General morphology
The larval body of
Wohlfahrtia nuba exhibits a vermiform morphology, featuring a tapered front and a wide posterior (Fig 1a). The anterior region of the larval body houses pseudocephalon, comprising two cephalic lobes. Following the anterior region are three thorax segments, seven abdomen segments and a caudal segment containing the posterior spiracles. Larval stages possess a three-dimensional cephalopharyngeal skeleton, necessitating the capture of two microscopic images: one before the coverslip and another after compression (Fig 1b, c, d). The anterior segment of the thorax features two broad anterior spiracles. The first and second larval instars bear morphological similarities to the third larval instar, irrespective of anterior and posterior spiracles, as well as variations in size and the degree of sclerotization.
First larval instar
The length of the first instar larvae, including the cephalo-pharyngeal skeleton, was measured to be 4.95±0.054 mm, with a width of 1.21 ± 0.038 mm (n = 20). The length of the cephalopharyngeal skeleton was 1.63±0.013 mm (n = 20). The weight of the first instar larvae was the lowest, measuring 12.18±0.248 mg (n = 10) (Table 1). The pseudocephalon displays a bilateral structure consisting of two cephalic lobes. Each cephalic lobe has a ventral maxillary palpus, a dorsal antennal complex and oral ridges in the middle on its anterior surface. A short antennal dome and a basal ring constitute the antennal complex. The oral ridges extend laterally to the ventral side of the pseudocephalon adjacent to the mouth aperture, forming a facial mask surrounding the mouth opening (Fig 1b).
The cephalopharyngeal skeleton has a pair of large, symmetrical and heavily sclerotized mouth hooks. Each mouth hook is connected to a basal dental sclerite with an anteriorly projecting chitinous process. These mouth hooks are highly curved with a pointed end directed ventrally, resembling a talon. The absence of the large median hook is observed (Fig 1b, c, d). A pair of anterior spiracles are on either side of the spiny band of the prothorax (Fig 1c). The anterior spiracles have eight papillae arranged in a row, forming an arch-like shape (Fig 1d). The anterior bands of posteriorly directed spines, form a complete circle on the dorsal side and are interrupted laterally along the margins of all abdominal segments (Fig 2c, d). Both the anterior and posterior bands feature elongated, pointed spines. The anal pads are prominently extended (Fig 1a, 2a). The posterior spiracular plates are more distantly spaced, with the peritreme lacking development on the ventral side. They feature a single aperture split in their upper portion and are shielded by thin, hair-like structures (Fig 2b).
Second larval instar
The second larval instar had a length of 17.83±0.049 mm, including the cephalopharyngeal skeleton and a width of 3.19±0.028 mm (n = 20). The second instar larvae showed a significant weight gain, increasing to more than six times the weight of the first instar larvae, reaching 84.87±0.396 mg (n = 10), which was highly significant (P<0.05). The length of the cephalopharyngeal skeleton increased by 1.82± 0.037 mm (n = 20) (Table 1).
The pseudocephalon in the second larval stage closely resembles that in the first stage. The only things that make them different are their level of sclerotization and size. The pair of mouth hooks exhibit increased sclerotization and pronounced tapering, resembling talons (Fig 3a, b and 4a). The mouth hooks are attached to the more extensive dental sclerite, which is hooked in shape (Fig 4a-c). The parastomal sclerite exhibits increased distinctness and elongation (Fig 4b). Additionally, the labial sclerite exhibits increased prominence and lateral elongation (Fig 5b). No morphological differences are observed in the dorsal arch between the first and second larval instars (Fig 1c, d, 4b).
