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Abstract

There is a developing interest in how immune genes may function in other physiological roles, and how traditionally non-immune peptides may, in fact, be active in immune contexts. In the absence of infection, the induction of the immune response is costly, and there are well-characterized trade-offs between immune defense and fitness. The agents behind these fitness costs are less understood. Here we implicate antimicrobial peptides (AMPs) as particularly costly effectors of immunity using an evolutionary framework. We describe the independent loss of AMPs in multiple lineages of Diptera (true flies), tying these observations back to life history. We then focus on the intriguing case of the glycine-rich AMP,Diptericin, and find several instances of loss, pseudogenization, and segregating null alleles. We suggest thatDiptericin may be a particularly toxic component of the Dipteran immune response lost in flies either with reduced pathogen pressure or other environmental factors. AsDiptericins have recently been described to have neurological roles, these findings parallel a developing interest in AMPs as potentially harmful neuropeptides, and AMPs in other roles beyond immunity.

Introduction

The innate immune system plays a vital role in host defense against pathogens. This is particularly true in invertebrates, which lack an adaptive immune system. Antimicrobial peptides (AMPs) are one of the main effector molecules of innate immunity in many organisms and, as such, they represent the front lines in the coevolutionary struggle between host and pathogen. AMPs are often cationic, amphipathic peptides that defend their hosts against infection by disrupting the cell membranes of invading microbes (1). However, the dose makes the poison, and AMPs can also be toxic to eukaryotic host cells under certain conditions. This suggests that host immunity needs to strike a delicate balance: AMPs need to be potent enough to quickly inhibit pathogenic microbes, but not so potent that they upset the balance of the microbiota or damage host tissue.

Indeed, many pathologies in humans have been observed when this balance is perturbed. These include chronic inflammatory skin or bowel diseases (2–4), and pulmonary infections including cystic fibrosis wherein reduced levels of β-Defensins and the cathelicidin LL-37 are associated with increased risk of infection (5–9). The cathelicidin LL-37 is implicated in autoimmune reactions because it can be toxic to white blood cells (10), induce inflammation in the nervous system (11), or even damage host tissues during anti-cancer responses (12). Recent studies have also suggested the Alzheimer's peptide Amyloid-beta is an AMP in the nervous system, and that Alzheimer's may in part be an infectious disease (13–15). These observations of AMPs as toxic agents are further supported by reduced lifespan inDrosophila fruit flies ubiquitously expressing AMPs in the brain (16), or systemically (17). During aging,Drosophila NF-κB signaling is also implicated in neurodegeneration with AMPs as prime suspects (18). Thus, AMP dysregulation can impose a significant threat to organismal health.

Insects, and particularlyDrosophila melanogaster, have been integral to unraveling the innate immune response, including the regulation of AMPs by the Toll and Imd NF-κB signaling pathways (19). Thus, far seven AMP gene families have been described inDrosophila:Defensin, Cecropin, Attacin, Diptericin, Drosocin, Metchnikowin, andDrosomycin. Another class of AMP-like effectors called theBomanins are also essential for Toll-mediated defense, however their antimicrobial properties await functional clarification (20,21).Drosophila AMP evolution is shaped both by balancing and diversifying selection at the sequence level (22,23). Following a duplication event and subsequent speciation,Drosophila Diptericins rapidly diverged into distinctDiptericin clades (24). In contrast, balancing selection seems to maintain a stable polymorphism amongst alleles that provide either moderate or poor protection against systemic infection withProvidencia rettgeri (P. rettgeri) (25). Why selection should favorDiptericin alleles that result in loss of immune competence is unclear. One possibility is that the immune-poorDiptericin allele is selected for through trade-offs between poor immune defense when infected and higher fitness when uninfected. Indeed, rareDiptericin null alleles are observed in North American populations (25), and patterns of duplication and loss inDiptericin and otherDrosophila AMPs have resulted in copy number variations amongst species (24,26–28).

