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. 2020 Nov 30;117(50):31979–31986. doi: 10.1073/pnas.2000860117

Multiple origins of obligate nematode and insect symbionts by a clade of bacteria closely related to plant pathogens

Vincent G Martinson a,b,1, Ryan M R Gawryluk c, Brent E Gowen c, Caitlin I Curtis c, John Jaenike a, Steve J Perlman c
PMCID: PMC7749356  PMID: 33257562

Significance

Obligate symbioses are intimate associations between species in which neither partner can live without the other. It is challenging to study how obligate symbioses arise because they are often ancient and it is difficult to uncover early or intermediate stages. We have discovered a nascent obligate symbiosis involving Howardula aoronymphium, a well-studied nematode parasite of Drosophila flies, and a bacterium related to Pectobacterium, a lineage of plant pathogens. Moreover, this nematode symbiont is a member of a widespread group of invertebrate host-associated microbes that has independently given rise to at least four obligate symbioses in nematodes and insects, making it an exciting model to study transitions to obligate symbiosis.

Keywords: Howardula, symbiosis, Drosophila, genome reduction, Sodalis

Abstract

Obligate symbioses involving intracellular bacteria have transformed eukaryotic life, from providing aerobic respiration and photosynthesis to enabling colonization of previously inaccessible niches, such as feeding on xylem and phloem, and surviving in deep-sea hydrothermal vents. A major challenge in the study of obligate symbioses is to understand how they arise. Because the best studied obligate symbioses are ancient, it is especially challenging to identify early or intermediate stages. Here we report the discovery of a nascent obligate symbiosis in Howardula aoronymphium, a well-studied nematode parasite of Drosophila flies. We have found that H. aoronymphium and its sister species harbor a maternally inherited intracellular bacterial symbiont. We never find the symbiont in nematode-free flies, and virtually all nematodes in the field and the laboratory are infected. Treating nematodes with antibiotics causes a severe reduction in fly infection success. The association is recent, as more distantly related insect-parasitic tylenchid nematodes do not host these endosymbionts. We also report that the Howardula nematode symbiont is a member of a widespread monophyletic group of invertebrate host-associated microbes that has independently given rise to at least four obligate symbioses, one in nematodes and three in insects, and that is sister to Pectobacterium, a lineage of plant pathogenic bacteria. Comparative genomic analysis of this group, which we name Candidatus Symbiopectobacterium, shows signatures of genome erosion characteristic of early stages of symbiosis, with the Howardula symbiont’s genome containing over a thousand predicted pseudogenes, comprising a third of its genome.

Intimate symbioses involving intracellular bacteria have transformed eukaryotic life (1, 2), with mitochondria and chloroplasts as canonical examples. More recent, yet still ancient, acquisitions of obligate bacterial intracellular endosymbionts have enabled colonization and radiation by animals into previously inaccessible niches, such as feeding on plant sap and animal blood (3), and surviving in deep-sea hydrothermal vents (4). Among the most difficult questions to resolve in the study of obligate symbiosis are how do obligate symbioses evolve, and where do obligate symbionts come from? This is particularly challenging because most of the obligate symbioses that have been studied are ancient, making it extremely difficult to identify early or intermediate stages.

One of the most common ways to acquire an obligate symbiont is via symbiont replacement (5). As a result of a lifestyle shaped by genetic drift, vertically transmitted obligate symbionts follow a syndrome of accumulation of deleterious mutations, leading to genome degradation and reduction (6). A common pattern is that they are replaced by other less broken symbionts that may then renew the cycle of genomic degradation (7). Here the symbiont, which is often descended from common facultative symbionts or parasites (8, 9), is fitted into an established and well-functioning symbiosis (i.e., with a “symbiont-experienced” host). For example, the symbiont Sodalis has independently given rise to numerous obligate nutritional symbioses in blood-feeding flies and lice, sap-feeding mealybugs, spittlebugs, hoppers, and grain-feeding weevils (9).