The lateral view of the cephalopharyngeal skeleton shows the parastomal sclerite’s connection to the intermediate sclerite. (Fig 4b, c). The width of the vertical plate is equal to one-third of the ventral cornua. The widths of the dorsal and ventral cornuae are identical and conical. However, the dorsal cornua is longer than the ventral cornua and has a posterior bifurcation (Fig 4a). The hypostomal sclerite is small (Fig 4b, c). The dorsal arch is short and curved. It does not extend to the parastomal sclerite (Fig 4c). The ventral view of the cephalopharyngeal skeleton reveals H-shaped intermediate sclerites, short parastomal sclerites and the labial sclerites. The vertical plate is heavily sclerotized (Fig 5a, b).
No morphological differences exist between the thoracic and abdominal segments of the first and second larval instars, except for size variations. A pair of more prominent anterior spiracles exhibits a rise in papillae, with approximately 10 organised in a row, forming an arch-like structure (Fig 4a, 5c). The anterior bands of spines that point backwards form a continuous ring around the edges of all thoracic segments (Fig 5c, d). Each abdominal segment possesses a pair of ventral creeping pads covered with spines (Fig 3c). Figures 3c and e illustrate the eighth abdominal segment, the caudal segment. The caudal segment of the second larval instar looks a lot like the first, but the posterior spiracles are more developed and sclerotized. Each posterior spiracle contains two internal slits within a peritreme. The peritreme lacks a ventral arch that is sclerotized. The button exhibits invisibility (Fig 3e, f).
Third larval instar
Sclerotization and size are the only morphological distinctions between the third and second instars. The length of the third larval instar, including the cephalopharyngeal skeleton, significantly increased (P<0.05) to 24.32±0.281 mm, with a width of 5.45 0.117 mm (n = 20). As shown in Table 1, there was a significant rise in weight, approximately 15 times greater than that of the first instar larvae, reaching 182.15±0.64 mg (n = 10), with a high degree of statistical significance (P<0.05). The length of the cephalopharyngeal skeleton was 2.51±0.041 mm (n = 20).
The pseudocephalon and cephalopharyngeal skeleton exhibit morphological similarities to those observed in the second larval stage, as depicted in Fig 6a-d, except for size and additional differentiation. The two mouth-hooks exhibit greater robustness, apical tapering, heavy sclerotization, increased thickness and more remarkable similarity. The dental sclerite is larger and hook-shaped than the one in the second larval instar. The parastomal sclerite exhibits greater prominence compared to the second larval instar. In the antennal complex, it was observed that the diameter and height of the basal ring are more significant than that of the antennal dome (Fig 7a, b). A center cluster of sensilla, comprising two basiconic and three coeloconic sensilla, forms the maxillary palpus. These sensilla are surrounded by several cuticular folds (Fig 7c, d). The oral ridges extend laterally to the ventral side of the pseudocephalon adjacent to the mouth aperture, forming a facial mask that surrounds the mouth opening (Fig 6b and 7b). Additionally, the mouth opening has a labial lobe in the middle, with labial organs on both sides (Fig 8a, b). The absence of the large median hook is observed. The dental sclerite is of considerable size and fused to the mouth hook’s base. Labrum is a reduced structure exhibiting four highly sclerotized teeth between the mouth hooks (Fig 8c). The cephalic spinose band, which consists of spines directed posteriorly, differentiates the cephalic region from the first segment of the thorax. The spines on the thoracic and abdominal segments exhibit a similar arrangement and pattern as observed in the preceding larval instars. The spines of the thoracic spinose band exhibit both single and double tips in their arrangement. The spines exhibit a tapering shape at the apex and a broad shape at the base. The spines are dorsally arranged in 7-9 rows and ventrally arranged in 9-10 rows (Fig 9a-c).
Similar to the first and second larval stages, each thoracic segment contains laterally a pair of anterior spiracles and Keilin’s organ ventrally (Fig 6a). These segments are bordered anteriorly by spiny bands with uniform, posterior-facing spines (Fig 6a). Additionally, each spiny band is accompanied by thoracic papillae on both sides (Fig 10a-d). An anterior spiracle pair of eleven papillae arranged in a row resembling an arch (Fig 9d). A pair of spiny creeping pads is present on the ventral side of each abdominal segment (Fig 10c).
