PacBio Hi-Fi genome assembly of Sipha maydis, a model for the study of multipartite mutualism in insects.

Dependence on multiple nutritional endosymbionts has evolved repeatedly in insects feeding on unbalanced diets. However, reference genomes for species hosting multi-symbiotic nutritional systems are lacking, even though they are essential for deciphering the processes governing cooperative life between insects and anatomically integrated symbionts. The cereal aphid Sipha maydis is a promising model for addressing these issues, as it has evolved a nutritional dependence on two bacterial endosymbionts that complement each other. In this study, we used PacBio High fidelity (HiFi) long-read sequencing to generate a highly contiguous genome assembly of S. maydis with a length of 410 Mb, 3,570 contigs with a contig N50 length of 187 kb, and BUSCO completeness of 95.5%. We identified 117 Mb of repetitive sequences, accounting for 29% of the genome assembly, and predicted 24,453 protein-coding genes, of which 2,541 were predicted enzymes included in an integrated metabolic network with the two aphid-associated endosymbionts. These resources provide valuable genetic and metabolic information for understanding the evolution and functioning of multi-symbiotic systems in insects.

Genomic insights into Spiroplasma endosymbionts that induce male-killing and protective phenotypes in the pea aphid.

The endosymbiotic bacteria Spiroplasma (Mollicutes) infect diverse plants and arthropods, and some of which induce male killing, where male hosts are killed during development. Male-killing Spiroplasma strains belong to either the phylogenetically distant Citri-Poulsonii or Ixodetis groups. In Drosophila flies, Spiroplasma poulsonii induces male killing via the Spaid toxin. While Spiroplasma ixodetis infects a wide range of insects and arachnids, little is known about the genetic basis of S. ixodetis-induced male killing. Here, we analyzed the genome of S. ixodetis strains in the pea aphid Acyrthosiphon pisum (Aphididae, Hemiptera). Genome sequencing constructed a complete genome of a male-killing strain, sAp269, consisting of a 1.5 Mb circular chromosome and an 80 Kb plasmid. sAp269 encoded putative virulence factors containing either ankyrin repeat, ovarian tumor-like deubiquitinase, or ribosome inactivating protein domains, but lacked the Spaid toxin. Further comparative genomics of Spiroplasma strains in A. pisum biotypes adapted to different host plants revealed their phylogenetic associations and the diversity of putative virulence factors. Although the mechanisms of S. ixodetis-induced male killing in pea aphids remain elusive, this study underlines the dynamic genome evolution of S. ixodetis and proposes independent acquisition events of male-killing mechanisms in insects.

Low prevalence of secondary endosymbionts in aphids sampled from rapeseed crops in Germany.

Peach-potato aphids, Myzus persicae Sulzer (Hemiptera:Aphididae), and cabbage aphids, Brevicoryne brassicae Linnaeus (Hemiptera:Aphididae), are herbivorous insects of significant agricultural importance. Aphids can harbour a range of non-essential (facultative) endosymbiotic bacteria that confer multiple costs and benefits to the host aphid. A key endosymbiont-derived phenotype is protection against parasitoid wasps, and this protective phenotype has been associated with several defensive enodsymbionts. In recent years greater emphasis has been placed on developing alternative pest management strategies, including the increased use of natural enemies such as parasitoids wasps. For the success of aphid control strategies to be estimated the presence of defensive endosymbionts that can potentially disrupt the success of biocontrol agents needs to be determined in natural aphid populations. Here, we sampled aphids and mummies (parasitised aphids) from an important rapeseed production region in Germany and used multiplex PCR assays to characterise the endosymbiont communities. We found that aphids rarely harboured facultative endosymbionts, with 3.6% of M. persicae and 0% of B. brassicae populations forming facultative endosymbiont associations. This is comparable with endosymbiont prevalence described for M. persicae populations surveyed in Australia, Europe, Chile, and USA where endosymbiont infection frequencies range form 0-2%, but is in contrast with observations from China where M. persicae populations have more abundant and diverse endosymbiotic communities (endosymbionts present in over 50% of aphid populations).

High specificity of symbiont-conferred resistance in an aphid-parasitoid field community.

