Detailed and Complete Descriptions of Immature Stages of Two Predatory Species of Eupeodes Osten Sacken, 1877 (Diptera: Syrphidae)

Abstract

With over 40 species, Eupeodes Osten Sacken, 1877, is a common aphidophagous hoverfly genus (Diptera: Syrphidae: Syrphinae) with a worldwide distribution. The immature stages of Eupeodes are well known, since 23 species have been reared, but only 8 species have been described. All known immature stages of Eupeodes species have predatory feeding habits, mainly on aphids and other soft-bodied Hemiptera, for which some of its species are commonly used as agents of biological control programs. In this work, the puparium of Eupeodes bucculatus (Rondani, 1857) is detailed and re-described, and the complete morphology of all immature stages of the life cycle of Eupeodes corollae (Fabricius, 1794) is documented by using stereomicroscope and scanning electron microscope techniques. An update to the taxonomic key for the known larvae/puparium of Eupeodes species is also provided.

1. Introduction

Syrphidae (Insecta: Diptera), commonly known as flower flies, syrphids, or hoverflies, has a worldwide distribution with 6674 species and 284 genera [1]. Nowadays, syrphids are divided into four subfamilies based on larval and adult morphology, molecular data, and the feeding habit of the immature stages: Eristalinae (phytophagous and saprophagous), Microdontinae (predatory), Pipizinae (predatory), and Syrphinae (predatory), with the latter presenting the largest number of species [2,3,4,5]. The larvae of almost one third of the immature stages prey on insects that are serious pest in crops, such as aphids (Hemiptera: Aphididae), and this is the reason why some syrphid species are widely used in biological and integrated pest control programs and commercialized as predator agents (e.g., Eupeodes corollae (Fabricius, 1794) or Sphaerophoria rueppellii (Wiedemann, 1820)) [6].

With over 40 species worldwide, the genus Eupeodes Osten Sacken, 1877, has been recently divided in two subgenera due to strong molecular evidence: Eupeodes (Eupeodes) and Eupeodes (Macrosyrphus) Matsumura in Matsumura & Adachi, 1917 [7]. At the moment, the immature stages of 23 species of Eupeodes have been reared, but only the immature stages of 8 species have been described [8,9,10,11,12]. Unfortunately, most descriptions do not provide fine details, thus there is a lack of information on comparative diagnosis of morphological characteristics for the correct species identification (e.g., Eupeodes bucculatus (Rondani, 1857) or E. corollae) [8,13,14,15].

Eupeodes bucculatus is one of the 21 species of the genus present in Europe [10]. This species has a lot of controversy regarding its identification, since in different works it was synonymized with Eupeodes latilunulatus (Collin, 1931) or Eupeodes luniger (Meigen, 1822) [10,16,17]. However, in Mazánek [18], it was reinstated as E. bucculatus based on the male genitalia, the lateral margins of tergite 5, and by the postocular orbit. The distribution range of this species is from Norway, Sweden, and Denmark, south to Portugal and Spain, and eastwards Ireland through central Europe [10]. The information regarding the immature stages is very vague, since the egg has been described by Kumar et al. (1987) [8] (by the name Metasyrphus latilunulatus), but the third larval stage and puparium are briefly described by Dixon [13] (by the name Syrphus latilunulatus), Dušek and Láska [14], and Kumar et al. [8]. According to Rojo et al. [9], the larvae of E. bucculatus (by the name E. latinulatus) primarily feed mainly on soft-bodied insects, such as aphids.

One of the most striking species of Eupeodes that can be found in Europe and that is widely used in biological control programs is E. corollae [6,10,19]. This species can be found in Iceland, Fennoscandia, and the Faroes and south to the Iberian Peninsula, the Mediterranean coast, Madeira, Canary Islands and North Africa, South Africa, and throughout Asia [10]. Eupeodes corollae is considered as a highly migratory species, with this being the reason of its wide distribution range [20]. The predator behavior of E. corollae larvae is well known, but the larval morphology is not completely detailed, since the description made by Dixon [13] (by the name Metasyrphus corollae) and Dušek and Láska [14] (by the name Syrphus corollae) lacks the morphological characteristics of utmost importance for its correct identification (e.g., head skeleton).

This study aims to substantially expand the knowledge of the morphology of the immature stages of the genus Eupeodes. The anterior respiratory process (ARP), the head skeleton, the posterior respiratory process (PRP), and the puparium of E. bucculatus as well as the complete life cycle and head skeleton E. corollae are characterized by using a stereomicroscope and scanning electron microscope (SEM). In addition, we provide an updated key for the third stage larva (l3) and puparium of the Eupeodes species.

