Anterior-posterior patterning of segments in Anopheles stephensi offers insights into the transition from sequential to simultaneous segmentation in holometabolous insects.

The gene regulatory network for segmentation in arthropods offers valuable insights into how networks evolve owing to the breadth of species examined and the extremely detailed knowledge gained in the model organism Drosophila melanogaster. These studies have shown that Drosophila's network represents a derived state that acquired changes to accelerate segment patterning, whereas most insects specify segments gradually as the embryo elongates. Such heterochronic shifts in segmentation have potentially emerged multiple times within holometabolous insects, resulting in many mechanistic variants and difficulties in isolating underlying commonalities that permit such shifts. Recent studies identified regulatory genes that work as timing factors, coordinating gene expression transitions during segmentation. These studies predict that changes in timing factor deployment explain shifts in segment patterning relative to other developmental events. Here, we test this hypothesis by characterizing the temporal and spatial expression of the pair-rule patterning genes in the malaria vector mosquito, Anopheles stephensi. This insect is a Dipteran (fly), like Drosophila, but represents an ancient divergence within this clade, offering a useful counterpart for evo-devo studies. In mosquito embryos, we observe anterior to posterior sequential addition of stripes for many pair-rule genes and a wave of broad timer gene expression across this axis. Segment polarity gene stripes are added sequentially in the wake of the timer gene wave and the full pattern is not complete until the embryo is fully elongated. This "progressive segmentation" mode in Anopheles displays commonalities with both Drosophila's rapid segmentation mechanism and sequential modes used by more distantly related insects.

even-skipped acts as a pair-rule gene in germ band stages of Tribolium development.

The pair-rule gene even-skipped (eve) is essential for insect segmentation, yet its function varies among insect clades. While loss of eve results in typical pair-rule phenotypes in Drosophila, knock-down of eve orthologs shows segmental, gap-like, or asegmental phenotypes in non-Drosophila insects. In Tribolium, knock-down of the eve ortholog (Tc-eve) resulted in a graded phenotypic series ranging from strong to weak, the most informative of which was intermediate phenotypes. The strong knock-down embryos displayed asegmental phenotypes and severely disorganized germ bands which have prevented determination of Tc-eve function in later stages. In order to understand the segmentation function of Tc-eve during later germ band elongation stages, we analyzed intermediate Tc-eveRNAi embryos in which germ band elongation was less affected. Most intermediate Tc-eveRNAi germ bands displayed segmentation defects with a double segmental periodicity in the abdomen. In these intermediate embryos, Tc-engrailed (Tc-en) stripes were ectopically expanded into large bands with a double segmental periodicity, while the remaining Tc-en stripes between the expanded Tc-en stripes were absent or barely formed. The expanded Tc-en stripes seemed to be activated by primary Tc-eve stripes and Tc-paired, both of which failed to resolve into secondary segmental stripes. The absence of Tc-en stripes appeared to be a consequence of the absence of the secondary stripes of Tc-runt that were required for the activation of Tc-en stripes. These results suggest that Tc-eve functions as a pair-rule gene at least in the germ band stages of Tribolium development.

Dermestes maculatus: an intermediate-germ beetle model system for evo-devo.

BACKGROUND: Understanding how genes change during evolution to direct the development of diverse body plans is a major goal of the evo-devo field. Achieving this will require the establishment of new model systems that represent key points in phylogeny. These new model systems must be amenable to laboratory culture, and molecular and functional approaches should be feasible. To date, studies of insects have been best represented by the model system Drosophila melanogaster. Given the enormous diversity represented by insect taxa, comparative studies within this clade will provide a wealth of information about the evolutionary potential and trajectories of alternative developmental strategies. RESULTS: Here we established the beetle Dermestes maculatus, a member of the speciose clade Coleoptera, as a new insect model system. We have maintained a continuously breeding culture in the lab and documented Dermestes maculatus embryogenesis using nuclear and phalloidin staining. Anterior segments are specified during the blastoderm stage before gastrulation, and posterior segments are added sequentially during germ band elongation. We isolated and studied the expression and function of the pair-rule segmentation gene paired in Dermestes maculatus. In this species, paired is expressed in stripes during both blastoderm and germ band stages: four primary stripes arise prior to gastrulation, confirming an intermediate-germ mode of development for this species. As in other insects, these primary stripes then split into secondary stripes. To study gene function, we established both embryonic and parental RNAi. Knockdown of Dmac-paired with either method resulted in pair-rule-like segmentation defects, including loss of Engrailed expression in alternate stripes. CONCLUSIONS: These studies establish basic approaches necessary to use Dermestes maculatus as a model system. Methods are now available for use of this intermediate-germ insect for future studies of the evolution of regulatory networks controlling insect segmentation, as well as of other processes in development and homeostasis. Consistent with the role of paired in long-germ Drosophila and shorter-germ Tribolium, paired functions as a pair-rule segmentation gene in Dermestes maculatus. Thus, paired retains pair-rule function in insects with different modes of segment addition.

Pair-rule patterning in the honeybee Apis mellifera: Expression of even-skipped combines traits known from beetles and fruitfly.

We have studied the binding pattern of antibody mAB 2B8 directed against even-skipped orthologous proteins (EVE) in honeybee embryos. Primary and secondary EVE stripes form in roughly anterior-to-posterior succession; there are 8 primary and 16 secondary stripes. The most posterior primary stripes appear only after the onset of gastrulation. The secondary stripes form by a splitting of primary stripes; they demarcate the parasegmental pattern. While these findings resemble EVE expression in long-germ beetles, the honeybee differs from both beetles and dipterans by two transient pair-rule traits in the parasegmental EVE pattern: the secondary stripes in head and thorax alternate in strength, yet out of register with the Drosophila pattern, and over the whole pattern the odd-numbered stripes vanish earlier than their even-numbered counterparts. As in Drosophila, however, the strong EVE stripes coincide with the weak engrailed (EN) stripes. These findings are taken to indicate that (1) honeybee and beetles share a conserved mode of EVE stripe formation whilst Drosophila has diverged in this respect, (2) honeybee and Drosophila have diverged from the beetles in specific pair-rule traits during the parasegmental expression of both EVE and EN, and (3) some of these traits differ in the register of segment pairing and thus may reflect regulatory divergences at the pair-rule level between dipterans and the honeybee.