Host-parasite coevolution is mediated by genetic interactions between the antagonists and may lead to reciprocal adaptation. In the black bean aphid, Aphis fabae fabae, resistance to parasitoids can be conferred by the heritable bacterial endosymbiont Hamiltonella defensa. H. defensa has been shown to be variably protective against different parasitoid species, and different genotypes of the black bean aphid's main parasitoid Lysiphlebus fabarum. However, these results were obtained using haphazard combinations of laboratory-reared insect lines with different origins, making it unclear how representative they are of natural, locally (co)adapted communities. We therefore comprehensively sampled the parasitoids of a natural A. f. fabae population and measured the ability of the five most abundant species to parasitize aphids carrying the locally prevalent H. defensa haplotypes. H. defensa provided resistance only against the dominant parasitoid L. fabarum (70% of all parasitoids), but not against less abundant parasitoids, and resistance to L. fabarum acted in a genotype-specific manner (G × G interactions between H. defensa and L. fabarum). These results confirm that strong species- and genotype-specificity of symbiont-conferred resistance is indeed a hallmark of wild A. f. fabae populations, and they are consistent with symbiont-mediated adaptation of aphids to the parasitoids posing the highest risk.

Bioefficacy of engineered Beauveria bassiana with scorpion neurotoxin, LqqIT1 against cotton mealybug, Phenacoccus solenopsis and cowpea aphid, Aphis craccivora.

Cotton mealybug, Phenacoccus solenopsis (Tinsley) and cowpea aphid Aphis craccivora (Koch) are notorious polyphagous, hemipteran sap sucking insect pests. A recombinant toxin gene 'LqqIT1' from the scorpion Leiurus quinquestriatus quinquestriatus (Ehrenberg) was cloned in the pAL1 fungal expression vector and then expressed in the entomopathogenic fungus Beauveria bassiana (Balasmo) using genetic modification techniques. The genetically transformed B. bassiana strain (BbLqqIT1-3) and its un-transformed parent strain (Bb-C) were screened to infect the third instar nymphs of P. solenopsis and first instar nymph of A. craccivora through leaf treatment and topical application (spray) method at 1 * 107 spores per ml concentration. The recombinant strain BbLqqIT1-3 was highly pathogenic against A. craccivora but non pathogenic to P. solenopsis. BbLqqIT1-3 induced 72 and 43.33% mortality in A. craccivora nymphs 96 h after leaf treatment and topical application, respectively. The nymphs of A. craccivora infected with BbLqqIT1-3 displayed classical neurotoxic symptoms such as sluggishness, solublize and liquification of the body. Crude soluble toxin protein, BbLqqIT1a-CSE and Bb-WT-CSE was extracted from the BbLqqIT1-3 and Bb-C, respectively using ammonium sulphate precipitation method, and their oral toxicity was analyzed at 5 µg/ml concentration. The survival of the studied insects was negatively affected by the crude soluble toxin extracts. The LT50 values of BbLqqIT1a-CSE against P. solenopsis and A. craccivora were 22.18 and 17.69 h, respectively. Exposure to crude soluble toxin extracts also accounted for the imbalance of ionic concentrations in the hemolymph of treated insects such as hyperpotassemia (3.53-8.18 meq/ml) in the P. solenopsis and hypopotassemia (7.52-0.47 meq/ml) in A. craccivora. The transformed fungus BbLqqIT1-3 strain exhibited promising results in invitro study.

Discovery of a novel circulation route of free-living Serratiasymbiotica mediated by predatory ladybird beetles.

Horizontal transmission of bacteria to varied hosts can maintain and even expand microbial niches. We previously found that the aphid gut bacterium Serratia symbiotica strain SsMj can be transmitted to ladybird beetles via predation, but whether the predator is a new host, a reservoir or a dead end of this bacterium is unknown. This study aims to provide a clear picture of SsMj circulation from aphids to plants and predators. We first found that SsMj in aphids and ladybirds was abundantly distributed not only in digestive tracts but also in droppings. We found no evidence for vertical transmission of SsMj to aphid offspring. Instead, we showed that it could be transmitted to conspecific aphids by sharing the same plant or contacting honeydews. The key finding of this study is that SsMj was transmitted from aphids to ladybirds through predation, while ladybirds could also transfer SsMj back to aphids, possibly through feces. Together, this evidence suggests that SsMj is able to survive in the digestive tracts and droppings of insects and to expand its host range with plants and predators as reservoirs.

Co-option of a conserved host glutamine transporter facilitates aphid/Buchnera metabolic integration.

