Abdelaziz Heddi

Antimicrobial Peptides Keep Insect Endosymbionts Under Control

A beetle species synthesizes an antimicrobial peptide to constrain a bacterial symbiont in specialized organs. Vertically transmitted endosymbionts persist for millions of years in invertebrates and play an important role in animal evolution. However, the functional basis underlying the maintenance of these long-term resident bacteria is unknown. We report that the weevil coleoptericin-A (ColA) antimicrobial peptide selectively targets endosymbionts within the bacteriocytes and regulates their growth through the inhibition of cell division. Silencing the colA gene with RNA interference resulted in a decrease in size of the giant filamentous endosymbionts, which escaped from the bacteriocytes and spread into insect tissues. Although this family of peptides is commonly linked with microbe clearance, this work shows that endosymbiosis benefits from ColA, suggesting that long-term host-symbiont coevolution might have shaped immune effectors for symbiont maintenance.

Lactobacillus plantarum strain maintains growth of infant mice during chronic undernutrition

Microbiota and infant development Malnutrition in children is a persistent challenge that is not always remedied by improvements in nutrition. This is because a characteristic community of gut microbes seems to mediate some of the pathology. Human gut microbes can be transplanted effectively into germ-free mice to recapitulate their associated phenotypes. Using this model, Blanton et al. found that the microbiota of healthy children relieved the harmful effects on growth caused by the microbiota of malnourished children. In infant mammals, chronic undernutrition results in growth hormone resistance and stunting. In mice, Schwarzer et al. showed that strains of Lactobacillus plantarum in the gut microbiota sustained growth hormone activity via signaling pathways in the liver, thus overcoming growth hormone resistance. Together these studies reveal that specific beneficial microbes could potentially be exploited to resolve undernutrition syndromes. Science, this issue p. 10.1126/science.aad3311, p. 854 The gut microbiota supports the growth of juvenile mice via growth hormone signaling. In most animal species, juvenile growth is marked by an exponential gain in body weight and size. Here we show that the microbiota of infant mice sustains both weight gain and longitudinal growth when mice are fed a standard laboratory mouse diet or a nutritionally depleted diet. We found that the intestinal microbiota interacts with the somatotropic hormone axis to drive systemic growth. Using monocolonized mouse models, we showed that selected lactobacilli promoted juvenile growth in a strain-dependent manner that recapitulated the microbiotas effect on growth and the somatotropic axis. These findings show that the hosts microbiota supports juvenile growth. Moreover, we discovered that lactobacilli strains buffered the adverse effects of chronic undernutrition on the postnatal growth of germ-free mice.

MOLECULAR CHARACTERIZATION OF THE PRINCIPAL SYMBIOTIC BACTERIA OF THE WEEVIL SITOPHILUS ORYZAE : A PECULIAR G + C CONTENT OF AN ENDOCYTOBIOTIC DNA

Abstract. The principal intracellular symbiotic bacteria of the cereal weevil Sitophilus oryzae were characterized using the sequence of the 16S rDNA gene (rrs gene) and G + C content analysis. Polymerase chain reaction amplification with universal eubacterial primers of the rrs gene showed a single expected sequence of 1,501 bp. Comparison of this sequence with the available database sequences placed the intracellular bacteria of S. oryzae as members of the Enterobacteriaceae family, closely related to the free-living bacteria, Erwinia herbicola and Escherichia coli, and the endocytobiotic bacteria of the tsetse fly and aphids. Moreover, by high-performance liquid chromatography, we measured the genomic G + C content of the S. oryzae principal endocytobiotes (SOPE) as 54%, while the known genomic G + C content of most intracellular bacteria is about 39.5%. Furthermore, based on the third codon position G + C content and the rrs gene G + C content, we demonstrated that most intracellular bacteria except SOPE are A + T biased irrespective of their phylogenetic position. Finally, using the hsp60 gene sequence, the codon usage of SOPE was compared with that of two phylogenetically closely related bacteria: E. coli, a free-living bacterium, and Buchnera aphidicola, the intracellular symbiotic bacteria of aphids. Taken together, these results show a peculiar and distinctly different DNA composition of SOPE with respect to the other obligate intracellular bacteria, and, combined with biological and biochemical data, they elucidate the evolution of symbiosis in S. oryzae.

