Development of common leaf-footed bug pests depends on the presence and identity of their environmentally-acquired symbionts

Many beneficial symbioses between bacteria and their terrestrial arthropod hosts are vertically transmitted from mother to offspring, ensuring the progeny acquire necessary partners. Unusually, in several families of coreoid and lygeoid bugs (Hemiptera), nymphs must instead ingest the beneficial symbiont, Burkholderia ( sensu lato ), from the environment early in development. We studied the effects of Burkholderia on development of two species of leaf-footed bug (Coreidae) in the genus Leptoglossus, L. zonatus and L. phyllopus. We found no evidence for vertical transmission of the symbiont, but found stark differences in performance between symbiotic and aposymbiotic individuals. Symbiotic nymphs grew more rapidly, were approximately four times more likely to survive to adulthood than aposymbiotic bugs, and were two times larger. These findings suggest that Burkholderia is an obligate symbiont for Leptoglossus species. We also tested for variation in fitness effects conferred by four symbiont isolates representing different species within Burkholderia ’s insect-associated Stinkbug Beneficial and Environmental (SBE) clade. While three isolates conferred similar benefits to hosts, nymphs associated with the fourth isolate grew more slowly and weighed significantly less as adults. The effects of the four isolates were similar for both Leptoglossus species. This work indicates that both Burkholderia acquisition and isolate identity play critical roles in the growth and development of Leptoglossus. Importance Leptoglossus zonatus and L. phyllopus are important polyphagous pests and both species have been well-studied, but generally without regard to their dependance on a bacterial symbiont. Our results indicate that the central role of Burkholderia in the biology of these insects, as well as in other leaf-footed bugs, should be considered in future studies of coreid life history, ecology and pest management. Our work suggests acquisition of Burkholderia is critical for the growth and development of Leptoglossus species. Further, we found that there was variation in performance outcomes according to symbiont identity, even among members of the Stinkbug Beneficial and Environmental clade. This suggests that although environmental acquisition of a symbiont can provide extraordinary flexibility in partner associations, it also carries a risk if the partner is sub-optimal.

Potato leafroll virus reduces Buchnera aphidocola titer and alters vector transcriptome responses

Viruses in the Luteoviridae family, such as Potato leafroll virus (PLRV), are transmitted by aphids in a circulative and nonpropagative mode. This means the virions enter the aphid body through the gut when they feed from infected plants and then the virions circulate through the hemolymph to enter the salivary glands before being released into the saliva. Although these viruses do not replicate in their insect vectors, previous studies have demonstrated viruliferous aphid behavior is altered and the obligate symbiont of aphids, Buchnera aphidocola, may be involved in transmission. Here we provide the transcriptome of green peach aphids (Myzus persicae) carrying PLRV and virus-free control aphids using Illumina sequencing. Over 150 million paired-end reads were obtained through Illumina sequencing, with an average of 19 million reads per library. The comparative analysis identified 134 differentially expressed genes (DEGs) between the M. persicae transcriptomes, including 64 and 70 genes that were up- and down-regulated in aphids carrying PLRV, respectively. Using functional classification in the GO databases, 80 of the DEGs were assigned to 391 functional subcategories at category level 2. The most highly up-regulated genes in aphids carrying PLRV were cytochrome p450s, genes related to cuticle production, and genes related to development, while genes related to heat shock proteins, histones, and histone modification were the most down-regulated. PLRV aphids had reduced Buchnera titer and lower abundance of several Buchnera transcripts related to stress responses and metabolism. These results suggest carrying PLRV may reduce both aphid and Buchnera genes in response to stress. This work provides valuable basis for further investigation into the complicated mechanisms of circulative and nonpropagative transmission.

Impact of heat stress on the fitness outcomes of symbiotic infection in aphids: a meta-analysis

Beneficial microorganisms shape the evolutionary trajectories of their hosts, facilitating or constraining the colonization of new ecological niches. One convincing example entails the responses of insect-microbe associations to rising temperatures. Indeed, the insect resilience to stressful high temperatures depends on the genetic identity of the obligate symbiont and the presence of heat protective facultative symbionts. With accumulating empirical evidence, there is a need of integrative studies to draw general patterns about the thermal sensitivity of insect-microbe associations, from an eco-evolutionary perspective. Focusing on aphid-bacteria mutualisms, this meta-analysis aims to quantify the context-dependent impacts of symbionts on host phenotype in benign or stressful heat conditions, across fitness traits, types of heat stress, and symbiont species. We found that warming lowered the benefits (parasitoid resistance) and costs (development, fecundity) of infection by facultative symbionts, which was overall mostly beneficial to the aphids under short-term heat stress (heat shock) rather than extended warming. Heat tolerant genotypes of the obligate symbiont Buchnera aphidicola and some facultative symbionts (Rickettsia sp., Serratia symbiotica) improved or maintained aphid fitness under heat stress. As phytophagous insects are central to terrestrial ecosystems, symbiont-mediated responses to increasing mean temperatures and frequency of heat waves in the context of climate change are key elements that may have cascading effects on food webs and there is an urgent need to continue accumulating data on other models. We discuss the implications of these conclusions for the general understanding of the cost-benefits balance and eco-evolutionary dynamics of insect-microbe associations faced with climate change.