Organisms across the tree of life colonize novel environments by partnering with bacterial symbionts. These symbioses are characterized by intimate integration of host/endosymbiont biology at multiple levels, including metabolically. Metabolic integration is particularly important for sap-feeding insects and their symbionts, which supplement nutritionally unbalanced host diets. Many studies reveal parallel evolution of host/endosymbiont metabolic complementarity in amino acid biosynthesis, raising questions about how amino acid metabolism is regulated, how regulatory mechanisms evolve, and the extent to which similar mechanisms evolve in different systems. In the aphid/Buchnera symbiosis, the transporter ApGLNT1 (Acyrthosiphon pisum glutamine transporter 1) supplies glutamine, an amino donor in transamination reactions, to bacteriocytes (where Buchnera reside) and is competitively inhibited by Buchnera-supplied arginine-consistent with a role regulating amino acid metabolism given host demand for Buchnera-produced amino acids. We examined how ApGLNT1 evolved a regulatory role by functionally characterizing orthologs in insects with and without endosymbionts. ApGLNT1 orthologs are functionally similar, and orthology searches coupled with homology modeling revealed that GLNT1 is ancient and structurally conserved across insects. Our results indicate that the ApGLNT1 symbiotic regulatory role is derived from its ancestral role and, in aphids, is likely facilitated by loss of arginine biosynthesis through the urea cycle. Given consistent loss of host arginine biosynthesis and retention of endosymbiont arginine supply, we hypothesize that GLNT1 is a general mechanism regulating amino acid metabolism in sap-feeding insects. This work fills a gap, highlighting the broad importance of co-option of ancestral proteins to novel contexts in the evolution of host/symbiont systems.

Mobile elements create strain-level variation in the services conferred by an aphid symbiont.

Heritable, facultative symbionts are common in arthropods, often functioning in host defence. Despite moderately reduced genomes, facultative symbionts retain evolutionary potential through mobile genetic elements (MGEs). MGEs form the primary basis of strain-level variation in genome content and architecture, and often correlate with variability in symbiont-mediated phenotypes. In pea aphids (Acyrthosiphon pisum), strain-level variation in the type of toxin-encoding bacteriophages (APSEs) carried by the bacterium Hamiltonella defensa correlates with strength of defence against parasitoids. However, co-inheritance creates difficulties for partitioning their relative contributions to aphid defence. Here we identified isolates of H. defensa that were nearly identical except for APSE type. When holding H. defensa genotype constant, protection levels corresponded to APSE virulence module type. Results further indicated that APSEs move repeatedly within some H. defensa clades providing a mechanism for rapid evolution in anti-parasitoid defences. Strain variation in H. defensa also correlates with the presence of a second symbiont Fukatsuia symbiotica. Predictions that nutritional interactions structured this coinfection were not supported by comparative genomics, but bacteriocin-containing plasmids unique to co-infecting strains may contribute to their common pairing. In conclusion, strain diversity, and joint capacities for horizontal transfer of MGEs and symbionts, are emergent players in the rapid evolution of arthropods.

Cryptic community structure and metabolic interactions among the heritable facultative symbionts of the pea aphid.

Most insects harbour influential, yet non-essential heritable microbes in their hemocoel. Communities of these symbionts exhibit low diversity. But their frequent multi-species nature raises intriguing questions on roles for symbiont-symbiont synergies in host adaptation, and on the stability of the symbiont communities, themselves. In this study, we build on knowledge of species-defined symbiont community structure across US populations of the pea aphid, Acyrthosiphon pisum. Through extensive symbiont genotyping, we show that pea aphids' microbiomes can be more precisely defined at the symbiont strain level, with strain variability shaping five out of nine previously reported co-infection trends. Field data provide a mixture of evidence for synergistic fitness effects and symbiont hitchhiking, revealing causes and consequences of these co-infection trends. To test whether within-host metabolic interactions predict common versus rare strain-defined communities, we leveraged the high relatedness of our dominant, community-defined symbiont strains vs. 12 pea aphid-derived Gammaproteobacteria with sequenced genomes. Genomic inference, using metabolic complementarity indices, revealed high potential for cooperation among one pair of symbionts-Serratia symbiotica and Rickettsiella viridis. Applying the expansion network algorithm, through additional use of pea aphid and obligate Buchnera symbiont genomes, Serratia and Rickettsiella emerged as the only symbiont community requiring both parties to expand holobiont metabolism. Through their joint expansion of the biotin biosynthesis pathway, these symbionts may span missing gaps, creating a multi-party mutualism within their nutrient-limited, phloem-feeding hosts. Recent, complementary gene inactivation, within the biotin pathways of Serratia and Rickettsiella, raises further questions on the origins of mutualisms and host-symbiont interdependencies.

