Evidence that hematophagous triatomine bugs may eat plants in the wild.

Blood feeding is a secondary adaptation in hematophagous bugs. Many proteins are secreted in the saliva that are devoted to coping with the host's defense and to process the blood meal. Digestive enzymes that are no longer required for a blood meal would be expected to be eventually lost. Yet, in many strictly hematophagous arthropods, α-amylase genes, which encode the enzymes that digest starch from plants, are still present and transcribed, including in the kissing bug Rhodnius prolixus (Hemiptera, Reduviidae) and its related species, which transmit the Chagas disease. We hypothesized that retaining α-amylase could be advantageous if the bugs occasionally consume plant tissues. We first checked that the α-amylase protein of Rhodnius robustus retains normal amylolytic activity. Then we surveyed hundreds of gut DNA extracts from the sylvatic R. robustus to detect traces of plants. We found plant DNA in 8% of the samples, mainly identified as Attalea palm trees, where R. robustus are usually found. We suggest that although of secondary importance in the blood-sucking bugs, α-amylase may be needed during occasional plant feeding and thus has been retained.

Structural and Functional Characterization of Drosophila melanogaster α-Amylase.

Insects rely on carbohydrates such as starch and glycogen as an energy supply for growth of larvae and for longevity. In this sense α-amylases have essential roles under extreme conditions, e.g., during nutritional or temperature stress, thereby contributing to survival of the insect. This makes them interesting targets for combating insect pests. Drosophila melanogaster α-amylase, DMA, which belongs to the glycoside hydrolase family 13, sub family 15, has been studied from an evolutionary, biochemical, and structural point of view. Our studies revealed that the DMA enzyme is active over a broad temperature and pH range, which is in agreement with the fluctuating environmental changes with which the insect is confronted. Crystal structures disclosed a new nearly fully solvated metal ion, only coordinated to the protein via Gln263. This residue is only conserved in the subgroup of D. melanogaster and may thus contribute to the enzyme adaptive response to large temperature variations. Studies of the effect of plant inhibitors and the pseudo-tetrasaccharide inhibitor acarbose on DMA activity, allowed us to underline the important role of the so-called flexible loop on activity/inhibition, but also to suggest that the inhibition modes of the wheat inhibitors WI-1 and WI-3 on DMA, are likely different.

Amyrel, a novel glucose-forming α-amylase from Drosophila with 4-α-glucanotransferase activity by disproportionation and hydrolysis of maltooligosaccharides.

The α-amylase paralogue Amyrel present in true flies (Diptera Muscomorpha) has been classified as a glycoside hydrolase in CAZy family GH13 on the basis of its primary structure. Here, we report that, in fact, Amyrel is currently unique among animals as it possesses both the hydrolytic α-amylase activity (EC 3.2.1.1) and a 4-α-glucanotransferase (EC 2.4.1.25) transglycosylation activity. Amyrel reacts specifically on α-(1-4) glycosidic bonds of starch and related polymers but produces a complex mixture of maltooligosaccharides, which is in sharp contrast with canonical animal α-amylases. With model maltooligosaccharides G2 (maltose) to G7, the Amyrel reaction starts by a disproportionation leading to Gn - 1 and Gn + 1 products, which by themselves become substrates for new disproportionation cycles. As a result, all detectable odd- and even-numbered maltooligosaccharides, at least up to G12, were observed. However, hydrolysis of these products proceeds simultaneously, as shown by p-nitrophenyl-tagged oligosaccharides and microcalorimetry, and upon prolonged reaction, glucose is the major end-product followed by maltose. The main structural determinant of these atypical activities was found to be a Gly-His-Gly-Ala deletion in the so-called flexible loop bordering the active site. Indeed, engineering this deletion in porcine pancreatic and Drosophila melanogaster α-amylases results in reaction patterns similar to those of Amyrel. It is proposed that this deletion provides more freedom to the substrate for subsites occupancy and allows a less-constrained action pattern resulting in versatile activities at the active site.

Draft nuclear genome and complete mitogenome of the Mediterranean corn borer, Sesamia nonagrioides, a major pest of maize.