Analysis of milk gland structure and function in Glossina morsitans : Milk protein production, symbiont populations and fecundity

A key process in the tsetse reproductive cycle is the transfer of essential nutrients and bacterial symbionts from mother to intrauterine offspring. The tissue mediating this transfer is the milk gland. This work focuses upon the localization and function of two milk proteins (milk gland protein (GmmMGP) and transferrin (GmmTsf)) and the tsetse endosymbionts (Sodalis and Wigglesworthia), in the context of milk gland physiology. Fluorescent in situ hybridization (FISH) and immunohistochemical analysis confirm that the milk gland secretory cells synthesize and secrete milk gland protein and transferrin. Knockdown of gmmmgp by double stranded RNA (dsRNA) mediated RNA interference results in reduction of tsetse fecundity, demonstrating its functional importance in larval nutrition and development. Bacterial species-specific in situ hybridizations of milk gland sections reveal large numbers of Sodalis and Wigglesworthia within the lumen of the milk gland. Sodalis is also localized within the cytoplasm of the secretory cells. Within the lumen, Wigglesworthia localize close to the channels leading to the milk storage reservoir of the milk gland secretory cells. We discuss the significance of the milk gland in larval nutrition and in transmission of symbiotic bacteria to developing offspring.

Coexistence of Wolbachia with Buchnera aphidicola and a Secondary Symbiont in the Aphid Cinara cedri

Intracellular symbiosis is very common in the insect world. For the aphid Cinara cedri, we have identified by electron microscopy three symbiotic bacteria that can be characterized by their different sizes, morphologies, and electrodensities. PCR amplification and sequencing of the 16S ribosomal DNA (rDNA) genes showed that, in addition to harboring Buchnera aphidicola, the primary endosymbiont of aphids, C. cedri harbors a secondary symbiont (S symbiont) that was previously found to be associated with aphids (PASS, or R type) and an alpha-proteobacterium that belongs to the Wolbachia genus. Using in situ hybridization with specific bacterial probes designed for symbiont 16S rDNA sequences, we have shown that Wolbachia was represented by only a few minute bacteria surrounding the S symbionts. Moreover, the observed B. aphidicola and the S symbionts had similar sizes and were housed in separate specific bacterial cells, the bacteriocytes. Interestingly, in contrast to the case for all aphids examined thus far, the S symbionts were shown to occupy a similarly sized or even larger bacteriocyte space than B. aphidicola. These findings, along with the facts that C. cedri harbors the B. aphidicola strain with the smallest bacterial genome and that the S symbionts infect all Cinara spp. analyzed so far, suggest the possibility of bacterial replacement in these species.

Successful horizontal transfer of Wolbachia symbionts between Trichogramma wasps

Rickettsial symbionts of the genus Wolbachia, harboured by many arthropod species, are implicated in feminization, cytoplasmic incompatibility and parthenogenesis phenomena. These symbionts induce thelytokous parthenogenesis in some egg parasitoids of the Trichogramma genus. In our study of these minute wasps, purified Wolbachia from an infected species, T. pretiosum, were transferred by microinjection into in vitro developed pupae of an uninfected species, T. dendrolimi. We believe this to be the first successful transfer of Wolbachia in parasitoids. The presence or absence of Wolbachia was determined using DAPI staining, PCR and ftsZ gene sequencing. An ftsZ gene fragment from microinjected T. dendrolimi was shown to be identical to that of T. pretiosum, confirming that transfer was successful. Wolbachia were still present in the recipient species 26 generations after the transfer, although only partial induction of thelytoky was observed. Therefore, in Trichogramma, density of symbionts or symbiont–host interactions may be involved in the expression of parthenogenesis. The successful transfer of the symbiont within the Trichogrammatidae, a group of insects used in biological control strategies, could increase their agronomic importance by manipulating their reproductive system.

