Research Article |
Corresponding author: James B. Woolley ( jimwoolley@tamu.edu ) Academic editor: Petr Janšta
© 2019 Xanthe A. Shirley, James B. Woolley, Keith R. Hopper, Nunzio Isidoro, Roberto Romani.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Shirley XA, Woolley JB, Hopper KR, Isidoro N, Romani R (2019) Evolution of glandular structures on the scape of males in the genus Aphelinus Dalman (Hymenoptera, Aphelinidae). Journal of Hymenoptera Research 72: 27-43. https://doi.org/10.3897/jhr.72.36356
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The pores and associated glands on male antennae in species of Hymenoptera are involved in mate recognition and are diverse and widespread among taxa. However, nothing has been published about these structures in species of Aphelinus (Chalcidoidea: Aphelinidae), a genus of parasitoid wasps with a long history in biological control. Images from scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of Aphelinus varipes revealed pores on the ventral side of the male scape that were connected to glands. A survey of the scapes of male antennae in 16 species in six species complexes of Aphelinus, as well as two outgroup species, Aphytis melinus and Centrodora sp., showed that pores were present in all except Centrodora sp. The pores varied in several characters: the shape of the structures that carried them, pore size, elevation of the cuticle surrounding the structures, the extent of a carina delimiting the area around the structures, and the number and position of pores. The shape of the pore-bearing structures, the elevation of cuticle around these structures, and the extent of the carina around them map well onto a molecular phylogeny of these Aphelinus species. Combinations of pore characters are diagnostic of species complexes, and in some cases, species of Aphelinus.
Aphelinus, antennal morphology, scanning electron microscopy, scape, sexual dimorphism, mate recognition, glands
Successful mating depends on discriminating between individuals of the same versus different species, as well as between potential mates that will yield progeny with high versus low fitness. Communication with pheromones that attract conspecifics and provide cues for their recognition is often a key component in the quest for mates by insects in general and Hymenoptera in particular (for review, see
The well-documented role of antennation in courtship triggered investigations into the morphology of antennae. Based on scanning electron microscopy (SEM) of antennae,
RSS and their associated glands have been found on male antennae in other chalcidoids: Trichogramma australicum Girault (Hymenoptera: Trichogrammatidae) (
Observations on courtship behavior in several species of Aphelinus Dalman (Hymenoptera: Aphelinidae) showed that males antennate during courtship (
We follow the species complex classification of
With TEM, we studied the structure of the cells underlying the pores in the male scape of A. varipes. With SEM, we studied male scapes of two to three specimens each from nine species of Aphelinus in six species complexes, along with species in two other genera of Aphelininae: Aphytis melinus Debach and an undetermined species of Centrodora Förster (Hymenoptera: Aphelinidae) (Table
Collection information and imaging technique for Aphelinus species (SEM = scanning electron microscopy, DICM = differential interference contrast microscopy, MP = Macropod macrophotography).
Species complex | Species | Year collected | Country | Host | Host plant | Collector | Permit, voucher | Imaging |
---|---|---|---|---|---|---|---|---|
subflavescens | A. perpallidus Gahan | 2009 | USA | Monelliopsis pecanensis | Carya illinoinesis | A. Dickey | TAMUIC voucher 733 | SEM |
asychis | A. asychis Walker | 2000 | France | Diuraphis noxia | Triticum sp. | N. Ramualde, D. Coutinot, J. Lopez | P526P-15-04274, AFr00_Dn | SEM |
A. sinensis Shirley & Woolley | 2002 | China | Aphis glycines | Glycine max | K. Hoelmer, K. Chen, W. Meikle | P526P-01-53096, AChAg | SEM | |
abdominalis | A. abdominalis (Dahlman) | 2014 | USA | – | – | Syngenta Bioline | P526P-15-04767, AbUSA_14 | SEM |
daucicola | A. daucicola Kurdjumov | 2012 | USA | Aphis helianthi | Daucus carota | C. Dieckhoff | P526P-15-04767, DUSA12_DE | SEM |
mali | A. coreae Hopper & Woolley | 2009 | Korea | Aphis glycines | Glycine max | K. Hoelmer | P526P-08-02142, MKor09_M | SEM |
A. glycinis Hopper & Woolley | 2007 | China | Aphis glycines | Glycine max | K. Hoelmer | P526P-01-72318, MCh04_Bj | DICM | |