The Mediterranean corn borer (Sesamia nonagrioides, Noctuidae, Lepidoptera) is a major pest of maize in Europe and Africa. Here, we report an assembly of the nuclear and mitochondrial genome of a pool of inbred males and females third-instar larvae, based on short- and long-read sequencing. The complete mitochondrial genome is 15,330 bp and contains all expected 13 and 24 protein-coding and RNA genes, respectively. The nuclear assembly is 1021 Mb, composed of 2553 scaffolds and it has an N50 of 1105 kb. It is more than twice larger than that of all Noctuidae species sequenced to date, mainly due to a higher repeat content. A total of 17,230 protein-coding genes were predicted, including 15,776 with InterPro domains. We provide detailed annotation of genes involved in sex determination (doublesex, insulin-like growth factor 2 mRNA-binding protein, and P-element somatic inhibitor) and of alpha-amylase genes possibly involved in interaction with parasitoid wasps. We found no evidence of recent horizontal transfer of bracovirus genes from parasitoid wasps. These genome assemblies provide a solid molecular basis to study insect genome evolution and to further develop biocontrol strategies against S. nonagrioides.

Horizontal Transfer and Gene Loss Shaped the Evolution of Alpha-Amylases in Bilaterians.

The subfamily GH13_1 of alpha-amylases is typical of Fungi, but it is also found in some unicellular eukaryotes (e.g., Amoebozoa, choanoflagellates) and non-bilaterian Metazoa. Since a previous study in 2007, GH13_1 amylases were considered ancestral to the Unikonts, including animals, except Bilateria, such that it was thought to have been lost in the ancestor of this clade. The only alpha-amylases known to be present in Bilateria so far belong to the GH13_15 and 24 subfamilies (commonly called bilaterian alpha-amylases) and were likely acquired by horizontal transfer from a proteobacterium. The taxonomic scope of Eukaryota genomes in databases has been greatly increased ever since 2007. We have surveyed GH13_1 sequences in recent data from ca. 1600 bilaterian species, 60 non-bilaterian animals and also in unicellular eukaryotes. As expected, we found a number of those sequences in non-bilaterians: Anthozoa (Cnidaria) and in sponges, confirming the previous observations, but none in jellyfishes and in Ctenophora. Our main and unexpected finding is that such fungal (also called Dictyo-type) amylases were also consistently retrieved in several bilaterian phyla: hemichordates (deuterostomes), brachiopods and related phyla, some molluscs and some annelids (protostomes). We discuss evolutionary hypotheses possibly explaining the scattered distribution of GH13_1 across bilaterians, namely, the retention of the ancestral gene in those phyla only and/or horizontal transfers from non-bilaterian donors.

Evolution of salivary glue genes in Drosophila species.

BACKGROUND: At the very end of the larval stage Drosophila expectorate a glue secreted by their salivary glands to attach themselves to a substrate while pupariating. The glue is a mixture of apparently unrelated proteins, some of which are highly glycosylated and possess internal repeats. Because species adhere to distinct substrates (i.e. leaves, wood, rotten fruits), glue genes are expected to evolve rapidly. RESULTS: We used available genome sequences and PCR-sequencing of regions of interest to investigate the glue genes in 20 Drosophila species. We discovered a new gene in addition to the seven glue genes annotated in D. melanogaster. We also identified a phase 1 intron at a conserved position present in five of the eight glue genes of D. melanogaster, suggesting a common origin for those glue genes. A slightly significant rate of gene turnover was inferred. Both the number of repeats and the repeat sequence were found to diverge rapidly, even between closely related species. We also detected high repeat number variation at the intrapopulation level in D. melanogaster. CONCLUSION: Most conspicuous signs of accelerated evolution are found in the repeat regions of several glue genes.

The Amylases of Insects.

Alpha-amylases are major digestive enzymes that act in the first step of maltopolysaccharide digestion. In insects, these enzymes have long been studied for applied as well as purely scientific purposes. In many species, amylases are produced by multiple gene copies. Rare species are devoid of Amy gene. They are predominantly secreted in the midgut but salivary expression is also frequent, with extraoral activity. Enzymological parameters are quite variable among insects, with visible trends according to phylogeny: Coleopteran amylases have acidic optimum activity, whereas dipteran amylases have neutral preference and lepidopteran ones have clear alkaline preference. The enzyme structure shows interesting variations shaped by evolutionary convergences, such as the recurrent loss of a loop involved in substrate handling. Many works have focused on the action of plant amylase inhibitors on pest insect amylases, in the frame of crop protection by transgenesis. It appears that sensitivity or resistance to inhibitors is finely tuned and very specific and that amylases and their inhibitors have coevolved. The multicopy feature of insect amylases appears to allow tissue-specific or stage-specific regulation, but also to broaden enzymological abilities, such as pH range, and to overcome plant inhibitory defenses.