A diversity of endosymbionts across Australian aphids and their persistence in aphid cultures.

There is increasing interest in the use of endosymbionts in pest control, which will benefit from the identification of endosymbionts from potential donor species for transfer to pest species. Here, we screened for endosymbionts in 123 Australian aphid samples across 32 species using 16S DNA metabarcoding. We then developed a qPCR method to validate the metabarcoding data set and to monitor endosymbiont persistence in aphid cultures. Pea aphids (Acyrthosiphon pisum) were frequently coinfected with Rickettsiella and Serratia, and glasshouse potato aphids (Aulacorthum solani) were coinfected with Regiella and Spiroplasma; other secondary endosymbionts detected in samples occurred by themselves. Hamiltonella, Rickettsia and Wolbachia were restricted to a single aphid species, whereas Regiella was found in multiple species. Rickettsiella, Hamiltonella and Serratia were stably maintained in laboratory cultures, although others were lost rapidly. The overall incidence of secondary endosymbionts in Australian samples tended to be lower than recorded from aphids overseas. These results indicate that aphid endosymbionts probably exhibit different levels of infectivity and vertical transmission efficiency across hosts, which may contribute to natural infection patterns. The rapid loss of some endosymbionts in cultures raises questions about factors that maintain them under field conditions, while endosymbionts that persisted in laboratory culture provide candidates for interspecific transfers.

Defensive symbiosis in the wild: Seasonal dynamics of parasitism risk and symbiont-conferred resistance.

Parasite-mediated selection can rapidly drive up resistance levels in host populations, but fixation of resistance traits may be prevented by costs of resistance. Black bean aphids (Aphis fabae) benefit from increased resistance to parasitoids when carrying the defensive bacterial endosymbiont Hamiltonella defensa. However, due to fitness costs that come with symbiont infection, symbiont-conferred resistance may result in either a net benefit or a net cost to the aphid host, depending on parasitoid presence as well as on the general ecological context. Balancing selection may therefore explain why in natural aphid populations, H. defensa is often found at intermediate frequencies. Here we present a 2-year field study where we set out to look for signatures of balancing selection in natural aphid populations. We collected temporally well-resolved data on the prevalence of H. defensa in A. f. fabae and estimated the risk imposed by parasitoids using sentinel hosts. Despite a marked and consistent early-summer peak in parasitism risk, and significant changes in symbiont prevalence over time, we found just a weak correlation between parasitism risk and H. defensa frequency dynamics. H. defensa prevalence in the populations under study was, in fact, better explained by the number of heat days that previous aphid generations were exposed to. Our study grants an unprecedentedly well-resolved insight into the dynamics of endosymbiont and parasitoid communities of A. f. fabae populations, and it adds to a growing body of empirical evidence suggesting that not only parasitism risk, but rather multifarious selection is shaping H. defensa prevalence in the wild.

A rapidly spreading deleterious aphid endosymbiont that uses horizontal as well as vertical transmission.

Endosymbiotic bacteria that live inside the cells of insects are typically only transmitted maternally and can spread by increasing host fitness and/or modifying reproduction in sexual hosts. Transinfections of Wolbachia endosymbionts are now being used to introduce useful phenotypes into sexual host populations, but there has been limited progress on applications using other endosymbionts and in asexual populations. Here, we develop a unique pathway to application in aphids by transferring the endosymbiont Rickettsiella viridis to the major crop pest Myzus persicae. Rickettsiella infection greatly reduced aphid fecundity, decreased heat tolerance, and modified aphid body color, from light to dark green. Despite inducing host fitness costs, Rickettsiella spread rapidly through caged aphid populations via plant-mediated horizontal transmission. The phenotypic effects of Rickettsiella were sensitive to temperature, with spread only occurring at 19 °C and not 25 °C. Body color modification was also lost at high temperatures despite Rickettsiella maintaining a high density. Rickettsiella shows the potential to spread through natural M. persicae populations by horizontal transmission and subsequent vertical transmission. Establishment of Rickettsiella in natural populations could reduce crop damage by modifying population age structure, reducing population growth and providing context-dependent effects on host fitness. Our results highlight the importance of plant-mediated horizontal transmission and interactions with temperature as drivers of endosymbiont spread in asexual insect populations.