Less studied are young obligate symbioses in host lineages that did not already house obligate symbionts (i.e., “symbiont-naive” hosts) (10). Some of the best known examples originate through host manipulation by the symbiont via addiction or reproductive control. Addiction or dependence may be a common route for obligate symbiosis (11), and one of the most famous examples occurred in the laboratory, on the timescale of years, where strains of Amoeba evolved to become entirely dependent on intracellular symbionts (12). Many maternally inherited symbionts of terrestrial arthropods induce parthenogenetic (i.e., all female) reproduction in their hosts (13); accumulation of deleterious mutations in genes required for sexual reproduction will result in hosts that are unable to reproduce if cured of their symbiont (14). However, despite advances in microbial surveys, there are still few examples of young obligate symbioses that result in novel host functions. One intriguing example involves spheroid bodies, nitrogen-fixing organelles found in rhopalodiacean diatoms, that originated from a single acquisition of a cyanobacterial symbiont as recently as ∼12 Mya (15, 16).

Here we report the discovery of a nascent obligate symbiosis in Howardula aoronymphium, a well-studied nematode parasite of Drosophila (17), most recently in the context of a defensive symbiosis. A common host species, Drosophila neotestacea, harbors a strain of the facultative inherited symbiont Spiroplasma that protects it against nematode-induced sterility (18). The protection provided by Spiroplasma is so strong that symbiont-infected flies are spreading across North America and replacing their uninfected counterparts (19). Surprisingly, we have found that H. aoronymphium itself harbors an intracellular bacterial symbiont that is related to Pectobacterium, a well-studied group of plant pathogens often vectored by insects. We also report that the nematode symbiont, which we name Candidatus Symbiopectobacterium (and hereafter Symbiopectobacterium), is a member of a widespread lineage of invertebrate symbionts that has independently given rise to at least four obligate symbioses, one in nematodes and three in insects, representing an exciting model for the study of obligate symbiosis.

Results

Virtually all H. aoronymphium in the Field and Laboratory Host Symbiopectobacterium.

We surveyed wild-caught Drosophila spp. across North America and Europe, and over multiple years, for the presence of H. aoronymphium and Symbiopectobacterium. Using specific primers for Symbiopectobacterium, virtually all Howardula-infected flies were also positive for the symbiont (74 of 79, Table 1; the 5 individuals that tested positive for Howardula but negative for Symbiopectobacterium may have contained dead or dying nematodes). The symbiont was never amplified from Drosophila not infected with Howardula. Our laboratory strain of H. aoronymphium, maintained in the laboratory for over 10 y, is also always infected with Symbiopectobacterium, and in a controlled laboratory infection of D. neotestacea we confirmed the perfect association of H. aoronymphium and Symbiopectobacterium; i.e., only nematode-infected flies are positive for the symbiont (Fig. 1A).

Table 1.

PCR survey for Symbiopectobacterium in wild-caught D. neotestacea

Howardula + Howardula −
Sym. + Sym. − Sym. + Sym. −
2013 20 0 0 20
2014 8 5 0 27
2015 17 0 0 23
2020 29 0 0 55
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Sym., Symbiopectobacterium; +, detected by PCR; −, not detected by PCR.

Fig. 1.

Fig. 1.

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Symbiopectobacterium is pervasive in H. aoronymphium. (A) Symbiopectobacterium relative abundance in D. neotestacea nonparasitized (HA−) or parasitized (HA+) with H. aoronymphium (qPCR measurements). (B–G) Localization of bacteria within larval Howardula using FISH microscopy; (B) background autofluorescence and (C) excitation of probe-labeled bacteria. Localization of Symbiopectobacterium to the Howardula hypodermis. Transmission electron microscopy image showing bacterial cells visible in a Howardula young motherworm (D and E) and juvenile (F and G) developing within a D. neotestacea individual.

Symbiopectobacterium Is an Intracellular Inherited Symbiont of H. aoronymphium.

