Yuichiro Suzuki

The developmental control of size in insects

The mechanisms that control the sizes of a body and its many parts remain among the great puzzles in developmental biology. Why do animals grow to a species‐specific body size, and how is the relative growth of their body parts controlled to so they grow to the right size, and in the correct proportion with body size, giving an animal its species‐characteristic shape? Control of size must involve mechanisms that somehow assess some aspect of size and are upstream of mechanisms that regulate growth. These mechanisms are now beginning to be understood in the insects, in particular in Manduca sexta and Drosophila melanogaster. The control of size requires control of the rate of growth and control of the cessation of growth. Growth is controlled by genetic and environmental factors. Insulin and ecdysone, their receptors, and intracellular signaling pathways are the principal genetic regulators of growth. The secretion of these growth hormones, in turn, is controlled by complex interactions of other endocrine and molecular mechanisms, by environmental factors such as nutrition, and by the physiological mechanisms that sense body size. Although the general mechanisms of growth regulation appear to be widely shared, the mechanisms that regulate final size can be quite diverse. WIREs Dev Biol 2014, 3:113–134. doi: 10.1002/wdev.124

The role of Broad in the development of Tribolium castaneum:implications for the evolution of the holometabolous insect pupa

The evolution of complete metamorphosis in insects is a key innovation that has led to the successful diversification of holometabolous insects, yet the origin of the pupa remains an enigma. Here, we analyzed the expression of the pupal specifier gene broad (br), and the effect on br of isoform-specific, double-stranded RNA-mediated silencing, in a basal holometabolous insect, the beetle Tribolium castaneum. All five isoforms are weakly expressed during the penultimate instar and highly expressed during the prepupal period of the final instar. Application of hydroprene, a juvenile hormone analog, during the penultimate instar caused a repeat of the penultimate br expression patterns, and the formation of supernumerary larvae. Use of dsRNA against the br core region, or against a pair of either the br-Z2 or br-Z3 isoform with the br-Z1 or br-Z4 isoform, produced mobile animals with well-differentiated adult-like appendages, but which retained larval-like urogomphi and epidermis. Disruption of either the br-Z2 or the br-Z3 isoform caused the formation of shorter wings. Disruption of both br-Z1 and br-Z4 caused the appearance of pupal traits in the adults, but disruption of br-Z5 had no morphological effect. Our findings show that the br isoform functions are broadly conserved within the Holometabola and suggest that evolution of br isoform expression may have played an important role in the evolution of the pupa in holometabolous insects.

Genetic basis of adaptive evolution of a polyphenism by genetic accommodation

Polyphenisms are evolved adaptations in which a genome produces discrete alternative phenotypes in different environments. In this study, the genetic basis of the evolution of a polyphenism by genetic accommodation was investigated. A polyphenic strain and a monophenic strain of Manduca sexta (L.) were crossed and the F1 offspring and backcross progeny were analysed. The larval colour polyphenism was found to be regulated by one sex‐linked gene of major effect and many smaller effect modifier genes. The finding shows that the mechanism of genetic accommodation relies on genetic changes that are consistent with the current view of the genetic basis of adaptive evolution.

Evolution of insect abdominal appendages: are prolegs homologous or convergent traits?

Abstract. Many insects possess abdominal prolegs, raising the question of whether these prolegs are homologous or convergent structures. One way to address this issue is to compare mechanisms controlling the development of prolegs in different insects. Segmental morphologies along the insect body are controlled by the regulatory activities of the Hox proteins, and one well-studied regulatory target is the Distal-less (Dll) gene, which is required for the development of distal limb structures in arthropods. In Drosophila abdominal segments, Dll transcription is prevented by Hox proteins of the Bithorax Complex (BX-C). In lepidopteran abdominal segments, circular holes lacking BX-C protein expression allow Dll to be expressed and prolegs to develop. For comparison, we examined protein expression patterns in two species of sawfly from the hymenopteran suborder Symphyta; these insects develop prolegs on all abdominal segments. Interestingly, sawfly prolegs did not express Dll protein at any time, and expressed BX-C proteins throughout development. These results suggest that sawfly prolegs lack distal elements that are present in lepidopteran prolegs. Consistent with this interpretation, the proximal determinant extradenticle (exd) was present in cell nuclei all of the way to the tip of the sawfly proleg, whereas it was not detectable in the nuclei of cells near the tip of the lepidopteran proleg. Our results support the hypothesis that larval prolegs have evolved independently in the Lepidoptera and Hymenoptera.