As AMP dysregulation can affect health, copy number variation may impose a significant challenge for the maintenance of optimal gene expression (29). Yet perhaps the most overt patterns in AMP evolution are duplication events affecting copy number, which is widespread in both humans and fruit flies (30–34). Therefore, conflict between maintenance of healthy expression levels and improved immune competence may drive patterns of AMP gain/loss or changes in expression patterns. In this model, we expect that AMPs are evolutionary liabilities in the absence of infection, and that host ecology and associated pathogen pressure will drive the evolution of AMP content both at the level of broad AMP gain/loss, and also of AMP expression: species with strong pathogen pressures would evolve to increase potential AMP production, while species whose ecologies involve less exposure to pathogens would be expected to reduce their AMP complement.

While characterizing pathogen pressure in different animal hosts is exceedingly difficult, we can use host ecology as a proxy for infectious pressures. The use of sterile food resources (such as plant sap) reduces the opportunities for microbes to inoculate hosts. There are several insects that spend large portions of their lives (larval, adult, or both) feeding on sterile or near sterile food resources—likely reducing the evolutionary benefits of AMPs and/or AMP induction. The pea aphid (which feeds on sterile plant phloem) is one such insect that has lost not just effectors, but also an entire NF-κB immune signaling pathway (35). Loss of immune signaling is also observed in plant-feedingTetranychus mites (36,37), as well as bed bugs (38) and body lice (39), suggesting blood-feeding may be a similarly clean feeding ecology. It should be noted that in some cases these hosts have intimate associations with endosymbionts, microbes that supplement nutrition or protect against infection. One argument to explain loss of immune signaling is that it is a direct consequence of endosymbiont presence to avoid negative consequences associated with chronic activation of the host immune response (40). However, cereal weevils live in sterile environments and have nutritional endosymbionts, but they instead utilize AMPs to regulate their symbiont populations (41). Thus, what factors of sterile lifestyles and/or endosymbionts promote immune gene loss remains poorly resolved.

As AMP copy number evolves rapidly, we suspected AMP evolution might respond to shifts in host ecology before entire immune pathways are affected. To test this, we surveyed Diptera (true flies) for AMP presence or absence and interpret this in the context of host ecology. Diptera are an extremely diverse lineage with equally diverse and unique ecologies and life histories, and boast numerous sequenced genomes and transcriptomes. We probed these diverse flies for classic AMP families described inDrosophila and other insects to better understand the forces driving AMP gain or loss. We further analyzedDrosophila copy number and sequence variation in conserved AMPs, tying these results back to life history. Globally, we describe a pattern suggesting AMPs are lost in Diptera lineages with more sterile life histories, with striking parallels to loss of immune signaling in other arthropods with sterile food resources. We also focus onDiptericin, which we suggest is a particularly costly AMP, describing distinct evolutionary patterns across ecologically diverseDrosophila and withinD. melanogaster.

Results

Some AMP Families Are Absent in Diptera Living in More Sterile Environments

Diptera diverged from other related holometabolous insects about 272 mya [timetree.org; Kumar et al. (42)] and diversified into extremely broad ecological habitats. We surveyed 31 Dipteran genomes as well as diverseDrosophila species for the presence of eight AMP/AMP-like families either described inDrosophila (Bomanins, Drosocin, Drosomycin, Metchnikowin) or characterized more broadly across Dipterans and other insects (Diptericins, Cecropins, Attacins, Defensins). We also annotated the feeding ecologies of these diverse flies to better understand which lineages may have reduced pathogen pressure owing to food resource use (Supplementary Data File 1). We performed an iterative reciprocal BLAST search using known AMPs against genomic or transcriptomic sequence. We found thatDrosocin, Metchnikowin, Bomanins, andDrosomycin are restricted toDrosophila and their close relatives (Figure 1). Using a lenient E-value threshold, we were able to recoverMetchnikowin from diverse mushroom-feedingDrosophila and perhaps other flies, and confirmed their identities by alignment and reciprocal BLAST (Figure S1), improving on previous annotations of immune genes in this lineage (24). The other AMP families show a broader taxonomic distribution (Figure 1).