Within an adult fly host, a single adult parasitic female nematode, commonly referred to as a motherworm at this stage, can produce around 500 juveniles that fill her body until they rupture her hypodermis, disseminate throughout the host fly hemocoel, and finally exit the fly through the intestinal or genital tract (20). Using fluorescence in situ hybridization (FISH) microscopy and transmission electron microscopy (TEM), we observed pervasive bacterial infection in nematode motherworms, juveniles developing within motherworms, and juveniles that have been released from motherworms (Fig. 1 B–G and SI Appendix, Fig. S1). In motherworms, this bacterium is situated intracellularly within hypertrophied hypodermal cells, below the outer layer of microvilli, that distinguish the parasitic female (SI Appendix, Fig. S1). Within juveniles, bacteria are present in areas consistent with hypodermal cells. Bacterial 16S ribosomal RNA (rRNA) amplicon sequencing of H. aoronymphium was dominated by Symbiopectobacterium, and there were no other potential symbiont sequences (SI Appendix, Fig. S2).

Antibiotic Treatment Decreases Howardula Parasitism Success.

We attempted to clear Symbiopectobacterium from H. aoronymphium by rearing nematodes on their host D. neotestacea in media with the antibiotics ampicillin or rifampin. Adult flies were then screened for nematodes and symbionts. The proportion of parasitized D. neotestacea was significantly lower with ampicillin (0.085 ± 0.03) and rifampin (no infection) compared to the control H. aoronymphium exposure (0.25 ± 0.9) (control-amp χ2 (1, n = 687) = 23.48, P < 0.0001; control-rif χ2 (1, n = 469) = 45.74, P < 0.0001) (Fig. 2A). Further, in a subset of flies that we dissected, Symbiopectobacterium was found in all flies that contained motherworms (25/25), while visually nonparasitized flies were almost all negative (41/45) (Fig. 2B). Some visually nonparasitized flies were PCR-positive for H. aoronymphium but lacked Symbiopectobacterium (10/14), possibly indicating unsuccessful parasitism. Thus, we are at present unable to generate symbiont-free nematodes.

Fig. 2.

Fig. 2.

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Experimental antibiotic exposure reduces or eliminates successful parasitism in H. aoronymphium. (A) Percent of adult Drosophila that were visually parasitized by Howardula with the addition of different antibiotics (control, n = 268; ampicillin, n = 399; rifampin, n = 181). (B) PCR survey for Howardula and Symbiopectobacterium in a subset of visually parasitized or nonparasitized Drosophila individuals. * χ2, P < 0.0001.

Symbiopectobacterium Is a Common Associate of a Clade of Drosophila-Parasitic Nematodes.

In order to determine if other insect-parasitic nematodes in the suborder Hexatylina are ancestrally associated with Symbiopectobacterium, we screened nematode species with a range of primers designed to amplify Symbiopectobacterium. We detected Symbiopectobacterium in two close relatives of H. aoronymphium that also parasitize Drosophila (21)—Howardula neocosmis and an unnamed Japanese Howardula sp. (12/12 and 1/1, respectively) (Table 2). The Symbiopectobacterium gyrA gene sequences amplified from these nematodes formed a strongly supported (100% bootstrap support) monophyletic clade, sharing >99% nucleotide sequence identity (SI Appendix, Fig. S3 A–C). In contrast, Symbiopectobacterium was not found in two species of Fergusobia (0/6 individuals), the lineage that is sister to Drosophila-parasitic Howardula (22), in Parasitylenchus nearcticus, a more distantly related tylenchid parasite of Drosophila flies (21) (0/3 individuals), or in three unnamed nematodes that infect sphaerocerid flies (0/13, 0/3, and 0/9), using a number of primers (gyrA, groEL, purK spacer, and 16S rRNA genes) designed to target Symbiopectobacterium, as well as universal 16S rRNA primers (Table 2 and SI Appendix, Table S1 and Figs. S2 and S3C).

Table 2.