Fig 11a, b and c show the caudal segment with a pair of large, strongly protruding pads called anal pads, along with posterior spiracles that are fully developed. Twelve cuticular papillae encircle the spiracular cavity. The cuticular papillae are categorized into three pairs on the dorsal and dorsolateral regions and three on the lateral and ventral regions (Fig 11e). The interband areas of larval body segments lack spines but exhibit small granular structures called warts. The spines within the spiny bands exhibit apical tapering and share a consistent outline. The posterior spiracle consists of three dorsoventrally oriented slits enclosed by an incomplete peritreme. The slits are slightly curved. The button is absent (Fig 11d, e and 12a-c).
Prepupae
The morphology of prepupae closely resembles that of third- instar larvae; however, minor morphological differences differentiate these stages. The posterior spiracle exhibits internalization while maintaining three spiracular slits, indicating an early adaptation for the prepupal stage. The observable spiracular retraction is a key distinction during puparium formation, indicating preparation for pupa formation. As prepupae transition, the spiracles retract and become less prominent compared to the third-instar larvae, indicating a significant morphological change. Additionally, external hardening occurs during puparium formation. The puparium length, including the cephalopharyngeal skeleton, significantly decreased (P<0.05) to 19.48±0.417 mm (n = 20), while the width significantly increased to 6.286±0.053 mm (n = 20). There was a significant reduction in average weight (P<0.05) to 123.74±0.443 mg (n = 10). The length of the cephalopharyngeal skeleton decreased by 1.94±0.019 mm (n = 20).
Larval stages have two large mouth hooks that are strongly curved, resembling talons and aligned in parallel. It is worth noting that the medium hook is absent. The mouth hook data presented in this study are consistent with the findings reported by several previous researchers
(Sukontason et al., 2010; Szpila, 2010;
Szpila et al., 2015; Ubero-Pascal et al., 2015). The cephalopharyngeal skeletons of
W. nuba, distinguished by the presence of two mouth hooks and the absence of a median mouth hook, differ from those of
Wohlfahrtia vigil, which possesses a single median mouth hook.
Wohlfahrtia pattoni and
Wohlfahrtia magnifica exhibit a median hook, as documented by
Hilton, (1973) and
Szpila et al., (2014).
The vertical plate is highly sclerotized and measures one-third the length of the ventral cornua. In comparison to the ventral cornuam the dorsal cornua is longer and bifurcated posteriorly.
Lopes and de Albuquerque, (1982) reported the presence of hooked spines on the anterior margin of
Cochliomyia macellaria and
Cochliomyia hominivorax.
Sukontason et al., (2005) presented a photograph of the cephalopharyngeal skeleton that closely resembles the one depicted.
Hilton, (1973) reported the larval cephaloskeleton of
W. pattoni, which is very similar to what is described for
W. nuba. However, the difference observed is shorter length of the ventral cornua in
W. nuba across all instars. The first larval stage exhibits a diminutive hypostomal sclerite, whereas
Wohlfahrtia opaca lacks a hypostomal sclerite entirely (
James and Gassner, 1947).
Ishijima, (1967) highlighted the dorsal cornua as a distinctive characteristic of the Sarcophagidae family. In contrast, a study by
Szpila and Pape, (2007) found that the dorsal cornua is not a reliable characteristic for distinguishing the family Sarcophagidae.
The examinations suggest a correlation between the level of sclerotization of cephalopharyngeal skeleton sclerites and the age of the larvae. It appears that this correlation becomes more pronounced for the mouth hooks, which taper at the tip and lose some of their extreme concavity. Consequently, this observation indicates that each hook becomes thicker overall as the larvae age. The parastomal sclerites exhibit distinct elongation and different-iation. These results are in line with those of
Vairo et al., (2015), who studied
Peckia (Sarcodexia) lambens and
Lopes and de Albuquerque, (1982), who used cephalopharyngeal skeleton configurations to classify Sarcophagidae.