Long-Term Evolutionary Stability of Bacterial Endosymbiosis in Curculionoidea: Additional Evidence of Symbiont Replacement in the Dryophthoridae Family

Bacterial intracellular symbiosis (endosymbiosis) is well documented in the insect world where it is believed to play a crucial role in adaptation and evolution. However, although Coleopteran insects are of huge ecological and economical interest, endosymbiont molecular analysis is limited to the Dryophthoridae family. Here, we have analyzed the intracellular symbiotic bacteria in 2 Hylobius species belonging to the Molytinae subfamily (Curculionoidea superfamily) that exhibit different features from the Dryophthoridae insects in terms of their ecology and geographical spanning. Fluorescence in situ hybridization has shown that both Hylobius species harbor rod-shaped pleiomorphic symbiotic bacteria in the oocyte and in the bacteria-bearing organ (the bacteriome), with a shape and location similar to those of the Dryophthoridae bacteriome. Phylogenetic analysis of the 16S ribosomal DNA gene sequences, using the heterogeneous model of DNA evolution, has placed the Hylobius spp. endosymbionts (H-group) at the basal position of the ancestral R-clade of Dryophthoridae endosymbionts named Candidatus Nardonella but relatively distant from the S-clade of Sitophilus spp. endosymbionts. Endosymbionts from the H-group and the R-clade evolved more quickly compared with free-living enteric bacteria and endosymbionts from the S- and D-clades of Dryophthoridae. They are AT biased (58.3% A + T), and they exhibit AT-rich insertions at the same position as previously described in the Candidatus Nardonella 16S rDNA sequence. Moreover, the host phylogenetic tree based on the mitochondrial COI gene was shown to be highly congruent with the H-group and the R-clade, the divergence of which was estimated to be around 125 MYA. These new molecular data show that endosymbiosis is old in Curculionids, going back at least to the common ancestor of Molytinae and Dryophthoridae, and is evolutionary stable, except in 2 Dryophthoridae clades, providing additional and independent supplementary evidence for endosymbiont replacement in these taxa.

Identification of the Weevil immune genes and their expression in the bacteriome tissue

BackgroundPersistent infections with mutualistic intracellular bacteria (endosymbionts) are well represented in insects and are considered to be a driving force in evolution. However, while pathogenic relationships have been well studied over the last decades very little is known about the recognition of the endosymbionts by the host immune system and the mechanism that limits their infection to the bacteria-bearing host tissue (the bacteriome).ResultsTo study bacteriome immune specificity, we first identified immune-relevant genes of the weevil Sitophilus zeamais by using suppressive subtractive hybridization (SSH) and then analyzed their full-length coding sequences obtained by RACE-PCR experiments. We then measured immune gene expression in the bacteriome, and in the aposymbiotic larvae following S. zeamais primary endosymbiont (SZPE) injection into the hemolymph, in order to consider the questions of bacteriome immune specificity and the insect humoral response to symbionts. We show that larval challenge with the endosymbiont results in a significant induction of antibacterial peptide genes, providing evidence that, outside the bacteriome, SZPE are recognized as microbial intruders by the host. In the bacteriome, gene expression analysis shows the overexpression of one antibacterial peptide from the coleoptericin family and, intriguingly, homologs to genes described as immune modulators (that is, PGRP-LB, Tollip) were also shown to be highly expressed in the bacteriome.ConclusionThe current data provide the first description of immune gene expression in the insect bacteriome. Compared with the insect humoral response to SZPE, the bacteriome expresses few genes among those investigated in this work. This local immune gene expression may help to maintain the endosymbiont in the bacteriome and prevent its invasion into insect tissues. Further investigations of the coleoptericin, the PGRP and the Tollip genes should elucidate the role of the host immune system in the maintenance and regulation of endosymbiosis.

