis and fusiform antenna this genus seems to be closely related to the genus Neosphecia Le Cerf, 1916, but it can be separated from it by the smoothly scaled vertex (vs vertex covered with hair-like scales in the compared genus) and venation of the forewing (veins R4 and R5 short stalked in Premelittia vs veins R4 and R5 separate basally in Neosphecia). It is easily distinguished from all other Melittiini, including species of New World so-called “Melittia” (
Presently this genus contains the single species: Premelittia rufescens Le Cerf, 1916.
The generic name Premelittia was first introduced in the legends of figures as the binominal “Premelittia pufescens nov. sp.” (
“Neosphecia combusta ♀ nov. sp.” —
Dalla Torre, Strand 1925: 136 (as a distinct genus);
(after
Like the two previous taxa, on the basis of the venation of the hindwing this genus belongs to the tribe Melittiini. By the fusiform antenna and undeveloped proboscis this genus is closely related to the genus Premelittia Le Cerf, 1916, stat. rev., but it can be distinguished the forewing venation (veins R4 and R5 shortly stalked in Premelittia vs veins R4 and R5 separate basally in Neosphecia). Neosphecia differs from Melittina by the undeveloped proboscis (well developed and functional in the compared genus), shape of the antenna (strongly clavate and short, not reaching the middle of the forewing in Melittina vs fusiform, long, slightly longer than half forewing in Neosphecia), and the venation of the forewing (veins R4 and R5 long stalked in Melittina vs veins R4 and R5 separate basally in Neosphecia). From all other genera of the Melittiini, including New World so-called “Melittia” (
Currently, we include two species in this genus: Neosphecia combusta Le Cerf, 1916 (type species) and N. cecidogena sp. nov.
Like the genus Premelittia (see above), the genus name Neosphecia was firstly introduced in the legends of the figures as the binomen “Neosphecia combusta nov. sp.” (
Male (holotype) (Fig.
Head with antenna dark brown to black dorso-externally and yellow ventro-externally; scapus yellow and narrowly lined with dark brown dorsally; frons entirely dark brown with purple-blue sheen; vertex black with dark-blue sheen and an admixture of individual white and yellow hair-like scales; proboscis completely undeveloped; labial palpus dark brown to black with an admixture of yellow scales dorsally and white, long, hair-like scales ventrally in distal half; occipital black with a few white scales dorsally.
Thorax with patagia dark brown to black with a small, yellow, transverse spot anterior-ventrally; tegula dark brown to black with yellow, hair-like scales distally; mesothorax entirely dark brown to black; metathorax dark brown to black with two tufts of yellow, hair-like scales laterally; thorax laterally dark gray-brown with bronze-violet sheen. Legs with neck plate dark brown to black; fore coxa dark brown to black with a narrow, yellow exterior margin; hind tibia dark yellow with an admixture of black elongated scales on basal half; spurs dark yellow with golden sheen and a few black scales exterior-basally; hind tarsus dark yellow with a dense admixture of elongated black scales dorso-externally. Forewing: dorsally dark brown with dark-violet sheen and an admixture of individual yellow-orange scales, more dense distally and at anal margin; transparent areas present but very small: anterior and posterior ones very narrow, external one divided into two very short cells; cilia dark brown to black with dark violet-purple sheen. Hindwing transparent; veins broadly covered with dark brown and a few yellow-orange scales; discal spot undeveloped; outer margin about as broad as cilia, dark yellow and narrowly dark brown distally; cilia dark brown with dark violet sheen.
Abdomen including anal tuft black with dark blue sheen and a few yellow scales at base of second tergite.
Male genitalia
(Fig.
Neosphecia cecidogena genitalia morphology under light microscopy A male (LMCI 319-84), general, ventral view (unrolled preparation, sensu
Female (paratype) (Fig.
Female genitalia
(Fig.
The type series practically invariable in individual size and in the coloration of various parts of the body and wings.
This new species looks like Melittina nigra Le Cerf, 1917 (type locality: “Brésil, ex E. Le Moult, Coll. F. Le Cerf”;
The species name, an adjective, is derived from a composition between the Portuguese “Cecidia” (a gall) and the suffix gena (derived from the Latin verb “gigno”, be born). Thus, the epithet refers to the cecidogenous habit of the new described clearwing moth.
All specimens were either dissected or reared from galls associated with Cayaponia pilosa (Vell.) Cogn. (Cucurbitaceae), from the Centro de Pesquisas e Conservação da Natureza Pró-Mata (CPCN Pró-Mata, 29°28'36"S, 50°10'01"W, São Francisco de Paula Municipality, Rio Grande do Sul State (RS), Brazil; 04–06.IV.2014, G.R.P. Moreira & R. Brito legs., LMCI 263; 21–24.VI.2016, G.R.P. Moreira, R. Brito, J. Fochezato legs, LMCI 306; 28–30.VI.2017, G.R.P. Moreira and J. Fochezato legs., LMCI 319; 01–02.VIII.2017, G.R.P. Moreira and J. Fochezato, LMCI 320; 20–23.III.2018, G.R.P. Moreira, V. Becker, A. Moser, R. Brito & J. Fochezato legs., LMCI 326.
Type material (all pinned-dried adults). Holotype: ♂ LMCI 319-83; Paratypes: 1♂, LMCI 319-84, with genitalia preparation on slide; 1♀, LMCI 263-52, with genitalia preparation on slide; 1♀, LMCI 319-82, donated to MCTP (64103); 1♀, LMCI 319-85.
