Scanning electron morphological studies of Tribolium confusum Jacquelin du Val (Coleopteran: Tenebrionidae)

Background: The confused flour beetle Tribolium confusum Jacquelin du Val (Coleopteran: Tenebrionidae) is the most destructive pest of stored products worldwide. It is the most common pest of wheat flour. Results: This study describes and illustrates the larvae, pupae, and adults of T. confusum using scanning electron microscopy. The first larval instars are 5.0–5.1 mm long and 0.5–0.6 mm wide whereas the last larval instars are 5. 75–6.9 mm long and 0.75–0.95 mm wide. Adults of T. confusum are reddish brown elongate beetles (4.0–4.5 mm in body length and 1.0–1.2 mm in width). Electron micrographs revealed the structure of the mouth parts during the larval, pupal, and adult stages as well as the structure of thoracic and abdominal appendages. Results indicated that the setiferous sex patches which were reported in males can often be used for sexing specimens. A specific feature of the first instar larvae of T. confusum is the extreme shortened antenna with a reduced number of antennomeres and the presence of well-developed and moderately long legs. Conclusion: SEM examination may help us not only discover and understand new morphological details as the pits with spine on the elytra and the spikes on the membrane wings which will facilitate the identification of this species but also clarify the functions of various body parts.


Background
Tenebrionidae is one of the most diverse families within Coleoptera (beetles) and is very difficult to classify (Aalbu, Triplehorn, Campbell, Brown, Somerby, et al., 2002).
Studies of immature and adult stages of insects are very important and useful for classification of particular groups which were seeing several times (Beutel & Friedrich, 2005).
The red flour beetle, Tribolium confusum (Coleoptera: Tenebrionidae), is the most widespread and withering major insect pest of stored cereals in the world. Hana (2013) had reported the percentage of damages of these insects ranging from 5 to 30% of the world's total agriculture production. Freeman (1973) had reported that the success of the spreading of these insects is due to different evolutionary adaptations to the actions of humans who transport them through the world and give a protective habitat for stored food stuffs.
The aim of the present work was to study the morphology of different stages of T. confusum to explore new morphological details which will facilitate the identification of different stages of this species.

Methods
All experiments were done according to the guidance and animal care ethics of Sohag University, Egypt. Larvae, pupae, and adults of T. confusum were collected from the flour in the laboratory and fixed in 70% alcohol. Pupae were examined using binocular microscope to differentiate between male and female pupae. Specimens were prepared for examination by subjecting them to dehydration series of 90%, 95%, and absolute alcohol. Specimens were then critically point dried and coated with gold using sputter coating for examination using a scanning electron microscope (SEM), JEOL, JSM 5300.