PCR survey for Symbiopectobacterium in wild-caught flies and parasitic nematodes

Nematode + Howardula −
Sym. + Sym. − Sym. + Sym. −
H. aoronymphium (England) (Host: Drosophila) 1 0 0 19
H. aoronymphium (Germany) (Host: Drosophila) 4 0 0 16
Howardula sp. B (Japan) (Host: Drosophila) 1 0 0 19
H. neocosmisa (Host: Drosophila) 11 0 – –
Fergusobia sp.a (Host: Fergusonina) 0 6 – –
Parasitylenchus nearcticusa (Host: Drosophila) 0 3 – –
Spelobia sphaerocerid parasite #1a (Host: Spelobia) 0 13 – –
Spelobia sphaerocerid parasite #2a (Host: Spelobia) 0 3 – –
Spelobia sphaerocerid parasite #3a (Host: Spelobia) 0 9 – –
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Sym., Symbiopectobacterium; +, detected by PCR; −, not detected by PCR; a, only nematode screened.

Symbiopectobacterium Is an Invertebrate-Associated Lineage Allied with the Plant-Associated Genus Pectobacterium.

A large number of gene sequences that had >97% sequence identity to the Howardula symbiont 16S rRNA gene were recovered from GenBank. Phylogenetic reconstruction revealed a monophyletic clade closely related to the genera Pectobacterium, Dickeya, and Brenneria (SI Appendix, Fig. S4). This clade included several known intracellular symbionts, including in Cimex bed bugs (23), Euscelidius leafhoppers (24), and Chilacis bulrush bugs (25); the remaining sequences were almost completely observed in association with invertebrates (SI Appendix, Table S2). Symbiopectobacterium is most commonly associated with insects from the order Hemiptera; however, these insects are taxonomically diverse, sharing a most recent common ancestor nearly 300 Mya, and ecologically diverse with disparate life history traits (e.g., blood-feeding, phloem-feeding, seed-feeding) (26).

Signatures of Independent Genome Erosion Across the Symbiopectobacterium Clade.

We sequenced the genomes of Symbiopectobacterium in nematodes and bulrush bugs and compared them with publicly available sequences of related symbionts in mealybugs (7), leafhoppers (27), bed bugs (28), and parasitoid wasps (29), as well as free-living Pectobacterium (30). Regardless of the threefold size difference between the genomes of Symbiopectobacterium in nematodes (4.5 Mb) and in bulrush bugs (1.5 Mb), both genomes harbored >90% of 203 single-copy ortholog (SiCO) gammaproteobacterial genes (31), indicating we captured nearly full chromosomes. Similarly, the Symbiopectobacterium genomes recovered from the mealybug and parasitoid wasp genome projects were nearly complete, containing 98% and 86% of the SiCO genes. Genomic fragments of the bed bug and leafhopper symbionts were incomplete and lacked most of the SiCO genes, but could be positively identified as members of the Symbiopectobacterium clade and were included in the genome tree (Figs. 3 and 4A).

Fig. 3.

Fig. 3.

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Phylogenetic tree of Gammaproteobacteria using the conserved set of 203 single-copy orthologous genes. The clade containing the Howardula symbiont Symbiopectobacterium is sister to the genera Pectobacterium, Dickeya, and Brenneria and other SREs. Taxonomic classification of the host organism of each member of the putative symbiont clade. Phylogeny was constructed with RAxML using 100 bootstrap replicates. The full phylogeny is available in SI Appendix, Fig. S5.

Fig. 4.

Fig. 4.

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Symbiopectobacterium genomes compared to their closest relative, P. carotovorum. (A) Comparison of genomic features. (B) Example of conserved gene order across Symbiopectobacterium species and P. carotovorum, highlighting the differential pseudogene events across the symbiont clade. Red lines indicate contig boundaries. (C) Analysis for central metabolic pathways across P. carotovorum and the symbiont clade. CoA, Coenzyme A; GC, GC-content.