Figure 1

One striking pattern is the absence ofCecropin in two Nematocerans: the plant-feeding Hessian flyMayetiola destructor and the oyster mushroom pestCoboldia fuscipes. TheCoboldia genome is a small, well-assembled genome (~100 Mbp, scaffold N50 = 242 Kbp) (43,44), andCecropins throughout Diptera share similar motifs from the N terminus to C terminus. As such, we interpret this absence ofCecropin in bothM. destructor (plant sap-feeding) andC. fuscipes (scavenger-feeding) as a likely true loss ofCecropin in this basal lineage. We also found a partialCecropin sequence truncated by a premature stop codon inHermetia illucens (scavenger-feeding). Finally, we did not recoverCecropin from the genome of the leafminerLiriomyza trifolii, an independent transcriptome ofL. trifolii pupae (a life stage when AMPs are often highly upregulated), or from a sequence read archive (SRA) file (GenBank accession:DRX064600) of the relatedLiriomyza chinensis. We see no immediate pattern in feeding ecology or life history that predictsCecropin loss, but we also failed to recover anAttacin fromLiriomyza, suggestingLiriomyza has lost AMPs from two gene families (Cecropins andAttacins). Called “leafminers,”Liriomyza larvae feed internally in the leaves of plants, an environment protected from external microbes by the immune system of the host plant; a more sterile food resource than most Dipterans. Moreover, we also failed to recoverDiptericin in three flower-feeding Tephritid species. Like the leafminers, these flower-feeding flies live in a protected environment owing to larval development inside budding flower inflorescences (45).

Within the genusDrosophila, we observed two unique instances of AMP gain/loss we note separately. First, the genome of the cosmopolitan flyDrosophila busckii encodes no less than nine intactDiptericin genes, and we further recovered three pseudogenes in theD. busckii Diptericin gene region (Figure S2); for context, otherDrosophila typically have only 2–3Diptericin genes (24).Drosophila busckii is a cosmopolitan generalist species in common association withD. melanogaster, howeverD. busckii arrives later to rotting fruits and compost relative toD. melanogaster (46,47). To favor the retention of so manyDiptericin copies suggests theDiptericin response is highly importantfor D. busckii ecology. Second, we found that one paralog of theAttacin A/B duplication event has been lost inDrosophila sechellia, a species closely-related toD. melanogaster.Drosophila sechellia is famous for its unique ecology, feeding on toxic morinda fruit that repels other flies (48). Beyond losing thisAttacin paralog, D. sechellia also lacks the ability to encapsulate and kill invading parasitoid wasps, associated with loss of function in immune genes involved in the melanization and stress responses (49). It seems likely that the toxins in morinda fruit would repel parasites such as wasps, reducing infectious pressure onD. sechellia. Thus, this ecology—already associated with loss of immune genes—may have additionally promoted loss of anAttacin as well.

Overall, we observe numerous instances of AMP loss across the Diptera phylogeny. The loss of Cecropins in ecologically diverse lineages is puzzling. For the mushroom pestC. fuscipes and Hessian flyM. destructor, either scavenging (C. fuscipes) or sap-feeding (M. destructor) could reflect an ancestral ecology promotingCecropin loss. These two last shared a common ancestor ~250 mya [Timetree; (42)], and transitions from generalist to specialist ecologies, and back again, have been inferred inDrosophila (50). However, more strikingly we observe AMP gene family loss in all three strictly plant-feeding fly lineages assessed (the Hessian flyM. destructor, Liriomyza leafminers, and flower-feeding Tephritids), reminiscent of immune gene loss in sap-feeding Pea Aphids.

Parallel Loss of Diptericins in Lineages With Divergent Ecology

In our screen of AMP conservation in Diptera, we were intrigued by the loss ofDiptericin in some Tephritid fruit flies and someDrosophila;Diptericin was previously shown to have rare null alleles segregating in a North AmericanD. melanogaster population (25). While assembly quality was variable amongst the Tephritid genomes, the absence ofDiptericin in three independent flower-feeding Tephritid species, but presence in all screened fruit-feeding Tephritid species suggestsDiptericin is lost in the flower-feeding Tephritid lineage.Diptericin is an AMP that has attracted a great deal of attention as the canonical readout of Imd signaling inD. melanogaster (19), and for its highly specific interaction withProvidencia rettgeri bacteria (25,51). Interestingly, theDiptericin sequence retained in the fruit-feeding Tephritids has converged on a DrosophilidDptB-like sequence (Figure 2A). Furthermore, it was previously reported thatDptB was pseudogenized in the mushroom-feedingDrosophila speciesD. guttifera and likely also inDrosophila neotestacea (D. neotestacea) (24). However, when we screened the recently-sequenced mushroom-feedingDrosophila innubila genome, we recovered intact coding sequence forDptB. It is possible that the intactDptB sequence inD. innubila could reflect thatDptB in mushroom-feeding flies was initially pseudogenized not due to loss of coding sequence, but rather due to mutations affecting gene expression. We therefore performed qPCR following infection to determine the expression profile ofDiptericins amongst mushroom-feeding flies and included outgroupDrosophila to inform our interpretations.

