Annotation of Hox cluster and Hox cofactor genes in the Asian citrus psyllid, Diaphorina citri, reveals novel features

Hox genes and their cofactors are essential developmental genes that specify regional identity in animals, including insects. A particularly interesting feature of Hox genes is their conserved arrangement in clusters in the same order in which they specify identity along the anterior-posterior axis. Among insects, breaks in the cluster have been reported in a few species, but these seem to be the exception rather than the rule. We have annotated the ten Hox genes of the Asian citrus psyllid, Diaphorina citri, and determined that there is a split in its Hox cluster between the Deformed and Sex combs reduced genes. This is the first time a break at this position has been observed in an insect Hox cluster. We have also annotated the D. citri orthologs of the Hox cofactor genes homothorax, PKNOX and extradenticle. Interestingly, we found an additional copy of extradenticle in D. citri that appears to be a retrogene. Expression data and sequence conservation suggest that the extradenticle retrogene may have retained the original extradenticle function and allowed the parental extradenticle gene to diverge.

The genome of a daddy-long-legs (Opiliones) illuminates the evolution of arachnid appendages

Chelicerate arthropods exhibit dynamic genome evolution, with ancient whole-genome duplication (WGD) events affecting several orders. Yet, genomes remain unavailable for a number of poorly studied orders, such as Opiliones (daddy-long-legs), which has hindered comparative study. We assembled the first harvestman draft genome for the species Phalangium opilio , which bears elongate, prehensile appendages, made possible by numerous distal articles called tarsomeres. Here, we show that the genome of P. opilio exhibits a single Hox cluster and no evidence of WGD. To investigate the developmental genetic basis for the quintessential trait of this group—the elongate legs—we interrogated the function of the Hox genes Deformed ( Dfd ) and Sex combs reduced ( Scr ), and a homologue of Epidermal growth factor receptor ( Egfr ). Knockdown of Dfd incurred homeotic transformation of two pairs of legs into pedipalps, with dramatic shortening of leg segments in the longest leg pair, whereas homeosis in L3 is only achieved upon double Dfd + Scr knockdown. Knockdown of Egfr incurred shortened appendages and the loss of tarsomeres. The similarity of Egfr loss-of-function phenotypic spectra in insects and this arachnid suggest that repeated cooption of EGFR signalling underlies the independent gains of supernumerary tarsomeres across the arthropod tree of life.

Hox genes limit germ cell formation in the short germ insect Gryllus bimaculatus

Hox genes are conserved transcription factor-encoding genes that specify the identity of body regions in bilaterally symmetrical animals. In the cricket Gryllus bimaculatus, a member of the hemimetabolous insect group Orthoptera, the induction of a subset of mesodermal cells to form the primordial germ cells (PGCs) is restricted to the second through the fourth abdominal segments (A2 to A4). In numerous insect species, the Hox genes Sex-combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), and abdominal-A (abd-A) jointly regulate the identities of middle and posterior body segments, suggesting that these genes may restrict PGC formation to specific abdominal segments in G. bimaculatus. Here we show that reducing transcript levels of some or all of these Hox genes results in supernumerary and/or ectopic PGCs, either individually or in segment-specific combinations, suggesting that the role of these Hox genes is to limit PGC development with respect to their number, segmental location, or both. These data provide evidence of a role for this ancient group of genes in PGC development.

Hox genes mediate the escalation of sexually antagonistic traits in water striders

Sexual conflict occurs when traits favoured in one sex impose fitness costs on the other sex. In the case of sexual conflict over mating rate, the sexes often undergo antagonistic coevolution and escalation of traits that enhance females' resistance to superfluous mating and traits that increase males' persistence. How this escalation in sexually antagonistic traits is established during ontogeny remains unclear. In the water strider Rhagovelia antilleana, male persistence traits consist of sex combs on the forelegs and multiple rows of spines and a thick femur in the rear legs. Female resistance traits consist of a prominent spike-like projection of the pronotum. RNAi knockdown against the Hox gene Sex Combs Reduced resulted in the reduction in both the sex comb in males and the pronotum projection in females. RNAi against the Hox gene Ultrabithorax resulted in the complete loss or reduction of all persistence traits in male rear legs. These results demonstrate that Hox genes can be involved in intra- and inter-locus sexual conflict and mediate escalation of sexually antagonistic traits.

