Research Article |
Corresponding author: Andrea Aracil ( and.aracilgisbert@gmail.com ) Academic editor: Burgert Muller
© 2019 Andrea Aracil, Celeste Pérez-Bañón, Ximo Mengual, Snežana Radenković, Gunilla Ståhls, Ante Vujić, Santos Rojo.
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:
Aracil A, Pérez-Bañón C, Mengual X, Radenković S, Ståhls G, Vujić A, Rojo S (2019) New information about the pre-imaginal morphology of genus Graptomyza (Diptera, Syrphidae, Volucellini): description of third-instar larva and re-description of puparium of G. signata (Walker, 1860). African Invertebrates 60(1): 15-30. https://doi.org/10.3897/afrinvertebr.60.31521
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Pre-imaginal morphology of the flower fly species Graptomyza signata (Walker) is described and figured in detail based on specimens collected on a decomposed Aloe-like plant in KwaZulu-Natal province, South Africa. Third-instar larva is described for the first time and the puparium morphology is re-described using both light (optical) and electron microscopy. The present work represents the second larval description for a species of the genus Graptomyza, after the description of the larva of G. alabeta Séguy. The immatures of these two Graptomyza species were examined and compared to the pre-imaginal stages of the other members of the tribe Volucellini, pointing out the possible diagnostic characters of the genus Graptomyza. Moreover, new DNA barcodes are provided for G. signata and deposited in the NCBI GenBank.
Graptomyza , Volucellini , flower flies, hoverflies, DNA barcoding, larval morphology, immatures
Graptomyza Wiedemann, 1820 (Diptera, Syrphidae) is a medium-size flower fly genus with 90 described valid species, with several more still undescribed, widespread in the Afrotropical, Oriental and Australasian Regions, including the Pacific (
In the Afrotropical Region, 19 Graptomyza species are known and some new species await formal description (
Most of the information of the genus is based on the analysis of the morphology and biology of adults, as very little is known about the pre-imaginal stages. Amongst all known Graptomyza species, immature stages have been described only for four species.
Graptomyza signata belongs to the varia species group (
The main objectives of the present work are to describe for the first time the larval morphology of the third-instar larva of G. signata and to re-describe the morphology of the puparium, together with the cephalopharyngeal skeleton, highlighting its diagnostic features. At the same time, new DNA barcodes for the species are provided.
Nine larvae of G. signata were collected on decomposed roots and stems of an Aloe-like plant in a nursery garden in Queen Elizabeth Park Nature Reserve, Pietermaritzburg (KwaZulu-Natal province), South Africa, in December 2012 (29°33.981'S, 30°19.194'E; 900 m elev.). Larvae were reared with the same plant tissue in which they were found until they pupated. Emerged adults were killed by freezing and pinned for identification and preservation. For adult identification, keys from
Terminology used for larval and pupal descriptions follows
The larval integument was coated thickly in dried decaying plant tissue, particularly the basal region of the posterior respiratory process and the three pairs of lappets at the end of the body. To remove this dry layer, the larvae were immersed in water for 12 hours. After this period, with the help of a binocular microscope, larvae were cleaned using brushes of different strength and thickness. Subsequently, the larvae were washed and preserved in 70% ethyl alcohol.
Debris, adhering to the puparium integument, was removed by placing the specimens in an ultrasonic cleaner for a few minutes. Cleaned specimens were mounted on stubs and examined with a scanning electron microscope (S3000N Hitachi) using variable-pressure (or low vacuum) mode. This technique allows a direct evaluation of the specimens without coating the samples with gold. These studies were conducted in the technical research services at the University of Alicante. Cephalopharyngeal skeletons were extracted from the puparia and cleared by immersion in a 10% solution of potassium hydroxide (KOH) for 5 minutes. After the clearing process, the structures were preserved in glycerine. Third-instar larvae, puparia and cephalopharyngeal skeleton morphology were analysed using a stereomicroscope (Leica M205C) and pictures were taken using a camera adapted to it (Leica DFC450). Dimensions of preserved specimens were measured using the ImageJ informatics tool (
DNA was extracted from a single leg of each of the two pinned specimens (lab codes GS2 and GS3) and one puparium of G. signata (of GS2) using the Phire Tissue Direct PCR master Mix #F-170S kit (Thermo Scientific Baltics UAB, Vilnius, Lithuania) following the Dilution and Storage protocol with some modifications. The Phire Tissue Direct PCR master Mix is designed to perform PCR directly from tissue samples with no prior DNA purification. The tissue sample was placed in an Eppendorf tube in 30 µl of Dilution Buffer, with 0.8 µl of DNA Release Additive. The tube was briefly vortexed and centrifuged, then 1) incubated at room temperature for about 20 min, 2) placed in +56 °C for 10 min and 3) placed in a pre-heated block at 98 °C for 2 min and finally centrifuged at 11 000 rpm for 1 min. One µl of supernatant was used in a 25 µl PCR reaction using the kit PCR master Mix. The cycling conditions were: initial denaturation at 98 °C for 5 min, 40 cycles of denaturation at 98 °C for 5 s, annealing at 49 °C for 30 s, extension at 72 °C for 20 s and final extension at 72 °C for 1 min. The mtDNA COI barcode of the 5’ region of COI (=COIa) was amplified with forward primer LCO1490 (5’-GCTCAACAAATCATAAAGATATTGG-3’) and reverse primer HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (
The obtained mtDNA COI sequences were individually compared against the BOLD systems v4 (boldsystems.org, accessed 16 October 2018) and the NCBI GenBank databases, using BLASTn, for comparison. Sequences produced in this study were deposited in the NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/), accession numbers MK415842 (GS2_Leg) and MK415843 (Gs3_Pupa).