Figure 2

We used a Bomanin (Bom791) as a positive control for infections more specific to the Toll pathway inD. melanogaster. We further intended to useDpt as a specific readout of Imd signaling, and as an independent control gene for assessingDptB expression. First, we found thatDptB is strongly induced inD. innubila, suggesting it is not pseudogenized as in sister lineages. However, we found thatDpt expression is highly variable acrossDrosophila species (Figure 2B).Dpt is more specifically induced by Gram-negative bacterial challenge inD. melanogaster, and indeed we see this pattern in the outgroup fliesDrosophila pseudoobscura (D. pseudoobscura) andDrosophila immigrans (D. immigrans) (Supplementary Data File 1), and also broadly inD. innubila. However,Dpt andDptB are similarly induced by either Gram-negative or Gram-positive bacterial challenge inD. virilis, and the same is true forDpt in bothDrosophila testacea (D. testacea) (not shown), andD. neotestacea (Figure 2B, and see Materials and methods). Using Sanger sequencing, we additionally confirmed thatDptB is pseudogenized inD. testacea by a premature stop codon, supporting its absence in theD. neotestacea transcriptome (GenBank accession:MN311476). The mutation affectingDptB in the Testacea group is distinct from the mutation in the Quinaria group speciesD. guttifera (Figure 2C), suggesting independent loss events.

Thus, the pseudogenization ofDptB-like genes in both flower-feeding Tephritids and two lineages of mushroom-feeding flies reflects that first there was convergent evolution towardDptB-like sequence in both Tephritids and Drosophilids. Thereafter, subsequent independent losses ofDptB-likeDiptericins occurred in lineages with ecologies that diverged from fruit-feeding. This pattern suggestsDptB may be attuned to fruit-feeding ecology, but not as useful in other ecological niches.

We can only speculate on how evolution shapes patterns ofDptB gain/loss: inD. melanogaster, functional characterization ofDptB was long-ignored in favor of its more potently-induced paralogDpt. However, recent studies have revealed that the twoDiptericins have markedly different activities in immunity and host physiology. First, Unckless et al. (25) showed that a specific serine allele inDpt confers defense againstP. rettgeri, however noDptB gene in any fly encodes a serine at this site. Alternately, Barajas-Azpeleta et al. (52) found thatDptB, but notDpt, is required for long-term memory formation. There are a number of overt structure and sequence differences amongstDpt andDptB (Figure 2D). First,Dpt encodes an 83-residue mature peptide with a proline-rich domain tailed by a glycine-rich Attacin-family domain. This 83-residue mature peptide is secreted following cleavage of the Dpt signal peptide and propeptide. On the other hand,DptB encodes a furin cleavage site (RVRR) between its proline-rich and glycine-rich domains, similar to other AMPs of the Attacin gene family (53); In Attacin C, this furin cleavage results in two secreted peptides, a proline-rich AMP (called MPAC) and a separate glycine-rich AMP (54). Furthermore, amongst the many sequence differences betweenDpt andDptB (seeFigure 2A) is the aforementioned serine residue of Dpt that confers defense againstP. rettgeri bacteria. InDpt genes, this residue is polymorphic (S/R/Q/N) inD. melanogaster and close relatives (25). However, DptB encodes a polymorphism for only Q/N at this site, including in convergentDptB-likeDiptericins of Tephritid fruit flies. Globally, inD. melanogaster, Dpt appears to be key in mediating defense againstP. rettgeri bacteria, whileDptB is uniquely required for memory formation. Accordingly, these twoDiptericins have overt differences in mature peptide products. Here we implicate host ecology as a likely determinant ofDiptericin evolution, and suggest that these overt differences may have ecology-dependent effects on fitness leading to distinct patterns of gain/loss.