Olive fruit fly and its obligate symbiont Candidatus Erwinia dacicola: Two new symbiont haplotypes in the Mediterranean basin

The olive fruit fly, specialized to become monophagous during several life stages, remains the most important olive tree pest with high direct production losses, but also affecting the quality, composition, and inherent properties of the olives. Thought to have originated in Africa is nowadays present wherever olive groves are grown. The olive fruit fly evolved to harbor a vertically transmitted and obligate bacterial symbiont -Candidatus Erwinia dacicola- leading thus to a tight evolutionary history between olive tree, fruit fly and obligate, vertical transmitted symbiotic bacterium. Considering this linkage, the genetic diversity (at a 16S fragment) of this obligate symbiont was added in the understanding of the distribution pattern of the holobiont at nine locations throughout four countries in the Mediterranean Basin. This was complemented with mitochondrial (four mtDNA fragments) and nuclear (ten microsatellites) data of the host. We focused on the previously established Iberian cluster for the B. oleae structure and hypothesised that the Tunisian samples would fall into a differentiated cluster. From the host point of view, we were unable to confirm this hypothesis. Looking at the symbiont, however, two new 16S haplotypes were found exclusively in the populations from Tunisia. This finding is discussed in the frame of host-symbiont specificity and transmission mode. To understand olive fruit fly population diversity and dispersion, the dynamics of the symbiont also needs to be taken into consideration, as it enables the fly to, so efficiently and uniquely, exploit the olive fruit resource.

Novel insights on obligate symbiont lifestyle and adaptation to chemosynthetic environment as revealed by the giant tubeworm genome

The mutualism between the giant tubeworm Riftia pachyptila and its endosymbiont Candidatus Endoriftia persephone has been extensively researched over the past 40 years. However, the lack of the host whole genome information has impeded the full comprehension of the genotype/phenotype interface in Riftia. Here we described the high-quality draft genome of Riftia, its complete mitogenome, and tissue-specific transcriptomic data. The Riftia genome presents signs of reductive evolution, with gene family contractions exceeding expansions. Expanded gene families are related to sulphur metabolism, detoxification, anti-oxidative stress, oxygen transport, immune system, and lysosomal digestion, reflecting evolutionary adaptations to the vent environment and endosymbiosis. Despite the derived body plan, the developmental gene repertoire in the gutless tubeworm is extremely conserved with the presence of a near intact and complete Hox cluster. Gene expression analyses establishes that the trophosome is a multi-functional organ marked by intracellular digestion of endosymbionts, storage of excretory products and haematopoietic functions. Overall, the plume and gonad tissues both in contact to the environment harbour highly expressed genes involved with cell cycle, programmed cell death, and immunity indicating a high cell turnover and defence mechanisms against pathogens. We posit that the innate immune system plays a more prominent role into the establishment of the symbiosis during the infection in the larval stage, rather than maintaining the symbiostasis in the trophosome. This genome bridges four decades of physiological research in Riftia, whilst simultaneously provides new insights into the development, whole organism functions and evolution in the giant tubeworm.

Strong within-host selection in a maternally inherited obligate symbiont: Buchnera and aphids

Numerous animal lineages have maternally inherited symbionts that are required for host reproduction and growth. Endosymbionts also pose a risk to their hosts because of the mutational decay of their genomes through genetic drift or to selfish mutations that favor symbiont fitness over host fitness. One model for heritable endosymbiosis is the association of aphids with their obligate bacterial symbiont, Buchnera. We experimentally established heteroplasmic pea aphid matrilines containing pairs of closely related Buchnera haplotypes and used deep sequencing of diagnostic markers to measure haplotype frequencies in successive host generations. These frequencies were used to estimate the effective population size of Buchnera within hosts (i.e., the transmission bottleneck size) and the extent of within-host selection. The within-host effective population size was in the range of 10 to 20, indicating a strong potential for genetic drift and fixation of deleterious mutations. Remarkably, closely related haplotypes were subject to strong within-host selection, with selection coefficients as high as 0.5 per aphid generation. In one case, the direction of selection depended on the thermal environment and went in the same direction as between-host selection. In another, a new mutant haplotype had a strong within-host advantage under both environments but had no discernible effect on host-level fitness under laboratory conditions. Thus, within-host selection can be strong, resulting in a rapid fixation of mutations with little impact on host-level fitness. Together, these results show that within-host selection can drive evolution of an obligate symbiont, accelerating sequence evolution.

The green peach aphid, Myzus persicae, transcriptome in response to a circulative, nonpropagative polerovirus, Potato leafroll virus.