Results
Adults of T. confusum are reddish brown elongate beetles varying from 4.0 to 4.5 mm in body length and from 1.0 to 1.2 mm in width (Fig. 1a). Males and females are morphologically indistinguishable by light microscopy. Using scanning electron microscopy, the setiferous sex patches which were reported in males ( Fig. 2b-d) can often be used for sexing specimens. The setiferous patch was found on the fore, middle, and hind femur of males. The patches lie in a nearly circular depression on the ventral surface of the femur of fore and hind femur, but small in size in the middle femur ( Fig. 2b-d). The setae present in the patch are nearly of the same size. The depression containing the setae is deeper and has almost vertical walls and a strong ring separates it from the surrounding integument (Fig. 2b). The setae are slope such that their tips project through the opening of the depression. The integument beyond the ring has a minisculpture composed of transverse polygons (Fig. 2b).
The head of adult T. confusum is visible from above, it has no beak, and the thorax is slightly parallel on its sides (Fig. 1a). Legs consist of coxa, trochanter, femur, tibia, and five tarsi in the fore and middle legs and four tarsi in the hind legs ending with two claws (Fig. 1b) which are simple, curved apically, and are equipped with sensillae (Fig. 2a). The antenna of adult T. confusum is composed of 11 annuli, the scape, the pedicle, and a flagellum of nine annuli (Fig. 3a). The annuli are cylindrical in shape and generally the length decreased near the distal end of the antennae (Fig. 3a).
The adult mandible is short, and the posterior edge have many spines (Fig. 3b). The base of the maxilla consists of two segments, the cardo contains a single sclerite which is semicircular in shape in adults; the main sclerite of the stipes is triangular and articulates with the cardo along its diameter (Fig. 4a). The galea has a brush of spines at its apex (Fig. 3b). The lacinia is short, not extending beyond the galea with bidentate apical (Fig. 4a). The maxillary palp consists of four segments (Fig. 4a). The distal palp segment is elongated and terminates in a field of sensory receptors ( Fig. 4a, b).
The adult labium is a midventral structure; its base consists of the mentum, which contains a single sclerite (Fig. 5a). Distal to the mentum is the prementum to which the lateral labial palps are tied (Fig. 5a). Between the labial palps is the ligula, a structure fringed with long distal setae (Fig. 5a). The adult labial palps consist of three segments and, like the maxillary palps, terminate in a field of sensillae (Fig. 5b).
The structure of last larval maxilla differs from the adult maxilla in having only three palp segments (Fig. 6a). Two larval endites are present in the two maxillae. In larval labium two segmented labial palps are present (Fig. 6a).
The antenna of the larval stage of T. confusum consists of three segments inserted in a membranous structure called antacorium which connects it to the head capsule. The antenna consists of a basal subcylindrical segment (antennomere I), a segment equipped with a lobe (antennomere II) and an apical segment (antennomere III) which is both tiny and elongated internally situated on the apex of antennomere II and bearing a long seta (Fig. 6b). Most last larval instars are varying from 5.75 to 6.9 mm long and 0.75 to 0.95 mm wide. The head, thorax, and abdomen are covered with minute spines (Fig. 7a). The first larval instars are 5.0-5.1 mm long and 0.5-0.6 mm wide. A specific feature of the first instar larvae is the extreme shortened antenna with a reduced number of antennomeres. Another feature is the presence of well-developed and moderately long legs (Fig. 8a, b) and (Fig. 9a).
General body shape of the larvae is as follows: elongate, cylindrical, body mostly white, weakly sclerotized surface with sparse vestiture of whitish setae, segment IX dorsally showing the labium which includes the mentum, prementum, ligula (asterisk), and the labial palps. b Higher magnification of the adult labial palp which consists of three segments terminates in a field of sensillae forming divided sclerite (pygidium) (Fig. 7a-d). Urogomphi are appendages of tergum IX of beetle larvae of T. confusum which are reported for the first and last larval instars (Fig. 7c, d). Segment X is not visible in dorsal view; it is inserted on the ventral side posterior to sternum IX and may be represented by two lobes (Fig. 7c, d) and (Fig. 9b) which is probably developed as a pygopod. In the larval stage, there were three pairs of legs each of them consists of five segments (Figs. 7a and 9a); these are coax, trochanter, femur, tibia, and tarsus, and a single claw is present in the first and last instar larvae.
The mature formed pupa is white to light yellow and the head is depressed beneath the pronotum (Fig. 10a). The male pupa measured about 3.25-4.15 mm in length (from the anterior edge of pronotum until before the urogomphi) and 0.95-1.25 mm in width and the female pupa measured about 3.6-4.0 mm in length and 1.0-0.25 mm in width. The pupal abdomen was conical and the last segment has two pointed structures, these are the urogomphi (Fig. 10b). The female genital papillae, which are much larger than those of the male, are two finger-like structures just anterior to the pointed urogomphi (Fig. 11a, c). The male papillae are small enough that they look like just fingertips (Fig. 10b). Pupae of beetle T. confusum have jaws called gin traps (Fig. 11b) on the lateral margin of their jointed abdominal segments from segment 1 to 6 (Fig. 11b, c).
A beetle's first line of defense against environmental factors is their mesothoracic elytra, rigid, and protective forewings. The original elytra of T. confusum revealed hexagonal structuring and their vestiture, setose decumbent, curved, smooth-sided, tapering to a point, and   Figure 13b shows that these spikes are randomly distributed and vertical on the surface of the wings.