Null Alleles of Diptericin Are Segregating in Wild Populations ofD. melanogaster

Our findings onDiptericin evolution coupled with recent descriptions of distinctDiptericin activities uniquely position this AMP family for providing insight into how conflicting roles in immunity and physiology can shape AMP evolution. Unckless et al. (25) reported the maintenance of two alternate alleles (serine/arginine) at residue 92 of the full length Dpt protein (residue 69 of the mature peptide) in wild populations ofD. melanogaster andD. simulans.Providencia rettgeri is a natural pathogen ofD. melanogaster (55), yet theDpt arginine allele is maintained by balancing selection in the wild despite being associated with poor immune defense againstP. rettgeri infection. Additionally, Unckless et al. (25) reported a rare null mutation in aD. melanogaster North American population (DGRP) (56) resulting in a premature stop codon affecting ~1% of the reference strains. Surprisingly, when we investigated a set of African populations (DPGP) (57), we found multiple independent null mutations segregating in different sub-populations throughout the Africa sampling range (Figure 3A). Even more surprising, the prevalence of these null mutations reaches over 20% in some populations and appears to follow a latitudinal cline (Figure 3B). As such, selective pressure onDpt may follow a clinal gradient in Africa, favoringDpt loss in southern African populations. Note that the cline crosses the equator and so may not be driven by climate alone. We also recover a similar, though not significant, trend for null alleles segregating in North American collections (Figure 3C).

Figure 3

These data suggest that despite the described importance ofDpt in defense against the ecologically relevant pathogenP. rettgeri, null alleles associated with extremely poor immune defense are actively segregating in wildD. melanogaster. This suggests that the evolutionary forces behindDiptericin loss are not entirely passive. Taken together with the loss ofDptB in other flies, instead this implicates active selection onDiptericins as peptides deleterious for fitness in alternate ecological conditions.

Discussion

AMPs must maintain a fine balance: being potent enough that they can kill harmful pathogens but not so harmful that they damage host tissues directly or by damaging beneficial components of the host's microbiome. It stands to reason that host ecology drives pathogen pressure and therefore might indirectly shape the complement of AMPs in a given host. In our survey of AMPs across Diptera and withinDrosophila presented above, we find some support for an adaptive loss of AMPs in hosts associated with more sterile habitats. There is increasing awareness that these classic immune molecules can play diverse physiological roles, and that evolution may be shaping AMP copy number and sequence due to selection on non-immune functions. When considering the internal plant parasites of the Tephritid family and the leaf miner, clear parallels can be drawn regarding sterile food resource use and other fluid-feeding arthropods that have similarly lost or re-organized their immunity genes, namely: aphids, some mites, bed bugs, and body lice (35–39). It may also be that these plant-parasitic flies have yet-uncharacterized bacterial endosymbionts that impose selection against certain AMPs, enabling their specialist lifestyle. We also describe multiple incidents ofDiptericin loss inDrosophila:DptB in some mushroom-feeding flies, a lineage with a specialist ecology whose microbiota differs drastically fromD. melanogaster (58), whileDpt null alleles are segregating in wildD. melanogaster populations.

The central question then becomes: why should immune-inducible AMPs, antimicrobial agents required for competent host defense, be lost so readily? We can think of two evolutionary scenarios that would lead to the loss of AMPs. First, when infection pressure is low, relaxed constraint on protein sequence and/or expression could lead to the accumulation of mutations that compromise protein function and eventually lead to pseudogenization and loss. This represents a neutral process where genetic drift is the force removing AMPs. The second evolutionary scenario is that AMPs are costly in the absence of infection, so when infection pressure is low, mutations that compromise function (indels, premature stop codons, cis-regulatory mutations) are actually selected for. If periods of low infection pressure persist long enough, those mutations can become fixed, and gene function is lost.