2. Materials and Methods

Examined material and adult/larva identification. Six third larvae of E. bucculatus were found preying on aphid colonies on shoots and flowers of Centaurea sp. (Asteraceae) located in Montseny Natural Park at Matagalls (Barcelona, Spain) by Javier Quinto in July 2024. This mountainous enclave is situated within at the Montseny Biosphere Reserve in the Catalan pre-coastal range that occupies an area of over 50,000 ha, characterized by the dominance of pine (Pinus sp.) and oak trees (Quercus sp.). The E. bucculatus larvae were reared under laboratory conditions in small plastic boxes containing leaves of pepper, Capsicum anuum L. (Solanaceae) with Myzus persicae (Sulzer, 1776) (Hemiptera: Aphididae). After 5–6 days, all six larvae pupated, and six adults (one male and five females) emerged from their puparium. Adults of E. bucculatus were identified by the authors and confirmed by Libor Mazánek. The eggs, larvae, and pupae of E. corollae used for the descriptions were provided by BioNostrum Pest Control Company^® (Alicante, Spain). All the material is deposited at the CEUA-CIBIO collection, University of Alicante (Spain).

Sample preparation and study. The larvae and puparium were cleaned in an ultrasonic bath for 7 min and carefully brushed to remove any dirt. The head skeleton was removed from the puparium by soaking it in hot 10% KOH for 60 min, and it was photographed in glycerin. General features of the egg, larva, puparium, and head skeleton were observed under a Leica M205 C binocular stereomicroscope (Leica Camera AG, Wetzlar, Germany). The measurement of the egg, larval body, puparium, and PRP followed the protocol of Orengo-Green et al. [21]. Photos were produced as stacks of individual images made with a camera Leica DMC 5400 (Leica Camera AG, Wetzlar, Germany) attached to a Leica M205 C. Stacks were created in Leica Application Suite Las X^®, v.4.12.0. (Leica Microsystems, Wetzlar, Germany). For the immature stages of E. corollae, a Tousimis Autosamdri 931 critical point dryer (Tousimis Research Corp, Rockville, MD, USA) was used. For a more detailed description of the morphology, a Jeol JSM-IT500HR SEM (JEOL Ltd., Tokyo, Japan) at 5 kV from the SSTT at the University of Alicante (Alicante, Spain) and a Jeol JSM-6500F SEM (JEOL Ltd., Tokyo, Japan) at 5 kV from the I2AT at the Mississippi State University (Starkville, MS, USA) were used. Two pupae of E. bucculatus and one sample of all the immature stages (egg, first larval stage, second larval stage, third larval stage, and pupa) of E. corollae were mounted on aluminum stubs with double-sided adhesive carbon tape. One pupa of E. bucculatus was cut in half, and the posterior part was mounted on aluminum stubs with double-sided adhesive carbon tape. The samples were sputter-coated in a Quorum 150 T ES Plus (Quorum Technologies Ltd., Lewes, UK) with a 30 nm layer of platinum.

Morphology terminology. The terminology used for the descriptions of the larva, puparium, and head skeleton follows Rotheray [22] and for the egg follows that of Chandler [23].

3. Results

3.1. Eupeodes bucculatus Puparium (Figure 1)

Description. Length: 5.87–6.1 mm, height: 2.93–3 mm, and width: 2.98–3.13 mm (n = 6). Tear-drop shape and flat ventrally, with one pair of poorly developed crochet-less locomotory organs on light cream spots in the 2nd to 7th abdominal segment. The thorax and the abdominal segments with a pubescence surface (Figure 2). A pair of small sclerotized anterior respiratory processes with a smooth surface in the prothorax (Figure 2). Pupal spiracles were absent. The 8th abdominal segment (anal segment) with one visible pair of lappets, located under the PRP. Color: light brown. Chaetotaxy: first to seventh abdominal segments with three pairs of sensilla with seta dorsally, laterally with five pairs of sensilla with seta, and ventrally with three pairs of sensilla without seta. The anal segment with five pairs of visible sensilla with seta (see

Table 1. Chaetotaxy of the puparium of Eupeodes bucculatus. Numbers are in pairs. Δ indicated sensilla with seta.

