Glyphidops flavifrons was reared in situ, between the months of March and May, 2014 on the Melendez campus of the University of Valle located in Santiago de Cali, Colombia (3°22.448'N; 76°32.084'W; 987 masl) found in the tropical dry forest life zone sensuHoldridge (1967).

The study area was composed of C. papaya trees and Tradescantia zebrina, with vegetation coverage varying between 58% and 70%.

For the breeding process, fresh C. papaya stems were cut into 30 cm long pieces and placed at the study site in a plastic container to protect them from other organism during decomposition (2–3 days). Afterwards the stems were exposed to adults of G. (O.) flavifrons population (previously identified) for four hours. The time of oviposition was recorded. The egg masses were individualized by one-ounce plastic containers with fragments of C. papaya of 8 mm, each container was labeled and covered with fine mesh to allow ventilation and prevent intrusion by other invertebrates. To prevent injury of the eggs, the number of eggs per egg mass was recorded after the maturation of it (24 hours later) (Olsen and Rickman 1963, Craig 1967). After hatching, the larvae were observed daily. The puparia were individualized in plastic containers containing a layer of sifted and sterilized soil.

For the developmental rate assessment, 30 eggs were separated and observed every four hours until hatching. After hatching, ten larvae were sacrificed, following the method proposed by Adams and Hall (2003). Ten larvae were sacrificed daily until pupation occurred. An observation of 15 puparia was made every 12 hours, until emergence.

Humidity and temperature data were compiled daily, at 15 minute intervals with the help of a Dickson Data Logger TP125.

Egg

Twenty-six eggs were set on hollow plates with distilled water. Polar diameter and respiratory filament length were measured. Description follows the terminology used by Olsen and Rickman (1963).

Larva

To ensure accuracy, larval body length was measured immediately after sacrifice (Adams and Hall 2003). Micro-preparation of cuticular surface and cephalopharyngeal skeleton was performed following the methodology suggested by Niederegger et al. (2011). Body length (lateral view), hypopharyngeal sclerite length, and mandibular area (mandibular sclerite + mouth hook) measurements were performed daily. Additionally, antennal variations, maxillary palp, antenomaxillary lobe, and spinulose areas were observed. Description follows the terminology used by Foote and Teskey (1991).

Puparium

Total length of puparium was measured and morphology of both anterior spiracles (prothoracic spiracle) and thorny areas were examined. To determine duration of pupariation, photographic records were performed every 15 minutes for 150 minutes after pupation initiated (Bunchu et al. 2012).

Measurements of egg, larva, and puparium were performed using tpsDig2, version 2.22 (Rohlf 2007).

Photographic records were performed using a Canon EOS Rebel T3i camera, adapted to a Nikon Eclipse E200 microscope. Photographic compilation was done using Helicon Focus software. Diagrams were constructed with Corel DRAW program.

Larval instars were determined by using a one-way ANOVA (confidence level: 95%). Post-ANOVA (Tukey comparisons) was used to calculate the variation of hypopharyngeal sclerite length and mandibular area throughout the observation period. Moreover, a cluster analysis was performed for each measurement (including body length) using Euclidean measures and neighbor-joining as a linkage method for each cluster.

Additionally, scatter plots were graphed for each of the measured variables to monitor their distribution over time. Box-plot graphs were used to compare distribution between the variables for each different larval instar (Ln). Figures and analyses were performed using Microsoft Excel 2013 and Minitab16 software.

Development time of Glyphidops (O.) flavifrons was determined under natural conditions, temperature mean 25.8 °C (maxim. 41.06 °C, minim. 18.6 °C) and relative humidity mean 69.38% (maxim. 82.9%, minim. 44.9%). The eggs hatched 58 ± 4 hours (n = 30) after being laid. The total larval development time was 10 ± 1 days (n = 15) and the puparium stage had a development time of 13 ± 2 days (n = 15).

ANOVA showed significant differences for values of mandibular area versus time (df = 9.40; F = 1829.61 and P = 0.000) and the hypopharyngeal sclerite length versus time (df = 9.40; F = 7870.85 and P = 0.000). Post-ANOVA of mandibular area showed four groups, and post-ANOVA of hypopharyngeal sclerite length showed three groups, thus confirming three distinct larval instars L₁, L₂ and L₃. Life spans of all larval instars are summarized in Table 1.

Scatter plots (Fig. 1a and c) show two distinct jumps in growth for the observed structures: the first between day 2 and 3 and the second between day 4 and 5. Furthermore, box-plot graphs (Fig. 1b and d) graphically support the findings from the Tukey test, by illustrating the variation in each structure’s measurements.

Figure 1. 

Scatterplot and Box-Plot: a, b mandibular area c, d hypopharyngeal sclerite length.