The role of canonical Wnt signaling in leg regeneration and metamorphosis in the red flour beetle Tribolium castaneum.

Many organisms across the Metazoa have regenerative abilities with potentially conserved genetic mechanisms that can enlighten both medicine and evolutionary studies. Here, the role of canonical Wnt signaling was examined in the red flour beetle Tribolium castaneum in order to explore its role during metamorphosis and larval leg regeneration. Double-stranded RNA mediated silencing of Wnt-1 signaling resulted in a loss of wings and appendages with a dramatic reduction in width, indicating that the Wnt-1 signaling pathway is necessary for proper post-embryonic appendage development in T. castaneum. Furthermore, disruption of canonical Wnt signaling led to the complete impairment of limb regeneration in T. castaneum. Our findings suggest that Wnt-1 signaling is a conserved mechanism for appendage development across all holometabolous insects and indicate that the role of Wnt-1 signaling in limb regeneration has been retained across all insects as various modes of limb development evolved. Importantly, this study shows that the availability of the genome sequence and the ease of performing leg ablations make Tribolium an excellent holometabolous insect model for studying regeneration.

Amino Acids and TOR Signaling Promote Prothoracic Gland Growth and the Initiation of Larval Molts in the Tobacco Hornworm Manduca sexta

Molting in arthropods is orchestrated by a series of endocrine changes that occur towards the end of an instar. However, little is understood about the mechanisms that trigger these endocrine changes. Here, nutritional inputs were manipulated to investigate the minimal nutritional inputs required for a Manduca sexta larva to initiate a molt. Amino acids were found to be necessary for a larva to molt, indicating the involvement of an amino acid sensitive pathway. Feeding rapamycin, an inhibitor of the target of rapamycin (TOR) signaling, delayed the onset of a molt and resulted in abnormally larger larvae. Rapamycin also suppressed the growth of the prothoracic glands relative to the whole body growth, and this was accompanied by suppression of ecdysone production and secretion. Higher doses of rapamycin also slowed the growth rate, indicating that TOR signaling also plays a role in systemic growth. TOR signaling therefore couples the nutritional status of the larva to the endocrine system to regulate the timing of a molt.

The role of juvenile hormone and insulin/TOR signaling in the growth of Manduca sexta

BackgroundIn many insect species, fitness trade-offs exist between maximizing body size and developmental speed. Understanding how various species evolve different life history strategies requires knowledge of the physiological mechanisms underlying the regulation of body size and developmental timing. Here the roles of juvenile hormone (JH) and insulin/target of rapamycin (TOR) signaling in the regulation of the final body size were examined in the tobacco hornworm, Manduca sexta.ResultsFeeding rapamycin to wild-type larvae decreased the growth rate but did not alter the peak size of the larvae. In contrast, feeding rapamycin to the JH-deficient black mutant larvae caused the larvae to significantly increase the peak size relative to the DMSO-fed control animals by lengthening the terminal growth period. Furthermore, the critical weight was unaltered by feeding rapamycin, indicating that in Manduca, the critical weight is not influenced by insulin/TOR signaling. In addition, post-critical weight starved black mutant Manduca given rapamycin underwent metamorphosis sooner than those that were fed, mimicking the “bail-out mechanism”.ConclusionsOur study demonstrates that JH masks the effects of insulin/TOR signaling in the determination of the final body size and that the critical weights in Drosophila and Manduca rely on distinct mechanisms that reflect different life history strategies. Our study also suggests that TOR signaling lengthens the terminal growth period in Manduca as it does in Drosophila, and that JH levels determine the relative contributions of nutrient- and body size-sensing pathways to metamorphic timing.