Several lines of evidence support the second, “selective loss” scenario. First, if relaxed constraint in the absence of infection drove AMP loss, we would expect the loss of AMPs to be somewhat random. However, we see convergent loss ofDptB-like genes in independent lineages with divergent ecologies, andDpt null alleles segregating in wild populations ofD. melanogaster. Of course, if AMPs are specific to a small suite of pathogens (e.g.,Dpt andP. rettgeri), perhaps those pathogens are relatively absent in some natural populations compared to others. This would also lead to increased loss of AMPs, butvia neutral processes. In the case ofDpt, however,Providencia rettgeri is distributed worldwide including throughout Africa (59,60). Therefore, it is unlikely to be completely absent from African populations ofD. melanogaster where null alleles are common. Instead, the specific loss ofDiptericins inDrosophila might reflect a deleterious consequence when dysregulated in non-immune tissues. For example, AMP expression increases dramatically in the head tissue of aging flies (18). This explanation seems more likely, as non-cell autonomousDptB is known to affect memory formation inD. melanogaster (52), evidence of directDiptericin impact on brain function. A second line of support for the “selective loss” scenario is the null allele cline observed in African populations alongside a parallel (though not significant) cline in North America. Such parallel clines are often used as evidence for selection acting on alleles (61–63). While neutral processes could lead to clines in null alleles as well, with the null allele spreading from an initial source population, the likelihood of this happening in parallel on two continents is small. Finally, there is growing evidence that several AMPs may inflict damage on host tissues. For instance, the cathelicidin LL-37 is toxic to leukocytes (64), and constitutive expression of AMPs reduces lifespan inDrosophila (17). These direct observations of deleterious effects strongly undermine the idea that neutral processes are driving the loss of AMPs, instead suggesting these molecules impose a significant effect on host fitness.

One exciting explanation for AMP fitness costs is the idea that AMPs are dysregulated through aging, leading to chronic inflammatory responses and eventually cell death. Additionally, the idea that AMPs may be active in the nervous system is an attractive recent hypothesis that demands more consideration (65,66), particularly to understand the roles these short peptides play in neuronal homeostasis (67). For instance, while implicated in Alzheimer's disease for decades, the specific nature of how Amyloid-beta contributes to dementia remains unclear (68). Understanding its role in immunity may shed light on the cause and progression of amyloid plaques (13), and reveal the true culprit(s) behind Alzheimer's progression; an interesting recent study found that Amyloid-beta binds to the human cathelicidin LL-37, forming heterodimers that reduce the toxicity of LL-37 to host cells (69). Alongside evidence that Alzheimer's may be an infectious disease (14), dysregulation of AMPs in the nervous system upon chronic infection could lead to host cell toxicity. Appreciating the role of AMPs in the nervous system, particularly during infection, may lead to breakthroughs in treating neurodegenerative disorders such as Alzheimer's disease or Parkinson's disease.

If indeed AMPs are deleterious in non-immune contexts, this may promote balancing selection in populations with dynamic immune pressures. Beyond AMPs, trade-offs between immunity and fitness are well-documented, implying that an immune system is advantageous only in the context of immune challenge, but otherwise is detrimental to reproductive fitness (70–73). As the front line of innate immunity, AMPs should be primary actors on this evolutionary stage, and selection for or against immunity genes should therefore act strongly on context-dependent AMPs. Recent studies report that both balancing and diversifying selection has shaped theDrosophila AMP arsenal (22–25), revising how we view AMPs as actors in host-pathogen interactions (28). If balancing selection is driven by trade-offs between alleles that provide increased resistance during infection but are costly when hosts are uninfected, this could explain the dynamic patterns of AMP gain and loss described here. By characterizing e.g.Diptericin loss throughout Diptera, we provide the beginnings of an immunological fossil record with extinct (pseudogenes) and extantDiptericin gene copies in different lineages. The observations of other AMP gene losses throughout Diptera extend this fossil record back in time, describing lineages with different stages of loss stemming from an ancestral immune-competent fly to derived lineages lacking subsets of certain AMPs.

Globally, we highlight how host ecology predicts AMP loss, and follow the evolution of AMP lineages throughout Diptera. We describe that selection on the innate immune system can act swiftly and directly on AMPs, implicating some AMPs as deleterious molecules in the absence of microbial challenges. These results could relate to the newly discovered role of AMP-like peptides in neurodegenerative diseases and autoimmune disorders. If so, our findings offer evolutionary signatures supporting the notion that trade-offs between immunity and fitness are mediated by costs related to the maintenance of autotoxic host AMPs.