	1st–7th Abdominal Segment	Anal Segment
Dorsally	3 Δ	2 Δ
Laterally	5 Δ	2 Δ
Ventrally	3	1 Δ

). Head skeleton (Figure 3): labium and labrum elongated and heavily sclerotized. Labrum fused in the front with a pointed end. Labium fused in the front with a sharp triangle pointed end, ventrally with a noticeable protuberance, in the ventral posterior end with a cone protuberance. A pair of mandibles between the labium and labrum. Tentorial bar heavily sclerotized. The bottom half of the dorsal cornu heavily sclerotized, and the upper half slightly sclerotized. The dorsal cornu not elongated posteriorly. The ventral cornu slightly sclerotized and almost twice the length of the dorsal cornu. The roof of the pharynx slightly sclerotized in the middle of the ventral cornu. A pair of heavily sclerotized triangular lateral lips located laterally of the head skeleton. The PRP (Figure 4) shiny and light brown with a noticeable transverse ridge as a constriction. Smooth surface. Divided by a groove as deep as half of the height. The length above the transverse ridge: 0.131–0.157 mm; length below the transverse ridge: 0.09–0.11 mm, width at the transverse ridge: 0.47–0.53 mm. The spiracular plate (Figure 4A, C) with a pair of dorsal spurs, a pair of ecdysial scars between the dorsa spur and the first spiracular opening, three pairs of straight spiracular openings each on top of a sclerotized carina, four pairs of single inter-spiracular setae located in a small sclerotized protuberance, and one pair of perispiracular glands between the spiracular opening I and II.

Figure 1. Puparium of Eupeodes bucculatus: (A) dorsal view; (B) lateral view. Arrow indicates the light cream spots.

Figure 1. Puparium of Eupeodes bucculatus: (A) dorsal view; (B) lateral view. Arrow indicates the light cream spots.

Figure 2. Prothorax and mesothorax of Eupeodes bucculatus puparium. Arrow indicates sensilla with seta; dash circle indicates the anterior respiratory process.

Figure 2. Prothorax and mesothorax of Eupeodes bucculatus puparium. Arrow indicates sensilla with seta; dash circle indicates the anterior respiratory process.

Figure 3. Lateral view of Eupeodes bucculatus. Dc, dorsal cornu; Lb, labium; Lm, labrum; Rp, roof the pharynx; Tb, tentorial bar; Ts, triangle sclerites; Vc, ventral cornu. Dash circle indicates the mandible position.

Figure 3. Lateral view of Eupeodes bucculatus. Dc, dorsal cornu; Lb, labium; Lm, labrum; Rp, roof the pharynx; Tb, tentorial bar; Ts, triangle sclerites; Vc, ventral cornu. Dash circle indicates the mandible position.

Figure 4. Posterior respiratory process of Eupeodes bucculatus: (A) Polar view, SEM image; (B) lateral view, SEM image; (C) polar view, stereomicroscope image. Legend: dash arrow indicates the perispiracular gland; dash circle indicates the dorsal spurs; * indicates the interspiracular-setae; I, II, III, spiracular openings; Ds, dorsal spurs; Es, ecdysial scars.

Figure 4. Posterior respiratory process of Eupeodes bucculatus: (A) Polar view, SEM image; (B) lateral view, SEM image; (C) polar view, stereomicroscope image. Legend: dash arrow indicates the perispiracular gland; dash circle indicates the dorsal spurs; * indicates the interspiracular-setae; I, II, III, spiracular openings; Ds, dorsal spurs; Es, ecdysial scars.

3.2. Eupeodes corollae Early Stages

3.2.1. Egg (Figure 5)

Description. Length: 0.97–1.23 mm, and the width: 0.476–0.53 mm (n = 7). Oval shape, with the dorsal tip concave due to the micropyle. The chorionic sculpturing consists of elevated elongated units with a porous surface and with lateral branches that contact with other units (Figure 5A). The units around the micropyle were protuberances with irregular forms, more porous when compared with the rest of the units (Figure 5B). Color (Figure 5C): white when recently laid and darker cream when older.

Figure 5. Eupeodes corollae egg: (A) Chorionic structures, SEM image; (B) dorsal tip, SEM image (arrow indicates the micropyle); (C) lateral view, stereomicroscope image.

Figure 5. Eupeodes corollae egg: (A) Chorionic structures, SEM image; (B) dorsal tip, SEM image (arrow indicates the micropyle); (C) lateral view, stereomicroscope image.