α-Amylase Mediates Host Acceptance in the Braconid Parasitoid Cotesia flavipes.

Foraging parasitoids use chemical signals in host recognition and selection processes. Although, the volatiles play a relevant role in the localization by parasitoids of their hosts feeding on plants, the host identification process for acceptance occurs mainly during contact between the parasitoid and its host where host products related to feeding activities, fecal pellets and oral secretions, play a crucial role. The purpose of this study was to identify the nature of the contact kairomone(s) that mediate the acceptance for oviposition of the parasitoid Cotesia flavipes Cameron (Hymenoptera, Braconidae), which was released in Kenya in 1993 to control the invasive crambid Chilo partellus (Swinhoe). Using host and non-hosts of C. flavipes, we showed that it is mainly the oral secretions of the larvae that harbour the active compound(s) that mediate host acceptance for oviposition by C. flavipes. Using an integration of behavioral observations and biochemical approaches, the active compound of the oral secretions was identified as an α-amylase. Using synthetized α-amylases from Drosophila melanogaster (an insect model for which syntheses of active and inactive α-amylases are available), we observed that the conformation of the enzyme rather than its catalytic site as well as its substrate and its degradation product is responsible for host acceptance and oviposition mediation of C. flavipes females. The results suggest that the α-amylase from oral secretions of the caterpillar host is a good candidate for an evolutionary solution to host acceptance for oviposition in C. flavipes.

A single amino-acid substitution toggles chloride dependence of the alpha-amylase paralog amyrel in Drosophila melanogaster and Drosophila virilis species.

In animals, most α-amylases are chloride-dependent enzymes. A chloride ion is required for allosteric activation and is coordinated by one asparagine and two arginine side chains. Whereas the asparagine and one arginine are strictly conserved, the main chloride binding arginine is replaced by a glutamine in some rare instances, resulting in the loss of chloride binding and activation. Amyrel is a distant paralogue of α-amylase in Diptera, which was not characterized biochemically to date. Amyrel shows both substitutions depending on the species. In Drosophila melanogaster, an arginine is present in the sequence but in Drosophila virilis, a glutamine occurs at this position. We have investigated basic enzymological parameters and the dependence to chloride of Amyrel of both species, produced in yeast, and in mutants substituting arginine to glutamine or glutamine to arginine. We found that the amylolytic activity of Amyrel is about thirty times weaker than the classical Drosophila α-amylase, and that the substitution of the arginine by a glutamine in D. melanogaster suppressed the chloride-dependence but was detrimental to activity. In contrast, changing the glutamine into an arginine rendered D. virilis Amyrel chloride-dependent, and interestingly, significantly increased its catalytic efficiency. These results show that the chloride ion is not mandatory for Amyrel but stimulates the reaction rate. The possible phylogenetic origin of the arginine/glutamine substitution is also discussed.

Gene make-up: rapid and massive intron gains after horizontal transfer of a bacterial α-amylase gene to Basidiomycetes.

BACKGROUND: Increasing genome data show that introns, a hallmark of eukaryotes, already existed at a high density in the last common ancestor of extant eukaryotes. However, intron content is highly variable among species. The tempo of intron gains and losses has been irregular and several factors may explain why some genomes are intron-poor whereas other are intron-rich. RESULTS: We studied the dynamics of intron gains and losses in an α-amylase gene, whose product breaks down starch and other polysaccharides. It was transferred from an Actinobacterium to an ancestor of Agaricomycotina. This gene underwent further duplications in several species. The results indicate a high rate of intron insertions soon after the gene settled in the fungal genome. A number of these oldest introns, regularly scattered along the gene, remained conserved. Subsequent gains and losses were lineage dependent, with a majority of losses. Moreover, a few species exhibited a high number of both specific intron gains and losses in recent periods. There was little sequence conservation around insertion sites, then probably little information for splicing, whereas splicing sites, inside introns, showed typical and conserved patterns. There was little variation of intron size. CONCLUSIONS: Since most Basidiomycetes have intron-rich genomes and this richness was ancestral in Fungi, long before the transfer event, we suggest that the new gene was shaped to comply with requirements of the splicing machinery, such as short exon and intron sizes, in order to be correctly processed.

