Flight or protection: the genes Ultrabithorax and apterous in the determination of membranous and sclerotized wings in insects.

Present-day pterygote insects have two pairs of wings, one in the mesothorax (T2), the other in the metathorax (T3), and both have diverged in structure and function in different groups. Studies in endopterygote and paraneopteran species have shown that the gene Ultrabithorax (Ubx) specifies the identity and wing structure in T3, whereas the gene apterous (ap) significantly contributes to forming modified T2 wings. We wondered whether these Ubx and ap mechanisms operate in the lineage of polyneopterans. To explore this possibility, we used the cockroach Blattella germanica (Polyneoptera and Blattodea), in which the T2 wings are sclerotized (tegmina), whereas those of the T3 are membranous. We found that Ubx determines the structure of T3 and the membranous wing, while ap significantly contributes to form the sclerotized T2 tegmina. These results along with the studies carried out on the beetle Tribolium castaneum by Tomoyasu and collaborators suggest that ap plays an important role in the sclerotization and melanization of the T2 wings in neopteran groups that have sclerotized forewings. In turn, the sclerotizing properties of ap demonstrated in beetles and cockroaches suggest that the origin of this function goes back to the emergence of Neoptera, in the mid Devonian.

Exploring integument transcriptomes, cuticle ultrastructure, and cuticular hydrocarbons profiles in eusocial and solitary bee species displaying heterochronic adult cuticle maturation.

Differences in the timing of exoskeleton melanization and sclerotization are evident when comparing eusocial and solitary bees. This cuticular maturation heterochrony may be associated with life style, considering that eusocial bees remain protected inside the nest for many days after emergence, while the solitary bees immediately start outside activities. To address this issue, we characterized gene expression using large-scale RNA sequencing (RNA-seq), and quantified cuticular hydrocarbon (CHC) through gas chromatography-mass spectrometry in comparative studies of the integument (cuticle plus its underlying epidermis) of two eusocial and a solitary bee species. In addition, we used transmission electron microscopy (TEM) for studying the developing cuticle of these and other three bee species also differing in life style. We found 13,200, 55,209 and 30,161 transcript types in the integument of the eusocial Apis mellifera and Frieseomelitta varia, and the solitary Centris analis, respectively. In general, structural cuticle proteins and chitin-related genes were upregulated in pharate-adults and newly-emerged bees whereas transcripts for odorant binding proteins, cytochrome P450 and antioxidant proteins were overrepresented in foragers. Consistent with our hypothesis, a distance correlation analysis based on the differentially expressed genes suggested delayed cuticle maturation in A. mellifera in comparison to the solitary bee. However, this was not confirmed in the comparison with F. varia. The expression profiles of 27 of 119 genes displaying functional attributes related to cuticle formation/differentiation were positively correlated between A. mellifera and F. varia, and negatively or non-correlated with C. analis, suggesting roles in cuticular maturation heterochrony. However, we also found transcript profiles positively correlated between each one of the eusocial species and C. analis. Gene co-expression networks greatly differed between the bee species, but we identified common gene interactions exclusively between the eusocial species. Except for F. varia, the TEM analysis is consistent with cuticle development timing adapted to the social or solitary life style. In support to our hypothesis, the absolute quantities of n-alkanes and unsaturated CHCs were significantly higher in foragers than in the earlier developmental phases of the eusocial bees, but did not discriminate newly-emerged from foragers in C. analis. By highlighting differences in integument gene expression, cuticle ultrastructure, and CHC profiles between eusocial and solitary bees, our data provided insights into the process of heterochronic cuticle maturation associated to the way of life.

A cuticle protein gene in the honeybee: expression during development and in relation to the ecdysteroid titer.