3.2.2. First Stage Larva (L1) (Figure 6)

Description. length: 2.35–3.12 mm, height: 0.68–0.87 mm, width: 0.70–0.87 mm (n = 5). Oval in the cross-section and flat ventrally. The 8th abdominal segment (=anal segment) with an undeveloped grasping bar and no visible lappets. Color: transparent. Chaetotaxy: prothorax with 11 sensilla without seta, mesothorax with 8 pairs of sensilla with a long black seta, and metathorax with 8 pairs of sensilla with a long black seta. First to the seventh abdominal segment with three pairs of sensilla with a long black seta dorsally, laterally with five pairs of sensilla that were long and black, and ventrally with three pairs of sensilla without seta. The anal segment with five pairs of sensilla with a long black seta and ventrally with three pairs of sensilla with small seta (see

Table 2. Chaetotaxy of the immature stages of Eupeodes corollae. Numbers are in pairs. Δ indicates sensilla with seta.

		Prothorax	Mesothorax	Metathorax	1st–7th Abdominal Segment	Anal Segment
	Dorsally	5	3 Δ	3 Δ	3 Δ	3 Δ
L1	Laterally	3	2 Δ	2 Δ	5 Δ	2 Δ
	Ventrally	3	3 Δ	3 Δ	3	3
	Dorsally	5	3 Δ	3 Δ	3 Δ	3 Δ
L2	Laterally	3	2 Δ	2 Δ	5 Δ	2 Δ
	Ventrally	3	3 Δ	3 Δ	3	3
	Dorsally	5Δ	3 Δ	3 Δ	3 Δ	3 Δ
L3	Laterally	3 Δ	2 Δ	2 Δ	5 Δ	2 Δ
	Ventrally	3 Δ	3 Δ	3 Δ	3	3

). Head skeleton: labium and labrum moderate sclerotized and elongated. The labrum fused in the front with a pointed end. The labium fused in the front with a sharp end, in the anterior ventral end with a small protuberance, and on the posterior ventral end with a bigger protuberance when compared with the anterior ventral end protuberance. Non-distinguishable mandibles. The tentorial bar moderate sclerotized. The dorsal cornu with the posterior half heavily sclerotized, not elongated. The ventral cornu with the anterior part heavily sclerotized, and the posterior part slightly sclerotized. The ventral cornu almost as twice the size of the dorsal cornu. The pair of triangular lateral lips were absent. PRP (Figure 7): not fused, and could be seen dorsally. Light brown color. Smooth surface, with no distinguishable transverse ridge. Spiracular plate with three pairs of undeveloped spiracular openings with triangular spikes. One pair of a perispiracular gland. Dorsal spur absent. Non-observable inter-spiracular setae. See Table 3 for a resume comparation between the larval stages.

Figure 6. Eupeodes corollae first stage larva, dorsal view. Dash circle indicates the posterior respiratory process.

Figure 6. Eupeodes corollae first stage larva, dorsal view. Dash circle indicates the posterior respiratory process.

Figure 7. Posterior respiratory process of a first stage larva of Eupeodes corollae: arrow indicates the perispiracular gland; I, II, III, spiracular openings.

Figure 7. Posterior respiratory process of a first stage larva of Eupeodes corollae: arrow indicates the perispiracular gland; I, II, III, spiracular openings.

3.2.3. Second Stage Larva (L2) (Figure 8)

Description. Length: 11.3–11.7 mm, height: 2.6–2.97 mm, and width: 2.54–2.88 mm (n = 5). Oval in the cross-section, flat ventrally, and truncated posteriorly. Prothorax with a pair of well-developed antenna-maxillary organs. Well-developed crochet-less locomotory organs from the 1st to the 7th abdominal segment. The anal segment with a well-developed grasping bar. One pair of lappets in the anal segment. The PRP could be seen dorsally and in parallel with the rest of the body. Color: light cream, dorsally with a pair of white lines in the middle from the 1st abdominal segment to the anal segment and laterally with intermittent white lines from the 1st abdominal segment to the anal segment. Chaetotaxy: prothorax with 11 pairs of observable sensilla without seta. The mesothorax and metathorax each with eight pairs of sensilla with seta. First to seventh abdominal segment with three pairs of sensilla with seta (Figure 13) dorsally, five pairs of sensilla with seta laterally, and ventrally with three pairs of sensilla without seta. The anal segment with eight pairs of sensilla with seta observable. See Table 2 for a resume. Head skeleton (Figure 9): labium and labrum elongated and heavily sclerotized. Labrum fused in the front with an apical rounded prominence. Labium fused in the front with a sharp triangle pointed end. The anterior ventral end of the labium with a small rounded protuberance, and in the posterior ventral end with a serrated protuberance. A pair of heavily sclerotized mandibles in the middle of the labium and labrum. Tentorial bar and tentorial arm heavily sclerotized. The bottom half of the dorsal cornu heavily sclerotized, and the upper half slightly sclerotized. Roof of the pharynx sclerotized. A pair of heavily sclerotized triangular lateral lips located laterally of the head skeleton. Dorsal cornu not elongated. Ventral cornu slightly sclerotized and almost twice the size of the dorsal cornu. PRP (Figure 10): brown color, slightly fused in the base. Smooth surface, and no transverse ridge was visible. Spiracular plate with three pairs of straight spiracular openings on top of a carinae. Carina I well-developed, and the rest of the carinae undeveloped. Carinae not sclerotized. Spiracular opening I is the biggest, and the spiracular opening II is the smallest. One pair of small dorsal spurs. A pair of ecdysial scars between the dorsal spur and the first spiracular openings. Four pairs of inter-spiracular seta and a pair of perispiracular glands between the first and second spiracular opening. See Table 3 for a resume comparation between the larval stages.