Enzymatic characterization of recombinant α-amylase in the Drosophila melanogaster species subgroup: is there an effect of specialization on digestive enzyme?

We performed a comparative study on the enzymological features of purified recombinant α-amylase of three species belonging to the Drosophila melanogaster species subgroup: D. melanogaster, D. erecta and D. sechellia. D. erecta and D. sechellia are specialist species, with host plant Pandanus candelabrum (Pandanaceae) and Morinda citrifolia (Rubiaceae), respectively. The temperature optima were around 57-60℃ for the three species. The pH optima were 7.2 for D. melanogaster, 8.2 for D. erecta and 8.5 for D. sechellia. The kcat and Km were also estimated for each species with different substrates. The specialist species D. erecta and D. sechellia display a higher affinity for starch than D. melanogaster. α-Amylase activity is higher on starch than on glycogen in all species. α-Amylases of D. erecta and D. sechellia have a higher activity on maltooligosaccharides (G6 and G7) than on starch, contrary to D. melanogaster. Such differences in the enzymological features between the species might reflect adaptation to different ecological niches and feeding habits.

Molecular adaptation and resilience of the insect's nuclear receptor USP.

BACKGROUND: The maintenance of biological systems requires plasticity and robustness. The function of the ecdysone receptor, a heterodimer composed of the nuclear receptors ECR (NR1H1) and USP (NR2B4), was maintained in insects despite a dramatic divergence that occurred during the emergence of Mecopterida. This receptor is therefore a good model to study the evolution of plasticity. We tested the hypothesis that selection has shaped the Ligand-Binding Domain (LBD) of USP during evolution of Mecopterida. RESULTS: We isolated usp and cox1 in several species of Drosophilidae, Tenebrionidae and Blattaria and estimated non-synonymous/synonymous rate ratios using maximum-likelihood methods and codon-based substitution models. Although the usp sequences were mainly under negative selection, we detected relaxation at residues located on the surface of the LBD within Mecopterida families. Using branch-site models, we also detected changes in selective constraints along three successive branches of the Mecopterida evolution. Residues located at the bottom of the ligand-binding pocket (LBP) underwent strong positive selection during the emergence of Mecopterida. This change is correlated with the acquisition of a large LBP filled by phospholipids that probably allowed the stabilisation of the new Mecopterida structure. Later, when the two subgroups of Mecopterida (Amphiesmenoptera: Lepidoptera, Trichoptera; Antliophora: Diptera, Mecoptera, Siphonaptera) diverged, the same positions became under purifying selection. Similarly, several positions of the heterodimerisation interface experienced positive selection during the emergence of Mecopterida, rapidly followed by a phase of constrained evolution. An enlargement of the heterodimerisation surface is specific for Mecopterida and was associated with a reinforcement of the obligatory partnership between ECR and USP, at the expense of homodimerisation. CONCLUSIONS: In order to explain the episodic mode of evolution of USP, we propose a model in which the molecular adaptation of this protein is seen as a process of resilience for the maintenance of the ecdysone receptor functionality.

Temperature adaptations in psychrophilic, mesophilic and thermophilic chloride-dependent alpha-amylases.

The functional and structural adaptations to temperature have been addressed in homologous chloride-dependent α-amylases from a psychrophilic Antarctic bacterium, the ectothermic fruit fly, the homeothermic pig and from a thermophilic actinomycete. This series covers nearly all temperatures encountered by living organisms. We report a striking continuum in the functional properties of these enzymes coupled to their structural stability and related to the thermal regime of the source organism. In particular, thermal stability recorded by intrinsic fluorescence, circular dichroism and differential scanning calorimetry appears to be a compromise between the requirement for a stable native state and the proper structural dynamics to sustain the function at the environmental/physiological temperatures. The thermodependence of activity, the kinetic parameters, the activations parameters and fluorescence quenching support these activity-stability relationships in the investigated α-amylases.