Phylogenetic analysis of 203 single-copy genes resulted in an overall branching pattern similar to previous publications of Gammaproteobacteria (27, 32) and highly supported the Symbiopectobacterium lineage within the plant-pathogenic “soft rot Enterobacteriaceae” as the sister clade to the genera Pectobacterium and Brenneria (Fig. 3 and SI Appendix, Fig. S5). Regardless of sequence divergence, species within the Symbiopectobacterium group shared large areas of gene order synteny with the closely related Pectobacterium carotovorum genome. Alignment within syntenic regions revealed that, although certain genomic rearrangements are shared, each genome has independent insertions, deletions, and mutations that have resulted in differential pseudogene formation (Fig. 4 B and C).

Compared to their closest relative, P. carotovorum, the Symbiopectobacterium clade members had signatures of genome erosion often associated with the early stages of symbiosis, including more pseudogenes, fewer transfer RNAs, shorter average coding sequence size, lower coding density, and decreased genome size (Fig. 4A). Despite their similarities, there is large variation among members of Symbiopectobacterium in these same genomic traits, suggesting that they are at different stages in the process of genomic reduction, which might be connected with the age of their association with their host invertebrate. Using sequence divergence measurements (33), we estimate that Symbiopectobacterium split from Pectobacterium 400–500 Kya, and the symbiosis events among Symbiopectobacterium species occurred independently <100 Kya (SI Appendix, Fig. S6).

Along with genomic erosion, the metabolic potential of Symbiopectobacterium members has changed greatly. Pseudogene formation or deletion has interrupted many of the amino acid and vitamin synthesis pathways. While Symbiopectobacterium in nematodes has maintained many genes associated with synthesis of amino acids and vitamins, chemotaxis, motility, and secretion systems, the bulrush bug symbiont has lost the majority of functions, except for basic DNA replication and repair, and biosynthesis of lysine and several vitamins.

Proposal of “Candidatus Symbiopectobacterium”

We propose the genus name “Candidatus Symbiopectobacterium” for the lineage of Enterobacteriaceae that forms a monophyletic clade sister to Pectobacterium (SI Appendix, Fig. S4) and whose members are commonly found in association with diverse invertebrates, including intracellular symbionts that are vertically transmitted within their host (i.e., symbionts of H. aoronymphium, Euscelidius variegatus, Cimex lectularius, Chilacis typhae, and Pseudococcus longispinus). While the 16S rRNA gene in Symbiopectobacterium lacks sufficient genetic diversity to differentiate it from sister genera at the historically used 95% similarity level, this lineage is clearly divergent in the phylogeny produced with the genome-wide single-copy ortholog genes, which is mirrored in their ecological associations. The symbionts of the bulrush bug and sycamore seed bug, Chilacis typhae and Belonochilus numenius, respectively, have previously been named Candidatus Rohrkolberia, which refers to the German word for bulrush (25, 34). It was recently pointed out that this name does not conform to the current naming standards laid out in the International Code of Nomenclature of Prokaryotes (35, 36) because the German word for bulrush is used instead of the Latin (Rule and Recommendation 6). Our proposal of Candidatus Symbiopectobacterium would supersede the genus Ca. Rohrkolberia; this name indicates both the widespread symbiotic nature of this lineage and its relatedness to Pectobacterium.

Discussion

We have discovered a nascent obligate symbiosis involving H. aoronymphium, a nematode parasite of Drosophila, and a bacterium from a cryptic but widespread lineage of endosymbionts, allied with Pectobacterium, and which we here name Candidatus Symbiopectobacterium. We present four different lines of evidence supporting obligate symbiosis. First, there is almost perfect concordance between Symbiopectobacterium and H. aoronymphium presence. We never detect symbionts in nematode-free flies, and virtually all wild-caught flies that test positive for H. aoronymphium DNA are also positive for Symbiopectobacterium. The very rare mismatches could be due to false negative PCR amplification, perhaps due to low-quality and/or low-titer DNA; for example, due to dead or dying nematodes within a fly. Second, microscopy revealed intracellular bacterial infection, including inside motherworms, juveniles developing inside motherworms, and shed juveniles. Third, closely related symbionts were also detected in the sister species H. neocosmis and a Japanese Howardula sp. Finally, treating nematodes with antibiotics caused a severe reduction in fly infection success, although we cannot rule out the possibility that the antibiotic directly affected the nematode.