Figure 8. Second stage larva of Eupeodes corollae: (A) dorsal view; (B) lateral view. Arrow indicates the grasping bar.

Figure 8. Second stage larva of Eupeodes corollae: (A) dorsal view; (B) lateral view. Arrow indicates the grasping bar.

Figure 9. Lateral view of the head skeleton of a second stage larva of Eupeodes corollae. Legend: Dc, dorsal cornu; Lb, labium; Lm, labrum; M, mandibles; Rp, roof of the pharynx; Ta, tentorial arm; Tb, tentorial bar; Ts triangle sclerites; Vc, ventral cornu.

Figure 9. Lateral view of the head skeleton of a second stage larva of Eupeodes corollae. Legend: Dc, dorsal cornu; Lb, labium; Lm, labrum; M, mandibles; Rp, roof of the pharynx; Ta, tentorial arm; Tb, tentorial bar; Ts triangle sclerites; Vc, ventral cornu.

Figure 10. Posterior respiratory process of a second stage larva of Eupeodes corollae: (A) dorsal view, stereomicroscope image; (B) polar view, SEM image. Legend: arrow indicates the perispiracular gland; dash circle indicates the dorsal spur; * indicates the inter-spiracular setae; Es, ecdysial scar; I, II, III, spiracular openings.

Figure 10. Posterior respiratory process of a second stage larva of Eupeodes corollae: (A) dorsal view, stereomicroscope image; (B) polar view, SEM image. Legend: arrow indicates the perispiracular gland; dash circle indicates the dorsal spur; * indicates the inter-spiracular setae; Es, ecdysial scar; I, II, III, spiracular openings.

3.2.4. Third Stage Larva (L3) (Figure 11)

Description. Length: 14.6–15.3 mm, height: 3.65–3.93 mm, and width: 3.45–3.88 mm (n = 5). Oval in the cross-section, flat ventrally, and truncated posteriorly. The prothorax with a pair of well-developed antenna-maxillary organs and a pair of light brown sclerotized ARP with two spiracular openings (Figure 12). Well-developed crochet-less locomotory organs from the 1st to the 7th abdominal segment. The anal segment with a well-developed grasping bar. One pair of lappets in the anal segment. The PRP not visible in dorsal view, slightly visible laterally, and visible ventrally. Color: light cream, dorsally with a pair of white lines in the middle from the 1st abdominal segment to the anal segment and laterally with a complete white line from the 1st abdominal segment to the anal segment. Chaetotaxy: same as the L2, except that the prothorax had 11 sensilla with seta (see Table 2). Head skeleton (Figure 14): same as the L2, except that the anterior ventral end of the labium with a rounded protuberance, and the posterior ventral end with a shark fin-shaped protuberance with a rounded tip. Dorsal cornu elongated posteriorly. The ventral cornu heavily sclerotized and almost twice the size of the dorsal cornu. PRP (Figure 15): same as the L2, except that it is fused with a small groove in the middle. Carinae highly sclerotized and well-developed. See Table 3 for a resume comparison between the larval stages.

Figure 11. Third stage larva of Eupeodes corollae: (A) dorsal view; (B) lateral view. Arrow indicates the grasping bar.

Figure 11. Third stage larva of Eupeodes corollae: (A) dorsal view; (B) lateral view. Arrow indicates the grasping bar.

Figure 12. Prothorax of a third stage larva of Eupeodes corollae. Dash circle indicates the anterior respiratory process.

Figure 12. Prothorax of a third stage larva of Eupeodes corollae. Dash circle indicates the anterior respiratory process.

Figure 13. Abdominal sensilla with seta of a third stage larva of Eupeodes corollae.

Figure 13. Abdominal sensilla with seta of a third stage larva of Eupeodes corollae.