Phylogenetic distribution of intron positions in alpha-amylase genes of bilateria suggests numerous gains and losses.

Most eukaryotes have at least some genes interrupted by introns. While it is well accepted that introns were already present at moderate density in the last eukaryote common ancestor, the conspicuous diversity of intron density among genomes suggests a complex evolutionary history, with marked differences between phyla. The question of the rates of intron gains and loss in the course of evolution and factors influencing them remains controversial. We have investigated a single gene family, alpha-amylase, in 55 species covering a variety of animal phyla. Comparison of intron positions across phyla suggests a complex history, with a likely ancestral intronless gene undergoing frequent intron loss and gain, leading to extant intron/exon structures that are highly variable, even among species from the same phylum. Because introns are known to play no regulatory role in this gene and there is no alternative splicing, the structural differences may be interpreted more easily: intron positions, sizes, losses or gains may be more likely related to factors linked to splicing mechanisms and requirements, and to recognition of introns and exons, or to more extrinsic factors, such as life cycle and population size. We have shown that intron losses outnumbered gains in recent periods, but that "resets" of intron positions occurred at the origin of several phyla, including vertebrates. Rates of gain and loss appear to be positively correlated. No phase preference was found. We also found evidence for parallel gains and for intron sliding. Presence of introns at given positions was correlated to a strong protosplice consensus sequence AG/G, which was much weaker in the absence of intron. In contrast, recent intron insertions were not associated with a specific sequence. In animal Amy genes, population size and generation time seem to have played only minor roles in shaping gene structures.

Polyphyly of the Zaprionus genus group (Diptera: Drosophilidae).

The Zaprionus genus group comprises three drosophilid genera (Zaprionus, Phorticella and Samoaia) that are thought to be related to the Drosophila immigrans species group. We revised the phylogenetic relationships among the three genera and their placement within the subfamily Drosophilinae using one mitochondrial (COII) and one nuclear (Amyrel) gene. The Bayesian tree inferred from concatenated amino acid sequences of the two genes strongly suggests the polyphyly of the Zaprionus genus group and of each of the genera Zaprionus and Phorticella. Paraphyly of the D.immigrans species group was also shown here; the quadrilineata subgroup formed the sister clade to the genus Samoaia. These results suggest the necessity of taxonomic revisions for some relevant genera and species groups included within the genus Drosophila.

Grafting the molecular phylogenetic tree with morphological branches to reconstruct the evolutionary history of the genus Zaprionus (Diptera: Drosophilidae).

A molecular phylogeny for the drosophilid genus Zaprionus was inferred using a mitochondrial (CO-II) and a nuclear (Amyrel) gene using 22 available species. The combined molecular tree does not support the current classification, dubbed phylogenetic, based entirely upon a morphocline of forefemoral ornamentation. For species for which DNA was not available, phylogenetic positioning was only assigned using morphological characters. In order to avoid conflict between DNA and morphology in the combined analyses (supermatrix method), we developed a new method in which few morphological characters were sampled according to an a priori homoplasy assessment on the consensus molecular tree. At each internal node of the tree, a number of synapomorphies was determined, and species with no molecular sequences were grafted thereon. Analogously to tree vocabulary, we called our method 'morphological grafting'. New species groups and complexes were then defined in the light of our findings. Further, divergence times were estimated under a relaxed molecular clock, and historical biogeography was reconstructed under a maximum likelihood model. Zaprionus appears to be of recent origin in the Oriental region during the Late Miocene ( approximately 10 MYA), and colonization of Africa started shortly after ( approximately 7 MYA) via the maritime route of the Indian Ocean Islands. Most of the morphological and ecological diversification took place, later, in Western Africa during the Quaternary cyclic climatic changes. Furthermore, some species became recent invaders, with one, Zaprionus indianus, has successfully invaded South and North America during the last decade.

Evolution of testicular architecture in the Drosophilidae: a role for sperm length.