Viruses in the Luteoviridae family, such as Potato leafroll virus (PLRV), are transmitted by aphids in a circulative and nonpropagative mode. This means the virions enter the aphid body through the gut when they feed from infected plants and then the virions circulate through the hemolymph to enter the salivary glands before being released into the saliva. Although these viruses do not replicate in their insect vectors, previous studies have demonstrated viruliferous aphid behavior is altered and the obligate symbiont of aphids, Buchnera aphidocola, may be involved in transmission. Here we provide the transcriptome of green peach aphids (Myzus persicae) carrying PLRV and virus-free control aphids using Illumina sequencing. Over 150 million paired-end reads were obtained through Illumina sequencing, with an average of 19 million reads per library. The comparative analysis identified 134 differentially expressed genes (DEGs) between the M. persicae transcriptomes, including 64 and 70 genes that were down- and up-regulated in aphids carrying PLRV, respectively. Using functional classification in the GO databases, 80 of the DEGs were assigned to 391 functional subcategories at category level 2. The most highly up-regulated genes in aphids carrying PLRV were cytochrome p450s, genes related to cuticle production, and genes related to development, while genes related to histone and histone modification were the most down-regulated. PLRV aphids had reduced Buchnera titer and lower abundance of several Buchnera transcripts related to stress responses and metabolism. These results suggest carrying PLRV may reduce both aphid and Buchnera genes in response to stress. This work provides valuable basis for further investigation into the complicated mechanisms of circulative and nonpropogative transmission.

Two Deoxythymidine Triphosphate Synthesis-Related Genes Regulate Obligate Symbiont Density and Reproduction in the Whitefly Bemisia tabaci MED

Deoxythymidine triphosphate (dTTP) is essential for DNA synthesis and cellular growth in all organisms. Here, genetic capacity analysis of the pyrimidine pathway in insects and their symbionts revealed that dTTP is a kind of metabolic input in several host insect/obligate symbiont symbiosis systems, including Bemisia tabaci MED/Candidatus Portiera aleyrodidarum (hereafter Portiera). As such, the roles of dTTP on both sides of the symbiosis system were investigated in B. tabaci MED/Portiera. Dietary RNA interference (RNAi) showed that suppressing dTTP production significantly reduced the density of Portiera, significantly repressed the expression levels of horizontally transferred essential amino acid (EAA) synthesis-related genes, and significantly decreased the reproduction of B. tabaci MED adults as well as the hatchability of their offspring. Our results revealed the regulatory role of dTTP in B. tabaci MED/Portiera and showed that dTTP synthesis-related genes could be potential targets for controlling B. tabaci as well as other sucking pests.

Population structure and reproductive performance in the sea anemone associated shrimp Ancylocaris brevicarpalis (Caridea: Palaemonidae)

The peacock-tail shrimp Ancylocaris brevicarpalis Schenkel, 1902, is an obligate symbiont of sea anemones and well known for its remarkable colouration. Yet, very little information is available about its population structure and life-history traits, including reproductive parameters (fecundity, embryo volume and reproductive output). A total of 574 individuals were collected from the Gulf of Mannar, Tamil Nadu, India between February 2017 and July 2018, out of which 214 were males (37.28%), 355 were females (61.84%), and 5 (0.87%) juveniles. The highest percentage of individuals were observed in the post-monsoon season (38.10%) followed by monsoon (34.85%), pre-monsoon (15.02%), and summer seasons (12.01%). The overall sex ratio was skewed towards female individuals (0.55 male: 1 female). Fecundity was higher in females carrying early-stage embryos and embryo volume did increase, but not statistically significantly from early to late stages. The reproductive output was negatively allometric to the mean female body weight. The present study provides first-of-its-kind information on the population as well as individual-level reproductive characteristics of A. brevicarpalis.

Re-discovery and novel contributions to morphology and multigene phylogeny of Myxophyllum steenstrupi (Ciliophora: Pleuronematida), an obligate symbiont of terrestrial pulmonates

Myxophyllum steenstrupi is a symbiotic ciliate living in the body slime and mantle cavity of terrestrial pulmonates (Gastropoda: Pulmonata). In the present study, M. steenstrupi was re-discovered after almost 30 years and characterized using an integrative morpho-molecular approach for the first time. Myxophyllum is distinguished by a broadly ovate, about 140 × 115 μm-sized body, a nuclear apparatus typically composed of seven macronuclear nodules and a single micronucleus, a central contractile vacuole, a shallow oral cavity situated in the posterior body region and dense somatic ciliature with extensive thigmotactic field. According to the present phylogenetic analyses of two mitochondrial and five nuclear markers, M. steenstrupi is classified in the predominantly free-living order Pleuronematida (Oligohymenophorea: Scuticociliatia). This order also encompasses other taxa isolated from molluscs and traditionally classified along with Myxophyllum in the order Thigmotrichida. The proper classifications of Myxophyllum was hampered by the dramatic remodelling of its oral apparatus (reduction of the paroral membrane and adoral organelles, formation of vestibular kineties), a transformation that was likely induced by its firm association with terrestrial gastropods. The present study also documents that various ciliate lineages independently became commensals or parasites of various aquatic and terrestrial molluscs.