A cDNA encoding a cuticle protein containing the R&R Consensus was characterized in the honeybee integument. AmelCPR14 developmental expression is distinguished by an on-off-on pattern, the transition from a low to a high level of transcripts occurring as the ecdysteroid titer is declining after the peak that triggers the onset of pharate (pupal and adult) development. The transcript is abundant during cuticle tanning and sclerotization, and persists even in the adult integument, suggesting that the corresponding protein is required for differentiation and maintenance of the adult cuticle. Such developmental pattern suggested that AmelCPR14 gene might be regulated by the titer of ecdysteroids. We confirmed this hypothesis using different experimental strategies. By tying a ligature in early pupae to prevent exposure of abdominal integument to a high ecdysteroid titer, we delayed the accumulation of AmelCPR14 transcripts in the abdominal integument. This is consistent with ecdysteroid priming being required in pupae for the increase in AmelCPR14 expression in pharate adults. By injecting 20-hydroxyecdysone (20E) in early pupae we demonstrated that hormone titer decay after the peak is critical for AmelCPR14 expression induction. Exposure of pupal integument in vitro to a 20E concentration mimicking the pupal ecdysteroid peak repressed AmelCPR14 expression, which was recovered by hormone removal. Taken together, these data are consistent with an ecdysteroid pulse (increase in hormone titer followed by its decline) being critical for a high AmelCPR14 gene expression in pharate adults.

Tergal and pleural structures contribute to the formation of ectopic prothoracic wings in cockroaches.

Wings were a fundamental morphological innovation for the adaptive radiation of insects, the most diversified group among all animals. Pterygote insects have two pairs of wings, the mesothoracic (T2) forewings and the metathoracic (T3) hindwings, whereas the prothorax (T1) is wingless. Using RNA interference approaches, we have found that the gene Sex combs reduced (Scr) determines the wingless identity of T1 in the cockroach Blattella germanica. Interference of Scr triggers the formation of ectopic wing structures in T1, which are formed from the expansion of the latero-posterior region of the pronotum, along with a contribution of the epimeron, a pleurite of T1. These data support the theory of a dual origin for insect wings, from pronotal (tergal origin theory) and pleural (pleural origin theory) structures and genes.

RNAi-Mediated Functional Analysis of Bursicon Genes Related to Adult Cuticle Formation and Tanning in the Honeybee, Apis mellifera.

Bursicon is a heterodimeric neurohormone that acts through a G protein-coupled receptor named rickets (rk), thus inducing an increase in cAMP and the activation of tyrosine hydroxylase, the rate-limiting enzyme in the cuticular tanning pathway. In insects, the role of bursicon in the post-ecdysial tanning of the adult cuticle and wing expansion is well characterized. Here we investigated the roles of the genes encoding the bursicon subunits during the adult cuticle development in the honeybee, Apis mellifera. RNAi-mediated knockdown of AmBurs α and AmBurs ß bursicon genes prevented the complete formation and tanning (melanization/sclerotization) of the adult cuticle. A thinner, much less tanned cuticle was produced, and ecdysis toward adult stage was impaired. Consistent with these results, the knockdown of bursicon transcripts also interfered in the expression of genes encoding its receptor, AmRk, structural cuticular proteins, and enzymes in the melanization/sclerotization pathway, thus evidencing roles for bursicon in adult cuticle formation and tanning. Moreover, the expression of AmBurs α, AmBurs ß and AmRk is contingent on the declining ecdysteroid titer that triggers the onset of adult cuticle synthesis and deposition. The search for transcripts of AmBurs α, AmBurs ß and candidate targets in RNA-seq libraries prepared with brains and integuments strengthened our data on transcript quantification through RT-qPCR. Together, our results support our premise that bursicon has roles in adult cuticle formation and tanning, and are in agreement with other recent studies pointing for roles during the pharate-adult stage, in addition to the classical post-ecdysial ones.

Ecdysteroid-dependent expression of the tweedle and peroxidase genes during adult cuticle formation in the honey bee, Apis mellifera.