Egg

Body length 1–1.24 mm (x = 1.16, n = 20); respiratory filament length 3.13–4.01 mm (x = 3.3; n = 20). Body semi-cylindrical, dorsally convex and dorso-ventrally flattened, with a blunt posterior region. Respiratory filament originates from the anterior region, as long as 3.21 times the egg body length (Fig. 2). Two longitudinally lateral hatching lines departing from the respiratory filament attachment point and dim gradually towards the 1/6th posterior region of the egg body. Chorion sculpted with cells varying from pentagonal to octagonal (Fig. 2b), forming a mesh-like pattern that is more conspicuous in the posterior 1/6th. Anterior quarter region of the egg body with a small elliptical tubercle band visible with 10× magnification (Fig. 2c).

Figure 2. 

Egg in latero-dorsal view. Details a posterior end b average area of the egg, note the hexagonal pattern of the corium c basal respiratory filament point, note the tubercles over the apical area of the egg body d apex of the respiratory filament. Scale bar: 1 mm.

Larva

Vermiform body, glabrous, light to semitransparent (Fig. 3a–c). Head: (Fig. 4a–c) retractable, with two pairs of appendages located antero-dorsally; maxillary palpus bulbous, with an apical sensory depression, containing four to five tubercules with bristles over them; antenna reduced, with two to three antennomeres; a pair of ventrally curved mouth hooks, each originating from a mandibular sclerite (Fig. 6a–c.); antenomaxillary strongly lobed: lobes with 28–30 oral or pseudo-tracheal bridges, each radiating from the labial lobe; epipharyngeal sclerite (Fig. 4a) U-shaped with projections joining ventro-posteriorly to the back margin of labial sclerite; labial sclerite (Fig. 4b and c), arrow-shaped, directed antero-ventrally; Thorax: Pro-, meso-, and metathorax well-defined. Hypopharyngeal sclerite H-shaped, as long as 5.5 times its width (lateral view), formed by two parallel bars connected by a strong bridge originated in the anterior half of the sclerite, “bridge” concave, forming a canal that links antero-dorsally with epipharyngeal sclerite. Tentoropharyngeal sclerite from before the anterior half of the prothorax to almost the previous anterior half of metathorax, with two pairs of parastomal bars extending dorsally and ventrally along the hypopharyngeal sclerite, dorsal pair as long as 0.8 times the length of hypopharyngeal sclerite and ventral pair as long as 0.3 times the length of hypopharyngeal sclerite; dorsal bridge dorsally dark, extending anteriorly subsequently ¼ of hypopharyngeal sclerite; ocular spherical depression conspicuously located below the dorsal bridge; ventral cornu fused to the pharynx, forming a cavity that connects posteriorly with the esophagus and anteriorly with the cibarium; dorsal cornu 0.3 times shorter than the ventral cornu. Anterior spiracles, located dorsally in the latero-posterior half of pro-thorax, palmiform (Fig. 6a and b) variable across instars. Abdomen eight segmented, each (except I and VIII) in ventral view with two spinulose areas, anterior area with three transverse rows of spines, first anteriorly directed and second and third posteriorly directed, posterior area with only one transverse row of spines anteriorly directed; abdominal segment I, with two posteriorly directed anterior ventral rows, first with 36 (± 1) small spinules and second with 35 (± 2) papillae as spinules (Fig. 4c1), posterior row with 28 (± 2) spines directed above; abdominal segment VIII with two spinulose areas, anterior area with three transverse arranged rows of spines, first row with 34 (± 3) spines, anteriorly directed and the following two rows with 29 (± 2) and 43 (± 2) spines, posteriorly directed, posterior spinulose area with three transverse rows, the first continuous and the following two discontinuous: first row with 27 (± 4) spines, anteriorly directed and discontinuous rows with 8 (± 2) spines on each side, posteriorly directed (Fig. 3c3). Ventro-posterior rounded anal plate with a longitudinal slit in the middle; two dorso-posterior spiracular plates, located on abdominal protrusions, each with four processes as fractals (branchy structure) and spiracular openings that vary in number and shape on each instar (Fig. 6c and d).

Figure 3. 

Larvae and puparium in lateral view and approach to the third larval instar spinulose areas. a L₁, b L₂ and c L₃d Puparium. (1) Spinulose area of the first abdominal segment, (2) posterior row of spines on abdominal segment I and anterior spinulose area on abdominal segment II, (3) posterior row of spines on segment VII and spinulose areas on abdominal segment VIII. an. pl., anal plate; p sp, posterior spiracles; Met. metathorax. Scale bar: 2 mm.

Figure 4. 