Matrix metalloproteinases (MMPs) are required for wound closure and healing during larval leg regeneration in the flour beetle, Tribolium castaneum

Regenerative abilities are found ubiquitously among many metazoan taxa. To compare mechanisms underlying the initial stages of limb regeneration between insects and vertebrates, the roles of matrix metalloproteinases (MMPs) and fibroblast growth factor (FGF) signaling were investigated in the red flour beetle, Tribolium castaneum. RNA interference-mediated knockdown of MMP2 expression delayed wound healing and subsequent leg regeneration. Additionally, pairwise knockdown of MMP1/2 and MMP2/3, but not MMP1/3, resulted in inhibition of wound closure. Wound healing on the dorsal epidermis after injury was also delayed when MMPs were silenced. Our findings show that functionally redundant MMPs play key roles during limb regeneration and wound healing in Tribolium. This MMP-mediated wound healing is necessary for the subsequent formation of a blastema. In contrast, silencing of FGF receptor did not interfere with the initial stages of leg regeneration despite the alterations in tanning of the cuticle. Thus, insects and vertebrates appear to employ similar developmental processes for the initial stages of wound closure during limb regeneration, while the role of FGF in limb regeneration appears to be unique to vertebrates.

Developmental coupling of larval and adult stages in a complex life cycle: insights from limb regeneration in the flour beetle, Tribolium castaneum.

BackgroundA complex life cycle, such as complete metamorphosis, is a key innovation that can promote diversification of species. The evolution of a morphologically distinct larval stage is thought to have enabled insects to occupy broader ecological niches and become the most diverse metazoan taxon, yet the extent to which larval and adult morphologies can evolve independently remains unknown. Perturbation of larval limb regeneration allows us to generate larval legs and antennae with altered limb morphologies, which may be used to explore the developmental continuity that might exist between larval and adult appendages. In this study, we determined the roles of several appendage patterning transcription factors, abrupt (ab), dachshund (dac), Distal-less (Dll), and spineless (ss), in the red flour beetle, Tribolium castaneum, during larval appendage regeneration. The functions of these genes in regenerating and non-regenerating limbs were compared using RNA interference.ResultsDuring limb regeneration, dac and ss were necessary to re-pattern the same larval structures as those patterned during embryogenesis. Removal of these two genes led to larval appendage patterning defects that were carried over to the adult legs. Surprisingly, even though maternal knockdown of ab had minimal effects on limb allocation and patterning in the embryo, it was necessary for blastema growth, an earlier phase of regeneration. Finally, knockdown of Dll prevented the blastema-like bumps from re-differentiating into appendages.ConclusionsOur results suggest that, similar to vertebrates, the re-patterning phase of Tribolium larval limb regeneration relies on the same genes that are used during embryonic limb patterning. Thus, the re-patterning phase of regeneration is likely to be regulated by taxon-specific patterning mechanisms. Furthermore, Ab and Dll appear to play important roles during blastema proliferation and re-differentiation, respectively. Finally, our results show that continuity exists between larval and adult limb patterning, and that larval and adult leg morphologies may be developmentally coupled. Thus, the evolution of imaginal discs may have been a key step towards completely removing any developmental constraints that existed between larval and adult phenotypes.

Constraint and developmental dissociation of phenotypic integration in a genetically accommodated trait.

SUMMARY The genetic accommodation of novel adaptive traits may be accompanied by the evolution of correlated traits that constrain adaptive evolution. Very little is known about the removal of maladaptive correlated traits. In the present study, body size was found to have evolved as a correlated trait during the artificial selection for a polyphenism and a monophenism, and the developmental basis underlying this correlated trait was investigated. The body size and coloration were found to be developmentally integrated by titers of the insect developmental hormone, juvenile hormone (JH). Attempts to uncouple the two traits resulted in the evolution of one of the body size determinants—the critical weight—but not the delay period whose evolution is constrained by JH titers. Thus, maladaptive correlated traits can be removed when multiple developmental modules exist, and the evolution of one or more of these modules is not constrained.