Figure 14. Lateral view of a head skeleton of a third stage larva of Eupeodes corollae. Legend: Dc, dorsal cornu, Lb, labium; Lm, labrum; M, mandibles; Rp, roof of the pharynx; Tb, tentorial bar; Ts, triangle sclerites; Vc, ventral cornu.

Figure 14. Lateral view of a head skeleton of a third stage larva of Eupeodes corollae. Legend: Dc, dorsal cornu, Lb, labium; Lm, labrum; M, mandibles; Rp, roof of the pharynx; Tb, tentorial bar; Ts, triangle sclerites; Vc, ventral cornu.

Figure 15. Polar view of the posterior respiratory process of a third stage larva of Eupeodes corollae: (A) stereomicroscope image; (B) SEM image. Legend: arrow indicates the perispiracular gland; dash circle indicates the dorsal spurs; * indicates the inter-spiracular setae; Es, ecdysial scar; I, II, III, spiracular opening.

Figure 15. Polar view of the posterior respiratory process of a third stage larva of Eupeodes corollae: (A) stereomicroscope image; (B) SEM image. Legend: arrow indicates the perispiracular gland; dash circle indicates the dorsal spurs; * indicates the inter-spiracular setae; Es, ecdysial scar; I, II, III, spiracular opening.

3.2.5. Puparium (Figure 16)

Description. Length: 14.3–15.1 mm, height: 5.78–6 mm, and width: 6.3–6.57 mm (n = 5). Oval shape and flat ventrally with the grasping bar highly visible. The PRP can be seen laterally and ventrally. The pupal spiracles were absent. Color: dark brown. Chaetotaxy: same as the third stage larva. Head skeleton: same as the L3. PRP: dark brown. The rest of the PRP morphology was same as the third stage larva.

Figure 16. Puparium of Eupeodes corollae: (A) dorsal view; (B) lateral view. Arrow indicates the grasping bar.

Figure 16. Puparium of Eupeodes corollae: (A) dorsal view; (B) lateral view. Arrow indicates the grasping bar.

Table 3. Principal morphological characters between the larval stages.

	Head Skeleton	PRP
L1	Pair of triangular lateral lips absent. Two small protuberances in the ventral part of the labium.	Not fused. Dorsal spurs absent. Undeveloped spiracular openings with triangular spikes.
L2	Pair of sclerotized triangular lateral lips. Labium with a small protuberance anteriorly and posteriorly with a serrated protuberance.	Slightly fuse in the base. Spiracular openings well developed. Carina I well developed; the rest are undeveloped. Small dorsal spurs present.
L3	Pair of sclerotized triangular lateral lips. Labium anteriorly with a rounded protuberance and posteriorly with a shark fin shape protuberance.	PRP fused with a small groove in the middle. All carinae highly sclerotized and well-develop.

Table 3. Principal morphological characters between the larval stages.

	Head Skeleton	PRP
L1	Pair of triangular lateral lips absent. Two small protuberances in the ventral part of the labium.	Not fused. Dorsal spurs absent. Undeveloped spiracular openings with triangular spikes.
L2	Pair of sclerotized triangular lateral lips. Labium with a small protuberance anteriorly and posteriorly with a serrated protuberance.	Slightly fuse in the base. Spiracular openings well developed. Carina I well developed; the rest are undeveloped. Small dorsal spurs present.
L3	Pair of sclerotized triangular lateral lips. Labium anteriorly with a rounded protuberance and posteriorly with a shark fin shape protuberance.	PRP fused with a small groove in the middle. All carinae highly sclerotized and well-develop.

3.3. Update of the L3/Puparia Taxonomic Key of Eupeodes Species

With the re-description of E. bucculatus and E. corollae, the taxonomic key of the L3/puparia of Eupeodes [12] will be complemented as follow:

1a.

PRP spiracular plate: dorsal spurs present............................................................................2

1b.

PRP spiracular plate: dorsal spurs not present.....................................................................5

2a.

PRP spiracular plate: all carinae of similar size and pointing to the center......................

..............................................................................................................................E. bucculatus

2b.

PRP spiracular plate: carinae II smaller than the rest or carina II and III equal in size

and pointing to the center.......................................................................................................3

3a.

PRP spiracular plate: carina III almost reaching the base of the PRP (see figure 18 in

Bhatia [24]).................................................................................................................E. luniger

3b.

PRP spiracular plate: carina III slightly protrudes from the spiracular plate or reaching

half of the PRP.........................................................................................................................4

4a.

Head skeleton: labium present two sharp point and ventrally with one protuberance...

.....................................................................................................E. nuba (Wiedemann, 1830)

4b.