Cephalopharyngeal skeleton. a dorsal view b side view c ventral view. ant, antenna.; ante sp, anterior spiracle; d brg., dorsal bridge; d corn, dorsal cornu; epiphr scl, epipharyngeal sclerite; esph, esophagus; hypophr scl, hypopharyngeal sclerite; lab lb, labial lobe; lab scl, labial sclerite; mand, mandibule; md th, mandibular tooth; mx plp, maxilar palp; pastm b, parastomal bar; phr, pharynx; ocl dp, ocular depression; tnt phgm, tentorial phragm; v corn, ventral corn. Scale bar: 3 mm.

Figure 5. 

Mandibles of each larval instar. a L₁b L₂ and c L₃. dnt grv, dental groove; mand scl, mandibular sclerite; mthhook, mouthhook; 2nd-dent., secondary dentition. Scale bar: 1.5 mm.

Figure 6. 

Spiracles of each larval instar. Anterior spiracle on L₂ (a, e) and L₃ (b, f). Posterior spiral on L₁-L₂ (c, g) and L₃ (d, h). Scale bar of drawings 0.15 mm. ecdys sc, ecdysial scar; spr dg, spiracular digit; spr op, spiracular opening; spr prc, spiracular process; spr trch, spiracular trachea; rm, rime.

Larval instar

L₁. From 1.22 to 2.52 mm (x = 1.76, n = 18) in length; antenna bi-segmented, apical segment oval; mandibular sclerite slightly sclerotized, elongated, three times longer than wide, dorsally articulated with the mouth hook, the latter with marked sclerotic outside and with 7–8 ventral teeth (Fig. 5a); anterior spiracles not observed (under light microscope); posterior spiracles with two semicircular spiracular openings (Fig. 6c) and four spiracular processes: two of them closely associated with spiracular openings, the other two free.

L₂. From 2.95 to 6.2 mm (x = 4.36; n = 22) in length; antenna with two antennomeres, distally oval; mandibular sclerite as long as 2.5 times wider, differentially sclerotized, antero-dorsally fused with mouth hook (Fig. 5b); ventral margin of mouth hook with 10 teeth; anterior spiracle 2.5 times longer than its greatest width, apical third visible as a small stump with 8–9 digital radiation and light interdigital recesses (Fig. 7e.); posterior spiracle as L₁.

Figure 7. 

External coloration of G. (O.) flavifrons puparium, up to 3 hours after beginning cuticular exclerotization. From right to left: top row 0, 15, 30, 45, 60; bottom row 75, 90, 105, 135, and 180 minutes after beginning pupation. Scale bar: 1 mm.

L₃. From 6 to 11.16 mm (x = 9.17; n = 51) in length; tri-segmented antenna, second antennomere truncated, with sclerotized apical border, third antennomere reduced 0.3 times length of second like a papilla; mandible uniformly sclerotized, mandibular sclerite completely fused to mouth hook, reddish-brown, without ventral teeth mouth hook, showing a slight groove in basal ⅓ (Fig. 6c.); anterior spiracle 0.8 times as long as its greatest width, with 9–11 digits (Fig. 7b.), each on average 1.7 times longer than its greatest width, fully everted completely inside out, ecdysial conspicuous scar, mesal to digital projections (Fig. 7d); posterior spiracles with defined ecdysial scar, four spiracular openings elongated, mitochondrial-shaped (Fig. 6d), arranged semi-circularly to the scar, each opening with a spiracular process associated medially to the outer margin, each spiracular process radiates in a filamentous way to a small sclerotized point.

Puparium

Coarctate; 5.48–7.49 mm (x = 5.93; n = 15) in length, reddish brown (Fig. 3d and Fig. 7) with transverse striations or wrinkles that are more prominent on segments VII and VIII; anterior spiracles situated frontally (side view), with 9–11 sclerotized digits; posterior spiracles with four spiracular openings and a poorly defined spiracular process.

Biology

The body of each egg is buried in the substrate and the filament is spread over the surface. As the female began laying eggs (reaching packages up to 20 eggs, one egg at a time), it was observed that numerous filaments were emerging and radiating from the same point. Along this process, the male remains close to the female and the mating happen continuously between laid eggs. See Suppl. material 2.

Just moments before the hatching, the larva is observed moving the body and the head, rubbing its mandibles against the inner wall of the egg and finally thrusting the corium. The larva emerges from the anterior part of the egg by using one of the two longitudinally lateral hatching lines. See Suppl. material 3.

The pupation took between 150 and 175 minutes (n = 15) (Fig. 7a–k). The emergence of the imago was passed through a circular suture that ran from the anterior spiracles halfway to the first abdominal segment, separating two plates, one ventral and one dorsal, where the latter was completely separated from the puparium.

Table 2 shows the main features found when comparing pre-imaginal stages described to date.