Only a handful of obligate symbioses involving nematodes and bacteria has been described, and these include Wolbachia in some filarial nematodes (37), Xiphinematobacter and Burkholderia in some plant-parasitic dagger nematodes (38, 39), Photorhabdus and Xenorhabdus in entomopathogenic nematodes (40), and Thiosymbion sulfur-oxidizing ectosymbionts of marine stilbonematines (41). In contrast to many nematode–bacterium associations, this symbiosis appears to be very young. We detected Symbiopectobacterium only in the closely related H. aoronymphium, H. neocosmis, and Japanese Howardula sp., which represents just a sliver of the insect-parasitic, and Drosophila-parasitic, diversity within the Hexatylina (21, 42, 43). We were unable to amplify Symbiopectobacterium DNA in the related nematodes Fergusobia sp., Parasitylenchus nearcticus, or three unnamed nematodes that infect sphaerocerid flies. Nor did we find any evidence of bacterial endosymbiont infection in electron microscopy studies of allied Hexatylina nematodes, including Deladenus, Thripinema, and Contortylenchus (44–46), and a very detailed and extensive study of H. husseyi (47) (note that the genus Howardula is not monophyletic) (SI Appendix, Fig. S3 B and C). As there is very little available insect-parasitic nematode DNA sequence, it is difficult to provide an estimate for the age of the Symbiopectobacterium–Howardula association. Fergusobia nematodes, the sister group of the Howardula clade that hosts Symbiopectobacterium, are obligate mutualists of fergusoninid gall-making flies, and the age of this family of flies has been estimated to be not more than 42 My (48, 49), providing a conservative upper limit to the age of the symbiosis. Our sequence divergence measurements, however, suggest that the symbiosis is much more recent, with Symbiopectobacterium splitting from Pectobacterium about a half a million years ago.

Symbiopectobacterium is a surprisingly widespread lineage of symbionts, closely related to Pectobacterium, Dickeya, and Brenneria, often referred to as “soft rot Enterobacteriaceae” (SREs). These SREs are common pathogens of plants that are often vectored by insects (50). The high sequence similarity shared between Symbiopectobacterium and Pectobacterium (e.g., 16S rRNA gene) may explain why this lineage has remained largely undetected despite the increase in amplicon surveys of invertebrates. Symbiopectobacterium symbionts are diverse and include at least four independently evolved obligate mutualistic symbioses—once in Drosophila-parasitic Howardula and three times as a nutritional symbiont in sap- and seed-feeding hemipteran insects, including a young symbiont replacement in Pseudococcus longispinus, the long-tailed mealybug (7). They have also independently colonized the lygaeoid seed bugs Chilacis typhae, the bulrush bug, and Belonochilus numenius, the sycamore seed bug (25, 34). They are also common facultative symbionts of insects, including in Cimex lectularius bed bugs (23), Dipetalogaster maximus and possibly related kissing bugs (51, 52), and Euscelidius variegatus leafhoppers (24, 53). This latter symbiont, called 'BEV' (Bacterium of Euscelidius variegatus), was an early model in insect symbiosis (53) that is noteworthy because it exhibits both transovarial and horizontal transmission and can be easily cultured on media outside the host. We also found some Symbiopectobacterium sequences in GenBank that are not associated with invertebrate hosts, but instead were identified in association with plants. These sequences may represent free-living Symbiopectobacterium strains that have repeatedly forged symbioses with invertebrate hosts; alternatively, new host–symbiont combinations may have become established via horizontal transmission of facultative symbionts. Symbiopectobacterium is the fourth example of a lineage of symbionts that is shared between insects and nematodes, joining Wolbachia (54), Cardinium (55), and Burkholderia (38, 56).