Discussion
Experiments carried out by Huet and lenoir-Rousseaux (1976) and Daly and Sokoloff (1965) on the tenebrionid beetle Tenebrio molitor demonstrated that the entire larval appendage developed to the adult appendage and that specific adult structures within each appendage generally arise by transformation of their corresponding larval structure. Thus on the basis of its relatively close relationship with Tenebrio (Daly & Sokoloff, 1965) on T. castaneum (David, Frank, Ariel, Moto, & Elizabeth, 2012), the adult mouth parts are assumed to be formed by transformation of their larval precursors. This contrasts with Drosophila, where larval appendages exist as small sensory organs and adult appendages develop from imaginal discs that are set aside during embryonic development (David et al., 2012). In the present study, a specific feature of the first instar larvae of T. confusum is the extreme shortened antenna with a reduced number of antennomeres; these results agree with that reported by Ross and Pothecary (1970), Beutel and Hornschemeyer (2002), and Margarita et al. (2015). Another feature is the presence of well-developed and moderately long legs; these results agree with that reported by Ross and Pothecary (1970) and Margarita et al. (2015).
Also, Steiner (2014) described the pupa of Glyptotus cribratus which is bearing large divergent urogomphi. The urogomphi of pupa of Cibdelis blaschkei are long, wrinkled at base, gradually tapered to divergent sharp apices.
In the present study, T. confusum on the first and last larval instar, segment X is not visible in dorsal view, it is inserted on the ventral side posterior to sternum IX and may represented by two lobes which are probably developed as a pygopod. These results agree with that reported by Margarita et al. (2015) who reported the structure of segment X for the first larval instar of Tenomerga mucida that the greatly reduced segment X is inserted on the ventral side posterior to sternum IX, it is represented by two pairs of flattened, sclerotised anal flaps, the larger flap bears two long setae, the smaller ventromesal flap one long seta, and several short ones along its rounded posterior edge; the latter are fused basally but divided by a deep median cleft.
In contrast to Steiner (2014) who reported the absence of pygopods on the mature larva of Glyptotus cribratus, the abdominal segment X is small, ventral, transverse, semi-circular, convex, three times wider than long, with  row of six fine setae across width. Also, Steiner (2014) described the mature larva of Cibdelis blaschkei in which abdominal segment X is small, ventral, transverse, semicircular, convex, 2.5× wider than long, with a row of six fine setae across width, and pygopods absent. Beutel and Hornschemeyer (2002) reported that the eversible ventral lobes of segment IX are composed of a weakly sclerotized proximal part and a largely membranous distal element is present in larvae of Micromalthus and Cupedidae (Lawrence, 1991). Beutel and Hornschemeyer (2002) reported that the eversible ventral lobes of segment IX may facilitate locomotion in galleries. Also, Beutel and Hornschemeyer (2002) reported that segment X is not visible externally in all larvae of Archostemata. It is exposed in larvae of most other groups of Coleopteran (e.g., Adephaga, Staphyliniformia) (Frank, 1991;Newton, 1991). Beutel and Hornschemeyer (2002) reported the absence of urogompi from tergum IX of all larvae of Archostemata but they are present in most larvae of Adephaga, in Torridincolidae (Beutel, 1999b) and in larvae of many groups of Polyphaga (e.g., Hydrophiloidea, Histeroidea, and Staphylinoidae) (Frank, 1991;Newton, 1991).
In the present study, the presence of setiferous patches on the fore, middle, and hind femur of the legs were reported in male of T. confusum. Their basic structure, comprising a depression-containing dense, fluted or grooved setae, and numerous pores, is similar to that found in the setiferous sex patches of other Coleoptera (Faustini & Halstead, 1982).

Conclusions
So, using SEM examination add some knowledge about morphological details of different stages of T. confusum, the setiferous sex patches in males, the structure of the mouth parts of adult and larval stage, the pits with spine on the elytra, and the spikes on the membrane wings which will facilitate the identification of T. confusum and may help to clarify the functions of various body parts.
Based on the current results, the future work will concern mainly with the morphological, histological, and biochemical studies of this species and others.