Materials and Methods

Survey of AMP Families in Published Diptera Genomes and Transcriptomes

We first conducted a thorough literature review to annotate the life histories of diverse Diptera. We then searched forDrosophila AMP families present in other Diptera using an iterative step-wise tBLASTn approach followed by manual curation; of note, we used an extremely lenient E-value for the shortest peptides (e.g., E <100 for Mtk, 26 residues long), followed by manual curation. In brief, we collected AMP genes from sequencedDrosophila and then BLASTed all available orthologs against outgroup genomes from Vicoso and Bachtrog (43). We collected all confirmed outgroup orthologs and re-performed this BLAST against any species where no match was found, until we ceased to recover new orthologs. To verify any patterns of loss we observed (e.g.,Dpt loss in Tephritid species), we further searched for outgroup sequence data (genomes, transcriptomes, or raw SRAs) to include in our analyses as independent databases. For some orthologs, only a partial sequence was recovered on a scaffold assembled with many gaps (NNNs). If sequence similarity was highly conserved we annotated these AMPs as “present” but do not include them in phylogenetic analyses as their information content was poor. All sequence databases used in this study are included inSupplementary Data File 1. Sanger sequencing results are deposited in GenBank under accessions:MN311474-MN311476.

To investigate sequence similarity and validate curated orthologs, we aligned sequences using MAFFT and performed phylogenetic analysis using Neighbor-joining (1000 bootstraps) and/or Maximum likelihood (100 or 500 bootstraps) methods implemented in Geneious R10 and the PhyML webserver (74). ForDiptericin evolution inFigure S3, sequences were also codon-aligned using MAFFT.

Fly Stocks and Strain Information

The following strains were used in this study for gene expression analysis and Sanger sequencing:D. melanogaster (DrosDel isow¹¹¹⁸),D. pseudoobscura, D. virilis, D. immigrans, D. innubila, D. testacea, andD. neotestacea.Drosophila pseudoobscura andD. immigrans were generously provided by Ben Longdon and correspond to strains used in Duxbury et al. (75).Drosophila innubila used in this study is the same as the genome-sequenced strain from Hill et al. (76).Drosophila virilis was a gift from Richard Benton corresponding to Sackton et al. (28). Steve Perlman kindly provided Testacea group flies. TheD. testacea strain used corresponds to the wild-typeD. testacea described in Keais et al. (77) cleared ofWolbachia symbionts by the Perlman lab. TheD. neotestacea strain is the same as used in Hanson et al. (24).Drosophila melanogaster, D. pseudoobscura, andD. virilis were reared on standard food medium forD. melanogaster and reared at 25°C.Drosophila immigrans, D. innubila, andD. neotestacea were reared on Nutri Fly^TM instant formulation supplemented with a piece ofAgaricus bisporus mushroom, and reared at 22°C. All species were kept at 22°C during the course of infection. All flies used in this study were previously shown to be negative forWolbachia, a common endosymbiont ofDrosophila.

Gene Expression Analysis

Infections, RNA extraction, and cDNA synthesis were performed as previously described (51). Pooled samples of 6 flies (3 males and 3 females) were used for each replicate experiment, and three repeats were performed (18 total flies per treatment per species). Flies were frozen either 6 hpi (Unchallenged,Escherichia coli (E. coli)] or 24hpi (M. luteus, C. albicans) at −20°C in TRIzol. Quantitative PCR was performed on a LightCycler 480 (Roche) in 96-well plates using Applied Biosystems PowerUP Master Mix. Error bars represent one standard deviation from the mean. Statistical analysis was performed using One-way ANOVA with Holm's-Sidakpost-hoc comparisons to the treatment that induced expression most in each species for each gene (marked as “ref”); e.g., theE. coli treatment was the point of comparison forDpt, andC. albicans for Bomanin inD. melanogaster.P-values are reported as: not significant = ns, <0.1 =^•, <0.05 =^* <0.01 =^** and <0.001 =^***.

We note the pattern ofDpt induction we observed conflicts with a previous report thatDpt is not inducible inD. neotestacea (24), which is likely explained by measuring alternateDpt isoforms. Primers used in this study, additional expression data for AMPs in different species, andD. neotestacea Dpt primer comparisons suggesting alternateDpt isoforms can be found inSupplementary Data File 1.