The evolution and distribution of Symbiopectobacterium is reminiscent of Sodalis, another widespread lineage of mostly facultative insect symbionts that has repeatedly given rise to obligate nutritional symbioses in sap-, seed- and blood-feeding hemipterans and flies (9); acquisition of some Sodalis has been estimated to have occurred very recently, ∼30 Kya (33). Like Sodalis, Symbiopectobacterium is an exciting model for studying the evolution and dynamics of symbiosis because it and its close relatives run the gamut, from free-living, well-studied, genetically tractable pathogens (57, 58) to cultivable facultative inherited symbionts, all the way to obligate symbionts. This diversity is also reflected in the dynamic genome evolution of this lineage. Similar to Sodalis (33), the Howardula symbiont genome exhibits the hallmark of recent symbiosis, as it is very large and full of pseudogenes.

So what is the role of Symbiopectobacterium in Howardula? There are a number of not mutually exclusive possibilities. The symbiont may be boosting nematode fitness, for example, by aiding in evading the Drosophila immune response, or providing a novel function, such as supplementing nutrition or metabolism. One possibility is that the nematode symbiont supplements heme. While nematodes are the only animals known to have lost the ability to synthesize heme (59), two lineages of animal-parasitic nematodes have independently acquired the gene for ferrochelatase, the last step in the synthesis of heme, via horizontal gene transfer from bacteria (60, 61); this enzyme, including conserved active sites, is also encoded in the nematode Symbiopectobacterium genome. Alternatively, the symbiont may persist as a result of addiction by the host (11); for example, by producing a persistent toxin and its antidote, such that symbiont removal is deleterious. The symbiont may also be providing an essential function that was lost by the host. Comparing genomes of related nematodes with and without Symbiopectobacterium, as well as transcriptome studies to uncover highly expressed symbiont genes, may provide some useful clues. Interestingly, the role of the obligate Wolbachia symbiont of filarial nematodes is still unclear, and metabolic, defensive, and addictive processes have all been invoked (11, 62–64), particularly because a number of species have lost Wolbachia without having gained new symbionts or genes (65, 66).

Materials and Methods

PCR Survey of Symbiopectobacterium in Insect-Parasitic Nematodes.

Drosophila flies collected at mushroom baits at sites in North America, Europe, and Asia were individually subjected to DNA extraction and screened with specific primers for Howardula parasitism and the presence of Symbiopectobacterium; additional samples were dissected to determine nematode infection, followed by screening, which was done blind. Additionally, Fergusonina flies, that are obligately associated with Fergusobia nematodes, were collected at eucalyptus trees and screened for Symbiopectobacterium. Sphaerocerid flies were collected at mushroom baits and dissected for nematodes, which were subsequently genotyped and screened for Symbiopectobacterium. Bacterial 16S rRNA amplicon sequencing was performed on H. aoronymphium and sphaerocerid nematode motherworms, along with their uninfected fly hosts, Drosophila falleni and Spelobia sp. Specific information about species sampled, including dates, localities, primers, and PCR conditions are available in SI Appendix, SI Materials and Methods.

Controlled Howardula and Symbiopectobacterium Laboratory Infection and qPCR.

D. neotestacea larvae were infected with H. aoronymphium. Upon emergence, Symbiopectobacterium titer was quantified in 2-d-old adult flies, using qPCR. Specific information about laboratory infection and primers and qPCR conditions is available in SI Appendix, SI Materials and Methods.

Antibiotic Exposure Bioassay.

To eliminate Symbiopectobacterium from H. aoronymphium and measure subsequent parasitism success, we exposed nematodes to ampicillin or rifampin and compared them to a control group. Exposure lasted the entire parasitic life cycle. Briefly, free-living juvenile nematodes were allowed to parasitize larval D. neotestacea and carry out development within fly pupae and adults as internal parasites in vials with antibiotic impregnated food. One week post eclosion, adult flies were dissected to ascertain nematode parasitism, and a subset were screened for Symbiopectobacterium. Each treatment was repeated four times, and the proportion of parasitism was assessed with a χ2 test. Detailed procedures for laboratory rearing and antibiotic treatment of H. aoronymphium are found in SI Appendix, SI Materials and Methods.