The labrum is a reduced structure exhibiting four highly sclerotized teeth between the two mouth hooks. This supports the findings of previous research by
Lopes and de Albuquerque, (1982) and
YuG and Dolin, (1988), who did not classify the labrum as a third-mouth hook. The researchers classified it as a prominent labrum with a distinct hook shape, which they employed as a distinguishing characteristic for the subfamily Paramacronychiinae. Although our findings differ from previous reports, it is essential to note the contrasting conclusions reached by
Schumann, (1976),
Valentyuk, (1971),
Lehrer and Fromunda, (1986) and more recently by
Szpila et al., (2014) regarding the labrum in
W. magnifica. The labrum has been proposed to function as an additional mouth hook by
Ruiz-Martinez et al., (1989) and 1990.
Zumpt, (1965) described the mouth hooks of the first larval instar of
Chrysomya bezziana as toothed.
Szpila et al., (2014) described the cephalo pharyngeal skeleton of
C. bezziana, revealing that the labrum is a tiny structure hidden between two mouth hooks and the tips do not have highly sclerotized teeth.
The pseudocephalon of
W. magnifica larvae, as described by
Szpila et al., (2014), is strikingly similar to the one mentioned in this article. The only difference is that the antennal dome is shorter than the basal circle’s diameter and height. The findings of
Vairo et al., (2015) align with our observations for
W. nuba.
Lopes and de Albuquerque, (1982) employed the term “pseudocephalon” for broad taxonomic categorization, whereas
Kirk-Spriggs, (2003) regarded it as an interspecific anatomical feature.
The number of anterior spiracular papillae shows variability across the developmental stages observed in
W. nuba. Each anterior spiracle typically consists of eight papillae in the first larval instar. This number increases to 10 papillae in the second larval instar. By the third larval instar, the spiracular papillae arrangement typically comprises 11 aligned rows resembling an arch. The findings presented in this study provide a different perspective than the observations made by
Hilton, (1973) regarding the number of papillae in the larval instars of
W. pattoni and
W. vigil. Hilton reported that these species possess 8–10 papillae in the second and third larval instars while lacking papillae in the first instar. Moreover,
Szpila et al., (2015) reported that the first larval stage of
W. nuba displays anterior spiracles with more than seven papillae, which aligns with the findings of
Leite and Guevara, (1993) regarding
C. hominivorax.
Mazza and Jörg, (1939) reported that the anterior spiracle of
C. hominivorax larvae exhibit a range of seven to ten papillae in the second instar and six to eleven papillae in the third instar.
The posterior spiracles’ morphology, peritreme shape and internal spiracular slit are frequently employed as family-specific characteristics
(Sukontason et al., 2010; Velásquez et al., 2010;
Ubero-Pascal et al., 2015). The findings presented in this study align with the results reported by
Hilton, (1973) regarding
W. pattoni. According to
Velásquez et al., (2010), the posterior spiracle slits in
Sarcophaga africa are straight, while in other flesh fly larvae, they are curved. Based on the taxonomic classification provided by
Ishijima, 1967 for
W. nuba larvae, the absence of a sclerotized ventral arch is a distinguishing feature of the posterior spiracles peritreme. According to
Richet et al., (2011), the larvae of
Sarcophaga caerulescens exhibit a well-sclerotized ventral arch. The arrangement and number of papillae encircling the spiracular cavity are frequently employed in larval taxonomy (
Szpila, 2010). Within
W. nuba, 12 cuticular papillae surround the spiracular cavity. The arrangement of the cuticular papillae follows a pattern observed by
Kano et al., (1951) and
Sanjean, (1957). There are six pairs: three on the dorsolateral and dorsal sides and three on the ventrolateral and ventral sides.