Attraction of Myzus persicae (Hemiptera: Aphididae) to Volatiles Emitted From the Entomopathogenic Fungus Beauveria bassiana.

Beauveria bassiana (Balsamo) Vuillemin infects a wide variety of insects, including the green peach aphid, Myzus persicae (Sulzer). Volatiles emitted from B. bassiana can act as semiochemical attractants or repellents, with most responses reported to date resulting in insects avoiding B. bassiana. Since insects can detect 'enemy-specific volatile compounds', we hypothesized the preference behavior of M. persicae would be influenced by volatile emissions from B. bassiana. We conducted Petri dish and Y-tube olfactometer bioassays to characterize the preference of M. persicae to B. bassiana strain GHA. During Petri dish bioassays, more apterous and alate M. persicae were recorded in the vicinity of agar colonized by B. bassiana compared to agar, or Fusarium proliferatum (Matsushima) Nirenberg and Ambrosiella grosmanniae Mayers, McNew, & Harrington as representatives of nonentomopathogenic fungi. Petri dish bioassays also determined that apterous and alate M. persicae preferred filter paper saturated with 1 × 107, 1 × 106, and 1 × 105B. bassiana conidia/ml compared to Tween 80. Y-tube bioassays documented that more apterous and alate M. persicae oriented upwind to volatiles from B. bassiana mycelia compared to agar. Apterous and alate Myzus persicae were also preferentially attracted to 1 × 107 and 1 × 106B. bassiana conidia/ml compared to Tween-80 during Y-tube bioassays. These results complement a previous finding that the mosquito Anopheles stephensi (Diptera: Culicidae) Liston is attracted to volatiles from B. bassiana. Future studies aimed at characterizing the olfactory mechanism leading to the attraction of M. persicae to B. bassiana could aid in optimizing lure-and-kill strategies.

Strong Linkage Between Symbiotic Bacterial Community and Host Age and Morph in a Hemipteran Social Insect.

The relationships between symbionts and insects are complex, and symbionts usually have diverse ecological and evolutionary effects on their hosts. The phloem sap-sucking aphids are good models to study the interactions between insects and symbiotic microorganisms. Although aphids usually exhibit remarkable life cycle complexity, most previous studies on symbiotic diversity sampled only apterous viviparous adult females or very few morphs. In this study, high-throughput 16S rDNA amplicon sequencing was used to assess the symbiotic bacterial communities of eleven morphs or developmental stages of the social aphid Pseudoregma bambucicola. We found there were significant differences in bacterial composition in response to different morphs and developmental stages, and for the first time, we revealed male aphids hosted very different symbiotic composition featured with low abundance of dominant symbionts but high diversity of total symbionts. The relative abundance of Pectobacterium showed relatively stable across different types of samples, while that of Wolbachia fluctuated greatly, indicating the former may have a consistent function in this species and the latter may provide specific function for certain morphs or developmental stages. Our study presents new evidence of complexity of symbiotic associations and indicates strong linkage between symbiotic bacterial community and host age and morph.

Implications of environmentally shaped microbial communities for insecticide resistance in Sitobion miscanthi.

Insect-associated bacteria play an important role in the resistance to pesticides, yet bacterial community compositions in wild insect host populations and the environmental factors that shape them are mostly elusive. In this study, Sitobion miscanthi (Takahashi) populations were collected from major wheat growing regions in China. Following high-throughput sequencing of 16S rRNA gene fragments, association analyses were performed within the bacterial community associated with S. miscanthi, as well as with population resistance levels to four commonly used pesticides and different environmental factors. We found that bacterial community structures differed in various regions, and that the abundances of dominant bacteria such as Buchnera, Candidatus Regiella, Candidatus Hamiltonella showed high variations. The resistance of S. miscanthi to avermectin and bifenthrin was shown to decline with increasing bacterial diversity. Meanwhile, with the increase of bacterial network modularity, the resistance of S. miscanthi populations to imidacloprid, avermectin and bifenthrin also increased correspondingly. In addition, correlation analysis indicated that altitude and air pressure had the strongest impact on bacterial community diversity and relative abundance, followed by humidity, rainfall and temperature. Overall, insights into such complex interactions between bacteria and their insect hosts offer new directions for biological pest control.

Toxicity of crude toxin protein produced by Cordyceps fumosorosea IF-1106 against Myzus persicae (Sulze).