A total of nine third-instar larvae were collected in South Africa: KwaZulu-Natal Prov., Pietermaritzburg, Queen Elizabeth Park Nature Reserve nursery garden on 15.12.2012, by two of the authors (Snežana Radenković and Santos Rojo). Two specimens were preserved for morphological studies while the seven remaining larvae were allowed to pupate. One of the pupae did not emerge and was preserved. One of the adults emerged on 5.1.2013 (female), three of them on 6.1.2013 (one female and two males) and two on 7.1.2013 (one female and one male). Adults were also collected during December on Euphorbia flowers in Royal Natal National Park, KwaZulu-Natal Prov., Drakensberg Mountain by AV.
Examined material
Adult specimens were identified as G. signata and they matched very closely the characters used in the key by
Overall characters
Light-brown in colour. Length 6.3 ± 0.3 mm, Width 2.2 ± 0.15 mm (N = 2). Dorso-ventrally flattened, broadly rounded anteriorly and slightly tapered posteriorly, with protruding breathing tube. Dorsal and lateral surface hairy, covered by long, thin and slightly sclerotised setae, increasing in size in the anal segment. Vestiture of the surface not forming a pattern of transverse rows (Fig.
Cephalopharyngeal skeleton
Mandibles and mandibular lobes internal (Fig.
Graptomyza signata larvae and puparium. A larvae, ventral view B larvae, dorsal view C prothorax of larvae, ventral view D cephalopharyngeal skeleton, dorsal view E cephalopharyngeal skeleton, lateral view F pupae not cleaned, dorsal view G puparium cleaned, lateral view. Abbreviations: Ac – Antennomaxillary complex; C – Cibarium; Ce – Clypeal sclerite; Dc – Dorsal cornu; Dl – Dorsal lip; L – Labrum; Ll – Lateral lip; Lpt – Lappets; M – Mandibles; Ml – Mandibular lobes; Oc – Oral cavity; T – Tentorium; Tbr – Tentorial bar; Tr – Transversal ridges; Vc – Ventral cornu; Vl – Ventral lip. Scale bars: 2 mm (A, B, F, G), 0.5 mm (C, E), 0.25 mm (D).
Pseudocephalon and thorax
Dorsal, ventral and lateral lips well developed. Lateral lips rounded and covered by a dense tuft of long and thin setae at apex, becoming thinner at base. Antenno-maxillary organs well developed, located between dorsal lip and dorsal surface of prothorax. The antenno-maxillary organs are formed by a pair of cylindrical-shaped structures, each with antenna and maxillary palpus clearly identified. Several satellite sensilla present on top of antennae and maxillary palpi. Ventral surface of anterior fold of prothorax covered by sclerotised and hook-shaped spicules with apex more sclerotised than basal part. The spicules face backwards and reach up dorsally to second sensilla of prothorax (Fig.
Abdomen
Primordia of pupal spiracles on dorsal surface of first abdominal segment. Prolegs barely developed, represented as transversal ridges without crochets, present in abdominal segments 1–7. Abdominal segments with dorsal pairs of segmental sensilla 1, 2 and 3 with small basal papillae, increasing in size towards segment 7. Dorso-lateral pairs of sensilla 4, 5 and 6 with well-developed papillae, all surrounded by apical setae, increasing in size towards the posterior end of the larva. Anal segment about one and a half times longer than 7th abdominal segment, divided in two sections: one anterior section with two pairs of dorso-lateral lappets bearing sensilla 1, 2 and 3; and one posterior section with one pair of ventral lappets with sensilla 4 at the base and sensilla 5 on the apex. First lappet slightly shorter (± 400–450 μm) than second and third lappets (± 500–550 μm), fully covered with long and thin setae and facing the posterior margin of the larvae (Fig.