Head skeleton: labium with only one sharp point and ventrally with two

protuberances..........................................................................................................E. corollae

5a.

Larval integument with a translucent part...........................................................................6

5b.

Larval integument without a translucent part.....................................................................7

6a.

Larval color: dorsally with pink-reddish, ochre and dark brown fat cells........................

...........................................................................................................................E. latifasciatus

6b.

Larval color: dorsally with dark done shaped papillae that forms aggregations.............

....................................................................................................................................E. nielseni

7a.

Larval color: opaque with longitudinal white stripes [11]............................E. americanus

7b.

Larval color: peach with a fine chestnut stripe...................................................E. confrater

4. Discussion

The immature stages of E. bucculatus (egg, L3, and puparium) and E. corollae (egg and L3) were briefly described by Kumar et al. [8], Dixon [13], Dušek and Láska [14], Kuznetsov [15], and Chandler [23]. In all the descriptions of the L3 and puparium, critical morphological characteristics are missing (head skeleton or PRP) or are poorly described based on color or size. In this work, we demonstrate the importance of looking for new diagnostic characters that can be used to differentiate Eupeodes immature stages at the species level.

To our knowledge, only the eggs of E. bucculatus, E. confrater (Wiedemann, 1830), E. latifasciatus, and E. luniger (under the name Syrphus luniger) are described [8,15,23,24]. The egg size and color of E. corollae is very similar to the other eggs of Eupeodes species. The morphological characteristics that can be used to distinguished it from the eggs of other Eupeodes species is the chorionic structure. However, only E. luniger has an accurate description of the chorionic structure [23,24], so we base our comparisons mainly on this species. Chandler [23] and Bhatia [24] mentioned that the units of the chorionic structure of E. luniger have a surface with crests and the branches touch the other units. In the case of E. corollae, the surface is porous, and the branches touch other units. When it is compared to other aphidophagous species, such as Episyrphus balteatus (De Geer, 1776) or Scaeva pyrastri (Linnaeus, 1758), a difference in the form and surface of the chorionic structure can be observed. While in E. corollae the chorionic structure is slim and porous, in E. balteatus and S. pyrastri, the structure is thick and smooth [23].

Regarding the L1 stage, the only available description within the genus Eupeodes is of E. luniger [24], which is very similar to that provided here. In both larvae, it can be seen the presence of long seta and the PRP is not fused. The principal difference between these two species is observed in the head skeleton, since it can be seen in E. luniger that the dorsal cornu is elongated and with a sharp end, while, in E. corollae, it is not elongated, and it has a rounded end. Another difference that can be observed is the relationship between the length of the ventral cornu in comparison with the dorsal cornu. In E. luniger, the dorsal cornu and ventral cornu are almost the same size, while, in E. corollae, the ventral cornu is almost twice the size as the dorsal cornu. The absence of the pair of triangular lateral lips is normal in this stage, as has been previously observed in other L1 aphidophagous, such as E. balteatus [25].

Regarding the L2, a clear difference can be observed in the PRP, since Bhatia [24] mentioned that the PRP of E. luniger is not fused in the base yet, whereas, in E. corollae, it was found to be slightly fused in the base. In the head skeleton, several differences can be highlighted. According to Bhatia [24], the anterior ventral part of the labium of the L2 of E. luniger does not present a protuberance, while, in its posterior ventral part, it presents a cone-shaped protuberance (see figure in Bhatia [24]). In our work, we observed two evident protuberances, one in the anterior ventral part of the labium and the other on the posterior ventral part of the labium. Another difference is the form of the dorsal cornu, since, in E. luniger, it has a pointed end, while E. corollae has a rounded end. It can be confirmed that the L2 stage marks the initiation of the triangular sclerite development, as has been observed in E. balteatus, E. corollae, and E. luniger [24,25].

The PRP is the most importance morphological characteristic to distinguish E. bucculatus and E. corollae from other L3/puparium of Eupeodes species. In both E. bucculatus and E. corollae the presence of dorsal spurs in the spiracular plate can be seen, as also occurs in E. luniger and E. nuba [12,24], while, in Eupeodes nielseni (Dušek & Láska, 1976) (under the name Metasyrphus nielseni), dorsal spurs are not present [26]. One of the possible reasons that this characteristic has been observed only in some of the species may be due the small size of the dorsal spurs. In our work, we illustrate the tiny size of such dorsal spurs in E. corollae and suggests that the spurs are a good diagnostic trait, likely present in other species but not yet studied or still not properly described with modern approaches. According to Lillo et al. [27], the immature stages of Eupeodes have a well-developed pair of dorsal spurs, with this being a characteristic to distinguish it from Lapposyrphus Dušek & Láska, 1967. However, as we mentioned before, the pair of dorsal spurs in E. corollae were very small, meaning that this characteristic cannot be used to differentiate immature stages between Eupeodes and Lapposyrphus.