A. rhamni Hopper & Woolley | 2005 | China | Aphis glycines | Rhamnus sp. | K. Hoelmer | P526P-01-72318, MCh07_Bj | DICM | |
mali Haldeman | 1985 | Australia | Eriosoma lanigerum | – | M. Carver, H.J. Banks | ANIC database no. 32 064862 | MP | |
varipes | A. atriplicis Kurdjumov | 2000 | Republic of Georgia | Diuraphis noxia | Triticum sp. | D. Coutinot | P526P-15-04274, VGg00_Dn | DICM |
A. certus Yasnosh | 2001 | Japan | Aphis glycines | Glycine max | R. O’Neil, D. Voegtlin | P526P-01-53096, VJp01_TU | SEM | |
near A. certus | 2009 | Korea | Aphis glycines | Glycine max | K. Hoelmer | P526P-08-02142, VKor09_M | DICM | |
A. hordei Kurdjumov | 2011 | France | Diuraphis noxia | Triticum sp. | G. Mercadier, M. Roche | P526P-15-04274, VFr11_Dn | DICM | |
A. kurdjumovi (Kurdjumov) | 2000 | Republic of Georgia | Rhopalosiphum padi | Triticum sp. | D. Coutinot | P526P-13-02503, VGg00_Rp | DICM | |
A. nigritus Howard | 2015 | USA | Melanaphis sacchari | Sorghum bicolor | – | P526P-15-04767, VUSA14_TX | DICM | |
A. varipes (Förster) | 2000 | France | Rhopalosiphum padi | Triticum sp. | N. Ramualde, D. Coutinot, J. Lopez | P526P-13-02503, VFr00_Rp | SEM |
For TEM, specimens were immersed in a solution of glutaraldehyde (2.5 ml/ 25 ml solution) and paraformaldehyde (1 g/25 ml solution) in 0.1M cacodylate buffer +5% sucrose, pH 7.2–7.3. The scape was removed from the rest of the antenna and cooled at 4 °C for 3 h. The specimens were placed in 0.1M cacodylate buffer +5% sucrose, pH 7.2–7.3, overnight at 4 °C, then the specimens were post-fixed in 1% OsO4 (osmium tetroxide) for 1 h at 4 °C and rinsed in the previous buffer. Dehydration in a graded ethanol series from 60% to 99% was followed by embedding in Epon-Araldite with propylene oxide as bridging solvent. Thin sections were made with a diamond knife DiATOME ultra 45° (DiATOME AG, Biel, Switzerland) on a LKB Bromma ultramicrotome (LKB®, Sweden), and mounted on formvar-coated, 50-mesh grids. The sections were stained with uranyl acetate (20 min, room temperature) and lead citrate (5 min, room temperature). Finally, the sections were investigated with a TEM Philips EM 208 (Thermo Fischer Scientific, Hillsboro, Oregon, USA). Digital images with 1376×1032 pixels, 8 bit, uncompressed greyscale in TIFF files were obtained using a high-resolution digital camera MegaViewIII (SIS) connected to the TEM.
For SEM, specimens were critical-point-dried (CPD) using a Tousimis Samdri-790 (Tousimis Research Corporation, Rockville, Maryland, USA), following the manufacturer’s protocol. After CPD, two to three males from each accession (Table
Voucher specimens of material examined have been deposited in the Texas A&M University Insect Collection with Voucher #733. Voucher numbers in Table
Five morphological traits of the RSS were recorded for 14 species of Aphelinus, as well as for Aphytis melinus (Table
Characters of release and spread structure on male scapes of Aphelinus species and Aphytis melinus.
Species complex | Species | Pore | Pore region | ||||
---|---|---|---|---|---|---|---|
Number | Sizea | Shapeb | Locationc | Carinad | Elevatione | ||
N.A. | Aphytis melinus | 2 | small | flat | proximal | complete | none |
subflavescens | Aphelinus perpallidus | 2 | large | crenulated ridge | proximal | complete | depressed |
asychis | A. asychis | 4 | small | flat | midpoint | none | elevated |
A. sinensis | 5 | small | flat | midpoint | none | elevated | |
abdominalis | A. abdominalis | 3 | small | cone, round top | distal | complete | depressed |
daucicola | A. daucicola | 5 | large | cone, flat top | proximal | complete | depressed |
mali | A. coreae | 2 | large | cone, flat top | midpoint | complete | depressed |
A. glycinis | 2, 3, 5 | large | cone, flat top | midpoint | complete | depressed | |
A. rhamni | 2, 3 | large | cone, flat top | midpoint | complete | depressed | |
varipes | A. atriplicis | 3 | large | cone, flat top | proximal | half | depressed |
A. certus | 3 | large | cone, flat top | midpoint | half | depressed | |
A. near certus | 3 | large | cone, flat top | midpoint | half | depressed | |
A. hordei | 3 | large | cone, flat top | midpoint | half | depressed | |
A. kurdjumovi | 3 | large | cone, flat top | midpoint | half | depressed | |
A. nigritus | 3 | large | cone, flat top | midpoint | half | depressed | |
A. varipes | 3 | large | cone, flat top | midpoint | half | depressed |
The male scape in A. varipes is characterized by the presence of cuticular modifications located on the ventral side, defining a specialised area (Fig.