Posterior respiratory process
Shiny, light brown to reddish in colour, sub-elliptical in cross section, length 1.0 ± 0.25 mm (2.5 times as long as wide). The length of the posterior respiratory process (prp) varies between larva and pupa due to the larval tissue partially covering the surface and the pupa with all this tissue retracted, exposing the entirety of the structure. In the middle of its length, there is a shallow transverse ridge. The ornamentation from base to transversal ridge presents faint transversal wrinkles more visible at base. The shape of apex (from transversal ridge to spiracular plate) is rectangular, with irregular spaced dents in basal three quarters and being smooth in apical quarter (Fig.
Chaetotaxy
Prothorax (P) with 11 pairs of sensilla, mesothorax (Ms) and metathorax (Mt) with eight pairs of sensilla. Abdominal segments 1–7 with nine pairs of sensilla, anal segment (A8) with eight pairs of sensilla and three pairs of lappets (Fig.
Map of the chaetotaxy of the third instar larva of Graptomyza signata in lateral view showing the positions of the sensilla group. P – Prothorax, Ms – Mesothorax, Mt – Metathorax, A1–A7 – Abdominal segments, A8 – Anal segment. Sp – Anterior spiracle, Ll – Lateral lip, Psp – Primordia of pupal spiracles, Lpt – Lappets.
Overall description
Ovate; dorsally gibbose; sub-cylindrical in cross-section. Anterior end truncated, tapered posteriorly and flattened ventrally. Dull, light brown to reddish in colour. Surface covered with dense pubescence, longer in posterior segments. Anal segment with three pairs of lappets. Rough integument with larval segmentation persisting as transverse folds and wrinkles (Fig.
Pupal spiracles
Sub-cylindrical structures of 0.94 ± 0.03 mm long and 0.14 ± 0.02 mm wide (N=4), curved backwards and with the end slightly tapered. Shiny, light brown to reddish. Approximately 80% of dorsal and lateral surface is covered with irregular spaced and oval to sub-elliptical tubercles which are barely prominent (Fig.
Three COI sequences were obtained from two raised adults and one from one of the puparia, the length of the sequences varying between 563 and 660 bp. There was only a single nucleotide difference between the two adult barcodes (99.8% similarity). The barcode of the adult and of its puparium were similar, except for two ambiguous base positions due to the lower quality of the DNA barcode from the puparium. The GenBank BLASTn query returned five Graptomyza barcodes with the highest similarities (≥ 91%). BOLD query results were similar, but yielded an additional two highly similar barcodes (≥ 98%) of unnamed and unpublished specimens.
The puparium of G. signata is very similar to the puparium of the three other Graptomyza species for which the puparium has been described. They all share a more or less developed setose vestiture and the presence of three pairs of lappets (unclear in G. mitis). The edge of the spiracular plates is more defined and irregular in G. alabeta, G. triangulifera and G. mitis than in G. signata. The shape of the spiracular openings differs between the four species: they are straight and run almost parallel in G. alabeta (Fig.
Graptomyza alabeta spiracles. A spiracular plate of Posterior Respiratory Process B detail of spiracular openings of spiracular plate of the Posterior Respiratory Process C Posterior Respiratory Process, dorsal view D pupal spiracle E detail of spiracular openings of pupal spiracle. Scale bars: 100 μm (A), 50 μm (B), 250 μm (C), 200 μm (D), 25 μm (E).
Known morphology of the larvae and puparia of Graptomyza share the diagnostic characteristics of Vollucellini, proposed by
The morphology of the cephalopharyngeal skeleton between the five species described varies in the size of the mandibles and mandibular lobes, the relative lengths of the dorsal and ventral cornua and the degree of sclerotisation of the clypeal sclerite. Dorsal cornu is generally rounded apically, always shorter than the ventral cornu. The puparium of G. triangulifera has the ventral cornu clearly shorter than the rest of described species.
Graptomyza larval morphology is very similar to the overall morphology of Brachyopa (Syrphidae, Eristalinae, Brachyopini) larvae, although they are not phylogenetically closely related (
Descriptions of immature stages and rearing data can contribute to the systematics of a group as useful taxonomic information to develop a sound classification and a likely evolutionary scenario (
The study was funded by the project of the European Union: Horizon 2020, Marie Skłodowska-Curie action, Research and Innovation Staff Exchange (RISE) Programme: FlyHigh – “Insect-plant relationships: insights into biodiversity and new applications”. Marina G. Krivosheina, Severtsov Institute of Ecology and Evolution, Moscow, Russia, is thanked for the lending of material of Graptomyza alabeta, used for comparison during the current study.