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First published online 2 January 2008
doi: 10.1242/dev.015263

Development 135, 569-577 (2008)
Published by The Company of Biologists 2008
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The role of Broad in the development of Tribolium castaneum: implications for the evolution of the holometabolous insect pupa

Yuichiro Suzuki, James W. Truman* and Lynn M. Riddiford*,{dagger}

Department of Biology, University of Washington, Box 351800, Seattle, WA 98195-1800, USA.


Figure 1
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  		Fig. 1. The structure of the Tribolium br gene inferred from the genome and the isolated mRNA of br isoforms identified in the present study. Five zinc-finger domains (br-Z1 through br-Z5) were found in close proximity to the br core region. mRNA of the br isoforms consisted of each of these isoform-specific domains alternatively spliced to the core region. An additional isoform was found that consisted of the br core region spliced to the br-Z1 and br-Z4 zinc-finger domains with an intron between them.

 

Figure 2
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  		Fig. 2. Effect of application of increasing hydroprene concentration during early pupal development on the phenotype of the adults. Second pupal cuticle is visible on the pronotum (white arrow) and the abdomen (black arrowhead). At higher doses, a second pupal cuticle is also visible on the head and ventral thorax (gray arrows), and the pronotum and the ventral abdomen (gray arrowheads) have mostly pupal cuticle. (Inset) Gin traps are visible on the dorsal side (black arrow).

 

Figure 3
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  		Fig. 3. Expression profiles of the br isoforms. (A) Expression profile of the br isoforms during development of the sixth instar and the seventh instar larvae as determined by RT-PCR. (B) Expression profile of the br isoforms during the development of the hydroprene-treated and the acetone-treated seventh instar larvae as determined by RT-PCR. The hydroprene-treated larvae molt to a supernumerary instar, and the duration of the seventh instar is truncated relative to the acetone-treated final instar larvae. Expression of rps3 is provided to verify equal loading. Cycle numbers for br-Z1, br-Z2, br-Z3, br-Z4, br-Z5 and rps3 are 38, 40, 34, 32, 35 and 37, respectively. D, day.

 

Figure 4
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  		Fig. 4. Phenotypes obtained from larvae injected with br core dsRNA. (A) Ventral and dorsal views of the whole body of the larva, and the br dsRNA- and water-injected controls. Also shown are ventral and dorsal views of a br dsRNA-injected supernumerary individual, the old cuticle of which was removed. (B) Diagram of the body parts of Tribolium discussed in the text (shaded in gray). (C) Comparisons of the abdominal wall at the lateral side showing the presence and absence of gin traps in the untreated larva, and in the br dsRNA- and water-injected control animals. (D-I) Comparisons of the external morphology of appendages in larvae, pupae (water-injected), br dsRNA-injected individuals and adults. (D) The urogomphi of br dsRNA-injected individuals tended to appear more larva-like. (E,F) Antennae (E) and hind legs (F) of br dsRNA-injected individuals look more adult-like, with distinct segments and forked claws at the end of the legs. (G) Magnified view of the tarsus of the hind leg. Arrowheads indicate claws. The pigmentation of the tarsal cuticle in the br dsRNA-treated animals is more like that seen in the larvae. (H) The maxilla of br dsRNA-treated animals has an adult-like morphology with larger palps (p), and a more defined galea (ga) and lacinia (ln), but it retains the larval cuticular pigmentation pattern. (I) The morphology of the mandible of the br dsRNA-treated animal is most like that of the adult.

 

Figure 5
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  		Fig. 5. Phenotypes obtained following injection of different br isoform dsRNA. (A) Water-injected control pupa. (B) Phenotype obtained from knockdown of the br core region. (C) Phenotype obtained from knockdown of all of the br isoforms. A close up of the dorsal abdomen is shown with the reduced gin traps indicated by arrowheads. (D-F) Phenotypes obtained from isoform-specific knockdown of br-Z2 (D), br-Z3 (E), and a combination of br-Z2 and br-Z3 (F). Arrowheads indicate the tips of the reduced wings. (G,H) Phenotypes obtained from isoform-specific elimination of br-Z4 (G) and br-Z1 (H). (I) Pupa injected with br-Z5 dsRNA looks normal. (J) Effect of br-Z2 and br-Z3 dsRNA injection on the wings. Gray line marks the position of the tip of the wings in the control animal. The double arrows indicate the length of wings on each pupa. (K) Effect of isoform-specific knockdown on the pupal tibia. Arrowheads point to the claws of the pupae; arrows indicate the slightly augmented demarcation of claw region in br-Z1 and br-Z4 pupae. (L) Gin traps are reduced in pupae given br-Z4 dsRNA. Double arrows show the length between the two gin trap projections. (M) Normal adult. (N) Adult given br-Z2 dsRNA. (O) Adult given br-Z4 dsRNA. (P) Close-up of the pupal-like cuticle on the dorsal thorax (arrowhead) and ventral abdomen (arrow) in br-Z1 and br-Z4 dsRNA-treated animals. (Q) A close-up of gin trap-like projections (arrow) seen on the dorsal adult abdomen in br-Z4 dsRNA-treated adults.

 

Figure 6
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  		Fig. 6. Phenotypes obtained following pair-wise knockdown of br isoforms. (A) Phenotypes obtained. Arrow points to the most-affected urogomphi (br-Z2/Z4 dsRNA-treated pupa). For comparison, pupae of a control animal and of an animal given br core dsRNA are shown. (B) Dorsal and ventral views of an untreated adult with the wings removed. (C,D) Dorsal (C) and ventral (D) sides of an adult obtained from larva given br-Z1 and br-Z4 dsRNA. Pupal-specific gin traps are clearly visible on the dorsal abdomen (inset). Arrowhead shows the small patch of adult cuticle at the tip of the abdomen; arrow indicates a region of wing lacking the dark brown pigmentation. (E) Dorsal and ventral views of an adult produced from a larva treated with 1.5 nmoles hydroprene during the prepupal stage.

 




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