Diptericin Evolution in Drosophila

We identified segregating putative null alleles inDrosophila melanogaster populations by visually inspecting alignments of re-sequenced individual inbred lines (Figures 3A,B) or pool-seq alignments (North American populations inFigure 3C) (57,78). We found three classes of putative null alleles: (a) premature stop codons, (b) deletions that disrupt core parts of the transcript (i.e., the start codon), and (c) deletions that are in frame but were associated with reduced immune defense againstP. rettgeri in prior studies (79). Thus, while the counts inFigure 3A for the USA represent individual inbred lines, those inFigure 3C represent the proportion of reads at a given site carrying the particular null allele.

Data availability statement

The datasets generated for this study can be found in the GenBank database, accession numbersMN311474-MN311476.

Author contributions

MH performed live specimen experiments. MH and RU screened and analyzed data. MH, BL, and RU wrote the manuscript.

Funding

This work was supported by the National Institutes of Health Grants R00GM114714 and R01AI139154 to RU.

Acknowledgments

We would like to thank Florent Masson, Joanne Chapman, Tom Hill and members of the Unckless lab for useful comments on the manuscript.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary material

The Supplementary Material for this article can be found online at:https://www.frontiersin.org/articles/10.3389/fimmu.2019.02620/full#supplementary-material

Alignment of Metchnikowin (Mtk) and Mtk-like sequences. We recoveredMtk genes from mushroom-feeding flies (brown highlight) that retain the major portion of theMtk mature peptide. We also recovered a clearMtk ortholog in the outgroup DrosophilidP. variegata (Pvar\Mtk), which resembles Mtk-like sequence in the Brachyceran flySolenopsis brevicornis (S. brevicornis) (Sbre\Mtk-like). Other Mtk-like sequences from Brachycerans are also shown, and the full open reading frame is shown for all sequences.

TheDrosophila busckii Diptericin gene region encodes 6 copies of the subgenus Drosophila-restrictedDpt (DptC clade, seeFigure S3) and 3 copies ofDptB. There are an additional 3Diptericin pseudogenes apparent in the gene region.

Maximum likelihood tree showing that TephritidDiptericins paraphyletically cluster within theDrosophila DptB clade, though bootstraps for exact sorting are poor (as expected of paraphyletic clustering); 100 bootstraps, where bootstraps <30 are not shown. MajorDpt clades are highlighted as follows: Blue,DptA; Red,DptC; Green,DptB; Teal, TephritidDpts.

Annotated life histories: Annotations of feeding ecology in larvae and adults of Diptera in this study. Used inFigure 1.Datasets used in this study: Datasets and their accesions and quality scores for diverse Diptera used in this study. Used inFigure 1. Primers used in this study: Primers used in qPCR analysis or Sanger sequencing. qPCR reactions were run with a 60°C annealing and extension phase. Used inFigure 2.qPCR data from alt. species: qPCR data from initial explorations using Drosophila pseudoobscura, Drosophila immigrans, and Drosophila testacea (1 Experiment). Remarked as “not shown” in manuscript.Dneo DptC primer comparison: Comparison of Dneo\Dpt induction as measured by primers from Hamilton et al. (2014) and universal Testacea group Dpt primers from this study. Revises interpretation of Hamilton et al. (2014) and (24).

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Summary

Keywords

innate immunity, antimicrobial peptide (AMP), molecular evolution, population genetics, diptericin, drosophila, diptera, attacin

Citation

Hanson MA, Lemaitre B and Unckless RL (2019) Dynamic Evolution of Antimicrobial Peptides Underscores Trade-Offs Between Immunity and Ecological Fitness.Front. Immunol. 10:2620. doi:10.3389/fimmu.2019.02620

Received

21 August 2019

Accepted

22 October 2019

Published

08 November 2019

Volume

10 - 2019

Edited by

Charles Lee Bevins, University of California, Davis, United States

Reviewed by

Abdelaziz Heddi, Institut National des Sciences Appliquées de Lyon (INSA Lyon), France; Neal Silverman, Worcester Foundation for Biomedical Research, United States

Updates

Check for updates

Copyright

*Correspondence: Mark A. Hansonmark.hanson@epfl.chRobert L. Uncklessunckless@ku.edu

Disclaimer

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