The entomopathogenic fungus Cordyceps fumosorosea IF-1106 is a potential biocontrol agent with high pathogenicity to the aphid Myzus persicae. We extracted the crude toxin protein from a liquid culture broth of an isolated C. fumosorosea strain using the ammonium sulfate precipitation method, and its toxicity to Myzus persicae was measured by injection, oral exposure, and topical exposure. The crude toxin protein of C. fumosorosea had insecticidal activity against M. persicae. Body cavity injection and oral exposure had significantly higher insecticidal activity against adults than contact sprays. The highest cumulative corrected mortality of adults after injection was 81.85 ± 13.45 %, and the highest cumulative corrected mortality of adults after ingestion was 85.45 ± 11.88 %. The proportion of plasmatocytes in adult blood lymphocytes reached the highest at 3 days after injection and feeding, and the proportion of granulocytes was the highest at 2 days after injection and feeding. These data confirmed the toxicity of the crude toxin protein of C. fumosorosea toxin to M. persicae and helped clarify the pathogenic mechanism of the strain. Population management of M. persicae may be possible by using a natural toxic compound produced by C. fumosorosea that is selective to this pest species.

Compartmentalized into Bacteriocytes but Highly Invasive: the Puzzling Case of the Co-Obligate Symbiont Serratia symbiotica in the Aphid Periphyllus lyropictus.

Dependence on multiple nutritional symbionts that form a metabolic unit has evolved many times in insects. Although it has been postulated that host dependence on these metabolically interconnected symbionts is sustained by their high degree of anatomical integration (these symbionts are often housed in distinct symbiotic cells, the bacteriocytes, assembled into a common symbiotic organ, the bacteriome), the developmental aspects of such multipartner systems have received little attention. Aphids of the subfamilies Chaitophorinae and Lachninae typically harbor disymbiotic systems in which the metabolic capabilities of the ancient obligate symbiont Buchnera aphidicola are complemented by those of a more recently acquired nutritional symbiont, often belonging to the species Serratia symbiotica. Here, we used microscopy approaches to finely characterize the tissue tropism and infection dynamics of the disymbiotic system formed by B. aphidicola and S. symbiotica in the Norway maple aphid Periphyllus lyropictus (Chaitophorinae). Our observations show that, in this aphid, the co-obligate symbiont S. symbiotica exhibits a dual lifestyle: intracellular by being housed in large syncytial bacteriocytes embedded between B. aphidicola-containing bacteriocytes in a well-organized compartmentalization pattern, and extracellular by massively invading the digestive tract and other tissues during embryogenesis. This is the first reported case of an obligate aphid symbiont that is internalized in bacteriocytes but simultaneously adopts an extracellular lifestyle. This unusual infection pattern for an obligate insect symbiont suggests that some bacteriocyte-associated obligate symbionts, despite their integration into a cooperative partnership, still exhibit invasive behavior and escape strict compartmentalization in bacteriocytes. IMPORTANCE Multipartner nutritional endosymbioses have evolved many times in insects. In Chaitophorinae aphids, the eroded metabolic capabilities of the ancient obligate symbiont B. aphidicola are complemented by those of more recently acquired symbionts. Here, we report the atypical case of the co-obligate S. symbiotica symbiont associated with P. lyropictus. This bacterium is compartmentalized into bacteriocytes nested into the ones harboring the more ancient symbiont B. aphidicola, reflecting metabolic convergences between the two symbionts. At the same time, S. symbiotica exhibits highly invasive behavior by colonizing various host tissues, including the digestive tract during embryogenesis. The discovery of this unusual phenotype for a co-obligate symbiont reveals a new face of multipartner nutritional endosymbiosis in insects. In particular, it shows that co-obligate symbionts can retain highly invasive traits and suggests that host dependence on these bacterial partners may evolve prior to their strict compartmentalization into specialized host structures.

Ploidy dynamics in aphid host cells harboring bacterial symbionts.