BACKGROUND: Evolutionary biologists have so far largely treated the testis as a black box with a certain size, a matching resource demand and a resulting sperm output. A better understanding of the way that the testis responds to selection may come from recent developments in theoretical biology aimed at understanding the factors that influence the evolution of tissue architecture (i.e. the logical organisation of a tissue). Here we perform a comparative analysis of aspects of testicular architecture of the fruit fly family Drosophilidae. Specifically, we collect published information on the number of first (or primary) spermatocytes in spermatogenesis, which allows to infer an important aspect of testicular architecture. RESULTS: We show that testicular architecture is much more variable (both within and between species) than is generally appreciated. Moreover, the number of first spermatocytes is strongly correlated to the sperm length, which is inversely related to the sperm production, and thus the workload of the testis. CONCLUSION: Our study clearly documents that tissue architecture can evolve, and that in the Drosophilidae it may do so in response to sexual selection. We conclude that the testis of the Drosophilidae is a promising model organ to test recent models of tissue architecture.

Evolution of a desaturase involved in female pheromonal cuticular hydrocarbon biosynthesis and courtship behavior in Drosophila.

Drosophila species exhibit polymorphism in female pheromonal cuticular hydrocarbons, with 7-monoenes produced in Drosophila simulans and 7,11-dienes in most populations of Drosophila melanogaster (5,9-dienes in several African populations). A female-biased desaturase, desatF, expressed only in D. melanogaster is involved in the synthesis of 7,11-dienes. We investigated the role of desatF in 5,9-diene flies. We constructed a 5,9-diene strain knock-down for desatF and showed that desatF is involved in 5,9-diene formation. We also studied D. melanogaster/D. simulans hybrids. These hybrid females produced dienes and received normal courtship from D. melanogaster males, but copulation success was reduced. With D. simulans males, courtship was decreased and no copulation occurred. Hybrids with a chromosomal deletion of the D. melanogaster desatF gene had no dienes and received normal courtship from D. simulans males; depending on the D. simulans parental strain, 7-19% of them succeeded in mating. D. simulans desatF promoter region shows 21-23% gaps and 86-89% identity with D. melanogaster promoter region, the coding region 93-94% identity, depending on the strain. These differences could explain the functional polymorphism of desatF observed between both species, contributing to different cuticular hydrocarbon profiles, that constitute an effective barrier between species.

Where do animal alpha-amylases come from? An interkingdom trip.

Alpha-amylases are widely found in eukaryotes and prokaryotes. Few amino acids are conserved among these organisms, but at an intra-kingdom level, conserved protein domains exist. In animals, numerous conserved stretches are considered as typical of animal alpha-amylases. Searching databases, we found no animal-type alpha-amylases outside the Bilateria. Instead, we found in the sponge Reniera sp. and in the sea anemone Nematostella vectensis, alpha-amylases whose most similar cognate was that of the amoeba Dictyostelium discoideum. We found that this "Dictyo-type" alpha-amylase was shared not only by these non-Bilaterian animals, but also by other Amoebozoa, Choanoflagellates, and Fungi. This suggested that the Dictyo-type alpha-amylase was present in the last common ancestor of Unikonts. The additional presence of the Dictyo-type in some Ciliates and Excavates, suggests that horizontal gene transfers may have occurred among Eukaryotes. We have also detected putative interkingdom transfers of amylase genes, which obscured the historical reconstitution. Several alternative scenarii are discussed.

Origin and evolution of the Amyrel gene in the alpha-amylase multigene family of Diptera.

Alpha-amylase genes often form multigene families in living organisms. In Diptera, a remote paralog, Amyrel, had been discovered in Drosophila, where this gene is currently used as a population and phylogenetic marker. The putative encoded protein has about 40% divergence with the classical amylases. We have searched the presence of the paralog in other families of Diptera to track its origin and understand its evolution. Amyrel was detected in a number of families of Muscomorpha (Brachycera-Cyclorrapha), suggesting an origin much older than previously thought. It has not been found elsewhere to date, and it is absent from the Anopheles gambiae genome. The intron-exon structures of the genes found so far suggest that the ancestral gene (before the duplication which gave rise to Amyrel) had two introns, and that subsequent, repeated and independent loss of one or both introns occurred in some Muscomorpha families. It seems that the Amyrel protein has experienced specific amino acid substitutions in regions generally well conserved in amylases, raising the possibility of peculiar, functional adaptations of this protein.