Genome Degeneration and Adaptation in a Nascent Stage of Symbiosis

Symbiotic associations between animals and microbes are ubiquitous in nature, with an estimated 15% of all insect species harboring intracellular bacterial symbionts. Most bacterial symbionts share many genomic features including small genomes, nucleotide composition bias, high coding density, and a paucity of mobile DNA, consistent with long-term host association. In this study, we focus on the early stages of genome degeneration in a recently derived insect-bacterial mutualistic intracellular association. We present the complete genome sequence and annotation of Sitophilus oryzae primary endosymbiont (SOPE). We also present the finished genome sequence and annotation of strain HS, a close free-living relative of SOPE and other insect symbionts of the Sodalis-allied clade, whose gene inventory is expected to closely resemble the putative ancestor of this group. Structural, functional, and evolutionary analyses indicate that SOPE has undergone extensive adaptation toward an insect-associated lifestyle in a very short time period. The genome of SOPE is large in size when compared with many ancient bacterial symbionts; however, almost half of the protein-coding genes in SOPE are pseudogenes. There is also evidence for relaxed selection on the remaining intact protein-coding genes. Comparative analyses of the whole-genome sequence of strain HS and SOPE highlight numerous genomic rearrangements, duplications, and deletions facilitated by a recent expansion of insertions sequence elements, some of which appear to have catalyzed adaptive changes. Functional metabolic predictions suggest that SOPE has lost the ability to synthesize several essential amino acids and vitamins. Analyses of the bacterial cell envelope and genes encoding secretion systems suggest that these structures and elements have become simplified in the transition to a mutualistic association.

Wolbachia symbiont infections induce strong cytoplasmic incompatibility in the tsetse fly Glossina morsitans.

Tsetse flies are vectors of the protozoan parasite African trypanosomes, which cause sleeping sickness disease in humans and nagana in livestock. Although there are no effective vaccines and efficacious drugs against this parasite, vector reduction methods have been successful in curbing the disease, especially for nagana. Potential vector control methods that do not involve use of chemicals is a genetic modification approach where flies engineered to be parasite resistant are allowed to replace their susceptible natural counterparts, and Sterile Insect technique (SIT) where males sterilized by chemical means are released to suppress female fecundity. The success of genetic modification approaches requires identification of strong drive systems to spread the desirable traits and the efficacy of SIT can be enhanced by identification of natural mating incompatibility. One such drive mechanism results from the cytoplasmic incompatibility (CI) phenomenon induced by the symbiont Wolbachia. CI can also be used to induce natural mating incompatibility between release males and natural populations. Although Wolbachia infections have been reported in tsetse, it has been a challenge to understand their functional biology as attempts to cure tsetse of Wolbachia infections by antibiotic treatment damages the obligate mutualistic symbiont (Wigglesworthia), without which the flies are sterile. Here, we developed aposymbiotic (symbiont-free) and fertile tsetse lines by dietary provisioning of tetracycline supplemented blood meals with yeast extract, which rescues Wigglesworthia-induced sterility. Our results reveal that Wolbachia infections confer strong CI during embryogenesis in Wolbachia-free (GmmApo) females when mated with Wolbachia-infected (GmmWt) males. These results are the first demonstration of the biological significance of Wolbachia infections in tsetse. Furthermore, when incorporated into a mathematical model, our results confirm that Wolbachia can be used successfully as a gene driver. This lays the foundation for new disease control methods including a population replacement approach with parasite resistant flies. Alternatively, the availability of males that are reproductively incompatible with natural populations can enhance the efficacy of the ongoing sterile insect technique (SIT) applications by eliminating the need for chemical irradiation.