Aphids have evolved bacteriocytes or symbiotic host cells that harbor the obligate mutualistic bacterium Buchnera aphidicola. Because of the large cell size (approximately 100 µm in diameter) of bacteriocytes and their pivotal role in nutritional symbiosis, researchers have considered that these cells are highly polyploid and assumed that bacteriocyte polyploidy may be essential for the symbiotic relationship between the aphid and the bacterium. However, little is known about the ploidy levels and dynamics of aphid bacteriocytes. Here, we quantitatively analyzed the ploidy levels in the bacteriocytes of the pea-aphid Acyrthosiphon pisum. Image-based fluorometry revealed the hyper polyploidy of the bacteriocytes ranging from 16- to 256-ploidy throughout the lifecycle. Bacteriocytes of adult parthenogenetic viviparous females were ranged between 64 and 128C DNA levels, while those of sexual morphs (oviparous females and males) were comprised of 64C, and 32-64C cells, respectively. During post-embryonic development of viviparous females, the ploidy level of bacteriocytes increased substantially, from 16 to 32C at birth to 128-256C in actively reproducing adults. These results suggest that the ploidy levels are dynamically regulated among phenotypes and during development. Our comprehensive and quantitative data provides a foundation for future studies to understand the functional roles and biological significance of the polyploidy of insect bacteriocytes.

Elucidation of host and symbiont contributions to peptidoglycan metabolism based on comparative genomics of eight aphid subfamilies and their Buchnera.

Pea aphids (Acyrthosiphon pisum) are insects containing genes of bacterial origin with putative functions in peptidoglycan (PGN) metabolism. Of these, rlpA1-5, amiD, and ldcA are highly expressed in bacteriocytes, specialized aphid cells that harbor the obligate bacterial symbiont Buchnera aphidicola, required for amino acid supplementation of the host's nutrient-poor diet. Despite genome reduction associated with endosymbiosis, pea aphid Buchnera retains genes for the synthesis of PGN while Buchnera of many other aphid species partially or completely lack these genes. To explore the evolution of aphid horizontally-transferred genes (HTGs) and to elucidate how host and symbiont genes contribute to PGN production, we sequenced genomes from four deeply branching lineages, such that paired aphid and Buchnera genomes are now available for 17 species representing eight subfamilies. We identified all host and symbiont genes putatively involved in PGN metabolism. Phylogenetic analyses indicate that each HTG family was present in the aphid shared ancestor, but that each underwent a unique pattern of gene loss or duplication in descendant lineages. While four aphid rlpA gene subfamilies show no relation to symbiont PGN gene repertoire, the loss of aphid amiD and ldcA HTGs coincides with the loss of symbiont PGN metabolism genes. In particular, the coincident loss of host amiD and symbiont murCEF in tribe Aphidini, in contrast to tribe Macrosiphini, suggests either 1) functional linkage between these host and symbiont genes, or 2) Aphidini has lost functional PGN synthesis and other retained PGN pathway genes are non-functional. To test these hypotheses experimentally, we used cell-wall labeling methods involving a d-alanine probe and found that both Macrosiphini and Aphidini retain Buchnera PGN synthesis. Our results imply that compensatory adaptations can preserve PGN synthesis despite the loss of some genes considered essential for this pathway, highlighting the importance of the cell wall in these symbioses.

Entomopathogenic fungi tested in planta on pepper and in field on sorghum, to control commercially important species of aphids.

Given the aphids high reproductive capacity, assessing their biocontrol by using entomopathogenic fungi is crucial; to determine their potential, fungi were tested in planta and in field conditions. Significant decrease of Myzus persicae (Sulzer) population was observed in planta after applying Beauveria bassiana (strain 7R), Trichoderma gamsii (strain Z) or Metarhizium brunneum (strain Meta Br1) at 1 × 107 or 1 × 108 conidia/mL on pepper plants. Significant differences of aphids' populations were detected between fungus concentration and control (F = 68.743, df = 6.980, P < 0.001), where M. brunneum at 1 × 108 conidia/mL reduced aphids population close to zero. At 20 °C, dead aphids' mycosis by B. bassiana and T. gamsii was 78% and 84%; at 25 °C was 83% and 88%; and at 30 °C was 75% and 79%, respectively. In field conditions, Mexican PTG4 and commercial GHA B. bassiana strains were tested [(1 × 106 conidia/mL + corn starch) seed treatments] against the Melanaphis sacchari (Zehntner) aphid populations, on naturally infested sorghum plants. Results showed that plant germination and emergence were not affected, whereas yield (grams of sugar/plant) was significantly higher among treated compared with untreated plants. The aphid population decreased in plants from PTG4 treated seeds; indeed, this treatment had a significant positive effect on the flowering index, whereas the stem fresh weight and juice volume was significantly increased among plants from GHA treated seeds. Taken together, tested strains can be used as a tool to control aphids' population on several crops such as pepper and even increase the yield in sorghum.