Post-translational modifications of Drosophila melanogaster HOX protein, Sex combs reduced

Homeotic selector (HOX) transcription factors (TFs) regulate gene expression that determines the identity of Drosophila segments along the anterior-posterior (A-P) axis. The current challenge with HOX proteins is understanding how they achieve their functional specificity while sharing a highly conserved homeodomain (HD) that recognize the same DNA binding sites. One mechanism proposed to regulate HOX activity is differential post-translational modification (PTM). As a first step in investigating this hypothesis, the sites of PTM on a Sex combs reduced protein fused to a triple tag (SCRTT) extracted from developing embryos were identified by Tandem Mass Spectrometry (MS/MS). The PTMs identified include phosphorylation at S185, S201, T315, S316, T317 and T324, acetylation at K218, S223, S227, K309, K434 and K439, formylation at K218, K309, K325, K341, K369, K434 and K439, methylation at S19, S166, K168 and T364, carboxylation at D108, K298, W307, K309, E323, K325 and K369, and hydroxylation at P22, Y87, P107, D108, D111, P269, P306, R310, N321, K325, Y334, R366, P392 and Y398. Of the 44 modifications, 18 map to functionally important regions of SCR. Besides a highly conserved DNA-binding HD, HOX proteins also have functionally important, evolutionarily conserved small motifs, which may be Short Linear Motifs (SLiMs). SLiMs are proposed to be preferential sites of phosphorylation. Although 6 of 7 phosphosites map to regions of predicted SLiMs, we find no support for the hypothesis that the individual S, T and Y residues of predicted SLiMs are phosphorylated more frequently than S, T and Y residues outside of predicted SLiMs.

Hox genes mediate the escalation of sexually antagonistic traits in water striders

Sexual conflict occurs when traits favoured in one sex impose fitness costs on the other sex. In the case of sexual conflict over mating rate, the sexes often undergo antagonistic coevolution and escalation of traits that enhance female’s resistance to mating and traits that increase male’s persistence. How this escalation in sexually antagonistic traits is established during ontogeny remains unclear. In the water strider Rhagovelia antilleana, male persistence traits consist of sex combs in the forelegs and multiple rows of spines and a thick femur in the rearlegs. Female resistance trait consists of a prominent spike-like projection of the pronotum. RNAi knockdown against the Hox gene Sex Combs Reduced resulted in the reduction of both the sex comb in males and the pronotum projection in females. RNAi against the Hox gene Ultrabithorax resulted in the complete loss or reduction of all persistence traits in male rearlegs. These results demonstrate that Hox genes can mediate sex-specific escalation of antagonistic traits along the body axis of both sexes.

Hox genes limit germ cell formation in the short germ insect Gryllus bimaculatus

Hox genes are conserved transcription factor-encoding genes that specify the identity of body regions in bilaterally symmetrical animals. In the cricket Gryllus bimaculatus, a member of the hemimetabolous insect group Orthoptera, the induction of a subset of mesodermal cells to form the primordial germ cells (PGCs) is restricted to the second through the fourth abdominal segments (A2-A4). In numerous insect species, the Hox genes Sex-combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx) and abdominal-A (abd-A) jointly regulate the identities of middle and posterior body segments, suggesting that these genes may restrict PGC formation to specific abdominal segments in G. bimaculatus. Here we show that all of these Hox genes, either individually or in segment-specific combinations, restrict PGC formation. Our data provides evidence for a segmental Hox code used to regulate the placement of PGC formation, reminiscent of the segmental Hox codes used in other arthropod groups to establish other aspects of segmental identity. These data also provide, to our knowledge, the first evidence for this ancient group of genes in determining PGC placement within the context of axial patterning in any animal studied thus far.