Cuticle renewal is a complex biological process that depends on the cross talk between hormone levels and gene expression. This study characterized the expression of two genes encoding cuticle proteins sharing the four conserved amino acid blocks of the Tweedle family, AmelTwdl1 and AmelTwdl2, and a gene encoding a cuticle peroxidase containing the Animal haem peroxidase domain, Ampxd, in the honey bee. Gene sequencing and annotation validated the formerly predicted tweedle genes, and revealed a novel gene, Ampxd, in the honey bee genome. Expression of these genes was studied in the context of the ecdysteroid-coordinated pupal-to-adult molt, and in different tissues. Higher transcript levels were detected in the integument after the ecdysteroid peak that induces apolysis, coinciding with the synthesis and deposition of the adult exoskeleton and its early differentiation. The effect of this hormone was confirmed in vivo by tying a ligature between the thorax and abdomen of early pupae to prevent the abdominal integument from coming in contact with ecdysteroids released from the prothoracic gland. This procedure impaired the natural increase in transcript levels in the abdominal integument. Both tweedle genes were expressed at higher levels in the empty gut than in the thoracic integument and trachea of pharate adults. In contrast, Ampxd transcripts were found in higher levels in the thoracic integument and trachea than in the gut. Together, the data strongly suggest that these three genes play roles in ecdysteroid-dependent exoskeleton construction and differentiation and also point to a possible role for the two tweedle genes in the formation of the cuticle (peritrophic membrane) that internally lines the gut.

Developmental characterization, function and regulation of a Laccase2 encoding gene in the honey bee, Apis mellifera (Hymenoptera, Apinae).

In insects, exoskeleton (cuticle) formation at each molt cycle includes complex biochemical pathways wherein the laccase enzymes (EC 1.10.3.2) may have a key role. We identified an Amlac2 gene that encodes a laccase2 in the honey bee, Apis mellifera, and investigated its function in exoskeleton differentiation. The Amlac2 gene consists of nine exons resulting in an ORF of 2193 nucleotides. The deduced translation product is a 731 amino acid protein of 81.5 kDa and a pI of 6.05. Amlac2 is highly expressed in the integument of pharate adults, and the expression precedes the onset of cuticle pigmentation and the intensification of sclerotization. In accordance with the temporal sequence of exoskeleton differentiation from anterior to posterior direction, the levels of Amlac2 transcript increase earlier in the thoracic than in the abdominal integument. The gene expression lasts even after the bees emerge from brood cells and begin activities in the nest, but declines after the transition to foraging stage, suggesting that maturation of the exoskeleton is completed at this stage. Post-transcriptional knockdown of Amlac2 gene expression resulted in structural abnormalities in the exoskeleton and drastically affected adult eclosion. By setting a ligature between the thorax and abdomen of early pupae we could delay the increase in hemolymph ecdysteroid levels in the abdomen. This severely impaired the increase in Amlac2 transcript levels and also the differentiation of the abdominal exoskeleton. Taken together, these results indicate that Amlac2 expression is controlled by ecdysteroids and has a critical role in the differentiation of the adult exoskeleton of honey bees.

Expression profile of a Laccase2 encoding gene during the metamorphic molt in Apis mellifera (Hymenoptera,Apidae)

Expression profile of a Laccase2 encoding gene during the metamorphic molt in Apis mellifera (Hymenoptera, Apidae). Metamorphosis in holometabolous insects occurs through two subsequent molting cycles: pupation (metamorphic molt) and adult differentiation (imaginal molt). The imaginal molt in Apis mellifera L. was recently investigated in both histological and physiological-molecular approaches. Although the metamorphic molt in this model bee is extremely important to development, it is not well-known yet. In the current study we used this stage as an ontogenetic scenario to investigate the transcriptional profile of the gene Amlac2, which encodes a laccase with an essential role in cuticle differentiation. Amlac2 expression in epidermis was contrasted with the hemolymph titer of ecdysteroid hormones and with the most evident morphological events occurring during cuticle renewal. RT-PCR semiquantitative analyses using integument samples revealed increased levels of Amlac2 transcripts right after apolysis and during the subsequent pharate period, and declining levels near pupal ecdysis. Compared with the expression of a cuticle protein gene, AmelCPR14, these results highlighted the importance of the ecdysteroid-induced apolysis as an ontogenetic marker of gene reactivation in epidermis for cuticle renewal. The obtained results strengthen the comprehension of metamorphosis in Apis mellifera. In addition, we reviewed the literature about the development of A. mellifera, and emphasize the importance of revising the terminology used to describe honey bee molting cycles.