Structure of the male scape in Aphelinus varipes. A–C Scanning electron microscope images showing in A the modified male scape (Sc) with the presence of a ventral carina on which three specialized structures (arrowheads) are observed B detail of the carina revealing three elevated areas (Ea) C close up of the previous in which a single apical pore (Po) per Ea is clearly visible, as well as secretion (Scr) oozing from the pore itself. D–H Transmission electron microscope images showing internal ultrastructural features of the male scape D cross section of the scape taken through one of the elevated areas, showing the secretory cell (Scl) that occupies about half of the scape internal volume; Scl has a large nucleus (Nu) located basally, a central cluster of electron-lucid secretory vesicles (Sv) and a straight evacuating duct (Ed) connected with the external pore (Po); the rest of the scape volume is occupied by muscles (Mu) and the antennal nerve (An) E close-up view of the previous image, showing the cuticular evacuating duct (Ed) running straight towards the external pore F cross section of the scape taken in a different view, showing the large secretory cell (Scl) with a centrally positioned end apparatus (Ea) surrounded by numerous secretory vesicles (Sv); the evacuating duct (Ed), muscles (Mu), and antennal nerve (An) can be seen G detail of the end apparatus (Ea), which appears perforated and surrounded by microvilli (Mv); secretory vesicles (Sv) are above H detail of the duct cell (Dc) characterised by a very reduced cytoplasm and a small nucleus (Nu); the duct cell is surrounded by the cytoplasm of the secretory cell, that reveals the presence of ribosomes (Ri) and mitochondria (Mt); the evacuating duct (Ed) is visible in cross section. Scale bars: 50 µm (A); 10 µm (B, D, F); 5 µm (C); 2.5 µm (E); 1 µm (G); 2 µm (H).
The morphology of these release-and-spread structures varies among Aphelinus species and Aphytis melinus (Figs
Mapping six characters of the release-and-spread structures (Table
There is considerable variation among Aphelinus species in the characters of the release-and-spread structures on male scapes. Although three characters show some homoplasy with CI values of 0.50 to 0.75 (pore number, size, and location), three characters have CI = 1.0 (supporting structure, elevation of cuticle, and extent of carina). Combinations of characters are diagnostic for species complexes of Aphelinus (Table
Aphelinus varipes males have specialized secretory structures on the scape, which are connected with the external pores. The glandular units have secretory cells releasing secretions in electron-lucid vesicles. These vesicles surround an end apparatus connected with the cuticular evacuating duct (produced by a duct cell) that allows the external release of the secretion. These features are typical of secretory cells belonging to class III, according to the classification of insect epidermal glands proposed by
In Aphelinidae, male antennal glands have been reported in Encarsia asterobemisiae Viggiani and Mazzone (Hymenoptera: Aphelinidae), Encarsia aurantii (Howard) and Encarsia opulenta (Silvestri) (
We thank Andreas Holzenburg and Mike Pendleton of Texas A&M Microscopy Center for assistance with SEM imaging. Thanks to Kim Carr, Texas A&M, for the preliminary male scape survey. Thanks also to Jewel Coffey, Bryant McDowell, and Itzel Cetina, Texas A&M, for help with specimen preparation. We thank Petr Jansta and Ovidiu Popovici for their excellent comments and suggestions for the manuscript. TEM and SEM images of A. varipes and SEM images of A. varipes were done at the Centro Universitario di Microscopia Elettronica (CUME; Università degli Studi di Perugia, Italy). This research was supported by the following grants from the National Science Foundation, USA: DEB 1257601 and DEB 1555790, and it was part of the M.S. thesis of XAS at Texas A&M University.