Microscopy to Localize the Symbiont of Howardula.

Laboratory-reared H. aoronymphium were dissected from adult Drosophila and nematode samples for TEM were prepared with Karnovsky's fixative before being embedded in Epon, whereas FISH samples were fixed in Carnoy’s solution for either TEM or FISH microscopy and probed with the general bacterial target, Eub339. Detailed procedures for TEM and FISH are available in SI Appendix, SI Materials and Methods.

Sequencing, Genome Binning, and Annotation of Nematode and Bulrush Bug Symbiopectobacterium.

DNA obtained from H. aoronymphium, dissected from laboratory-raised Drosophila putrida, was sequenced with Illumina HiSeq technology, and genome assembly was performed in UniCycler (67). The Symbiopectobacterium genome was separated from the nematode and fly genomes using Blobtools (68) and the de Bruijn graph visualization tool Bandage (69) (SI Appendix, Fig. S7). Similar sequencing and assembly methods were employed for the symbiont genome of the bulrush bug (Chilacis typhae). Gene and metabolic pathways were annotated with Prokaryotic Genomes Annotation Pipeline (70, 71) and Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server (72) for each genome, and gene synteny comparisons were made in Geneious 11.1.5 (https://www.geneious.com) with Mauve (73). Detailed description of tissues, extraction, sequence data, and assembly parameters used for genomic analyses are available in SI Appendix, SI Materials and Methods.

Supplementary Material

Supplementary File
pnas.2000860117.sapp.pdf (1.9MB, pdf)

Acknowledgments

We thank Sonja Scheffer for providing Fergusobia nematodes/Fergusonina flies; Evan Tandy for maintaining Drosophila stocks; Alexandria Marshall and Finn Hamilton for help in the early stages of the project; and Ben Parker and Ellen Martinson for constructive conversations. This study was supported by NSF Grant 1144581 (to J.J.) and grants from the Natural Sciences and Engineering Research Council of Canada (Discovery Grant Program) and the Swiss National Science Foundation (Sinergia Grant CRSII3_154396) (to S.J.P.).

Footnotes

The authors declare no competing interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2000860117/-/DCSupplemental.

Data Accessibility.

Symbiopectobacterium genomes from this study (SyHa, PRJNA415854; SyCt, PRJNA521717) and from previous studies (SyPl, PRJNA510127; SyDa, PRJNA510132; SyCl, PRJNA510131); bacterial 16S rRNA (MK943676, MT859673, MT859692, MT859693), gyrA (MN175990–MN175995, MT860065), and groEL (MT860063, MT860064) genes; nematode 18S (MN175314–MN175319, MT863735–MT863741) and CO1 (MN167829–MN167835) genes; fly CO1 (MT863696–MT863702) genes; and 16S rRNA amplicon datasets (PRJNA655365) are available in GenBank. Raw reads for the SyHa and SyCt genomes are available at the Sequence Read Archive under the same bioprojects.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary File
pnas.2000860117.sapp.pdf (1.9MB, pdf)

Data Availability Statement

Symbiopectobacterium genomes from this study (SyHa, PRJNA415854; SyCt, PRJNA521717) and from previous studies (SyPl, PRJNA510127; SyDa, PRJNA510132; SyCl, PRJNA510131); bacterial 16S rRNA (MK943676, MT859673, MT859692, MT859693), gyrA (MN175990–MN175995, MT860065), and groEL (MT860063, MT860064) genes; nematode 18S (MN175314–MN175319, MT863735–MT863741) and CO1 (MN167829–MN167835) genes; fly CO1 (MT863696–MT863702) genes; and 16S rRNA amplicon datasets (PRJNA655365) are available in GenBank. Raw reads for the SyHa and SyCt genomes are available at the Sequence Read Archive under the same bioprojects.

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