According to Rotheray [26], the spiracular plate of E. nielseni is fused in the center (see figure 12 in Rotheray [26]), while, in the other immature stages of Eupeodes, it is not fused. In E. luniger, the spiracular opening III is the largest, almost reaching the base of the PRP, while the spiracular opening II is the smallest [24], while in E. bucculatus, E. corollae, E. nielseni, and E. nuba the spiracular openings does not reach the base. In E. bucculatus, all the spiracular openings are similar in size, and, in E. corollae, the first spiracular opening is the biggest, and the second is the smallest. In E. nielseni, the first spiracular opening is the smallest one [26], and, in E. nuba, they decreased in size, with the first one being the biggest, and the third one being the smallest [12]. In Orengo-Green et al. [12], it is mentioned that the size and direction of the carinae are the most important morphological characters, and we concluded that this assumption is correct. Since some species shared the size or the directions, these morphological characteristics must be taken as a whole and not individually.

In the head skeleton, the principal difference can be observed in the labium, since, in E. nuba, it presents two sharp points, while, in E. bucculatus and E. corollae, only one sharp point is present. Another difference is with the protuberance that is present in the ventral part of the labium. In E. bucculatus and E. corollae, two protuberances can be observed, while, in E. nuba, only one can be observed. The absence of pupal spiracles in the puparium stage of Eupeodes species is in line with Rotheray [22] mentioning that the Syrphinae does not develop them.

As we mentioned before, the egg, L3, and puparium of E. bucculatus were described briefly by Kumar et al. [8] and Dixon [13] under the name Syrphus latinulatus. Comparisons between the L3 and puparium were conducted, since the puparium always conserves many morphological characters of the L3 [28]. Both descriptions focused on the surface of the L3/puparium and on the PRP. When we compared the surface description of Dixon [13] with our description, we noticed a match in the color and in the presence of sensilla with seta. However, when we observed the description of Kumar et al. [8], our description does not match in the color, since he mentioned that it turns dark brown until emergence, and, in our case, it remained light brown throughout the process. In addition, Kumar et al. [8] did not mention the light cream spots on the ventral side. For the PRP description, Dixon [13] mentioned that it presents a noticeable transverse ridge, with three pairs of spiracular openings in the top of a carinae and the presence of dorsal spurs. All these characters were observed in our work. However, the description of Kumar et al. [8] does not match with Dixon [13] or with our work, since he mentioned that the carinae are located in the dorsal and ventral side of the spiracular plate and of different sizes (see figure 26 in Kumar et al. [8]). In contrast, in our work, we found that all carinae had similar sizes and were only located dorsally of the spiracular plate. Based on such differences, we conclude that the description of Kumar et al. [8] does not match that of E. bucculatus but of other species of Eupeodes.

A description of the egg and L3 of E. corollae has been made by Dixon [13], Dušek and Láska [14], Kuznetsov [15], and Chandler [23]. The egg description of Kuznetsov [15] is mainly based on size and color, and it matches the description we report here. We used Chandler’s [23] description as the comparison description, who described that chorionic structures have a crested surface without branches (see figure 3c in Chandler [23]), but we saw that it has a porous surface and branches that touch other units. One possible explanation lies in the use of differential technical approaches, as Chandler [23] only used a stereomicroscope, whereas we explored further fine details through SEM. Regarding the L3/puparium, a brief but very similar description was reported by Dixon [14] and Dušek and Láska [14], since both mentioned that the spiracular openings II and III are almost parallel, although Dixon [13] went further and mentioned the existence of a prominent pair of dorsal spurs. Our work matches with the previous descriptions conducted; however, we demonstrate that dorsal spurs were small and difficult to observe.

This study highlights how the implementation of modern microscopy techniques provides highly accurate and complementary diagnostic characteristics to former descriptions and, ultimately, determines the validity of the original descriptions. Further studies are needed since the majority of the immature stages of Eupeodes species still remain unknown, and, among the known species, many of them are briefly described, or the life-stages remain incomplete. This study makes a great contribution to the identification of the diagnostic characteristics of the genus Eupeodes, so it can be distinguished from other syrphid larvae. To achieve these results, in-depth field surveys are required to locate Eupeodes larvae from species with hitherto unknown immature stages.