Non-type material. Adults (pinned-dried): 1♂, with genitalia preparation on slide, LMCI 319-81; 1♀, with genitalia preparation on slide, LMCI 306-19. Immature stages (fixed in Dietrich’s fluid and preserved in 70% ethanol): ca 30 eggs, dissected from female during genitalia preparation, LMCI 263-52b; 2 last instar larvae (LMCI 263-49 and 326-148); 2 pupae (LMCI 263-51 and 309-02); 12 dissected, mature galls (LMCI 263-35); 5 empty, senescent galls with pupal exuviae (LMCI 319-86). Also, 6 last instar larvae, preserved in 100% ethanol at −20 °C, used for DNA extraction (4 specimens, LMCI 263-33; 2 specimens, 326-146), and 2 last instar larvae preparations, mounted in Canada balsam on a slide (LMCI 263-42, 43).
Eggs (Fig.
Last instar larva (Figs
Neosphecia cecidogena last larval instar under light microscopy A cephalic chaetotaxy, frontal view B thoracic and abdominal chaetotaxy, lateral C, D head in detail, anterior and lateral, respectively. E last abdominal segments in detail, dorsal. Scale bars: 200 µm (A, D); 0.4 mm (E); 0.5 mm (C); 1 mm (B).
Morphology of Neosphecia cecidogena last larval instar under scanning electron microscopy A head, antero-dorsal view B antenna, anterior C stemmata, lateral D maxillae and labium, ventral E spineret, lateral F prothoracic shield, dorsal G meso- and metathoracic segments, dorsal H prothoracic leg, posterior I tarsal claw in detail, posterior J prothoracic spiracle, lateral K second to fourth abdominal segments, lateral L abdominal callus in detail, ventral. Scale bars: 20 µm (E, I, J); 40 µm (B), 100 µm (C, D, H, L); 0.5 mm (A, G, K).
Chaetotaxy (Fig.
Pupa (Figs
Neosphecia cecidogena pupal morphology under scanning electron microscopy A, C head, ventral and lateral views, respectively B clipeal seta in detail D third to sixth abdominal segments, lateral E fourth abdominal spiracle, lateral F spines of fourth abdominal segment in detail, lateral G last abdominal segments, posterior H spine of last abdominal segment, mesal. Scale bars: 40 µm (H); 50 µm (B, E); 100 µm (F); 200 µm (C, G); 0.4 mm (A); 0.5 mm (D), respectively.
This new species is known only from the type locality, the humid forest portions of the CPCN Pró-Mata, São Francisco de Paula municipality, Rio Grande do Sul State, Brazil.
Galls of N. cecidogena have been found only in association with Cayaponia pilosa (Vell.) Cogn. (Cucurbitaceae), which is distributed in the ombrophilous Atlantic Forest of southern Brazil (from Minas Gerais to Rio Grande do Sul State) (
Natural history of Neosphecia cecidogena on Cayaponia pilosa A host plant at the type locality B young fruit, lateral C fully developed gall on axillary region, lateral D, E basal portion of median-sized gall sectioned transversally, showing yellowish nutritive tissue (pointed by closed arrows) (D) longitudinally sectioned medium-sized galls, showing larval feeding scars on nutritive tissue (some are indicated by open arrows) F longitudinally sectioned mature gall, with last instar larva (asterisk) inside (closed and open arrows indicate frass and operculum, respectively) G transversally sectioned, senescent gall showing detached operculum (indicated by seta) and internal wall covered by silk H longitudinally sectioned senescent gall showing internal silk covering (proximal limit pointed by closed arrow) I senescent, overwintering gall, lateral (seta indicates direction of adult emergence) J distal portion of senescent gall, showing pupal exuvium left partially protruded after adult emergence (marked with asterisk). Scale bars: 2 mm (G, H); 3mm (D–F); 4mm (J); 6 mm (I); 9 mm (C); 1 cm (B); 9 cm (A).
The unilocular, cylindrical galls of N. cecidogena measure on average (± standard deviation) 3.44 ± 2.51 cm (n = 9) in length when mature. They appear individually and from the beginning develop externally on axillary buds of C. pilosa vines. Contrary to the oval C. pilosa fruits (Fig.
A few C. pilosa plants have been found at the type locality bearing from one to five N. cecidogena galls per plant. Field collections carried out during five consecutive years at the type locality indicated that it is a univoltine species, larvae growing during the summer when young galls are seen on C. pilosa vines. Fully developed galls containing last instar larvae have been collected mainly during autumn. When brought to the laboratory, these remained larvae during the winter, apparently in a diapause state. Pupation in this case occurred during the first week of September and emergence a few days later during early spring. The absence of a proboscis suggests that adults of N. cecidogena are not active feeders. The appearance of a substantial number of corionated eggs in the abdomen of dissected females shortly after emergence in the laboratory indicates that reproduction occurs early in adult life, and thus, adults may not live long.
A few molecular phylogenies have been proposed for Sesiidae, particularly including Melittini (
Premelittia and Melittina are poorly known monotypic genera, with data available restricted to the original taxonomic description, and observations on morphology made a posteriori based also on the adult female holotypes only by
Sesiid eggs have been described as ventrally flat, being laid with the longitudinal axis parallel to the substrate and having the lower surface less ornamented than the upper one (
Unlike larvae of other sesiids that have pseudopodia bearing crochets (
Pupae of N. cecidogena showed the general integument morphology usually found in the family (
Finally, the galls described here appeared figured twice in association with Cayaponia spp. in a survey carried out at the type locality by
We thank the Instituto de Meio Ambiente/PUC-RS (PROMATA, São Francisco de Paula) for allowing us to collect the specimens in areas under their care and for providing assistance with field work. We acknowledge the staff members of CME/