Wolbachia are a genera of alpha-proteobacteria, and infect the cell cytoplasm of over 80% of Lepidoptera species. The endosymbionts can alter host physiology, causing cytoplasmic incompatibility and female-biased sex ratios. This would reduce the fitness of the host population, and potentially hinder conservation efforts. This would be particularly problematic if virulent strains could host-switch and infect other vulnerable local species. Lepidoptera were collected from the Cambridge University Botanic Garden and had DNA amplified by PCR using primers for a cytochrome C oxidase 1 mitochondrial gene in the Lepidoptera host, and a wsp surface protein gene in the Wolbachia. 25 individuals out of 48 collected were infected with Wolbachia. Phylogenies were reconstructed using a neighbour joining, maximum likelihood method, also utilising DNA from the NCBI database. Constructed phylogenies were more similar than expected by chance, indicating vertical transfer. However, the phylogenies were not identical, and sequences from the Wolbachia often matched Wolbachia sequenced in different genera. This suggests horizontal transfer, or convergent evolution is occurring. The use of managed Wolbachia infections has been proposed as a method to control vector-transmitted diseases, however the implications of this depend, in part, on the potential spread of Wolbachia to other hosts. The conservation of moths in the UK may be less successful if there are many virulent pathogens which are easily the spread between individuals, and species. If this is a case, the separation of certain populations may need to be considered.
Wolbachia inhabits the cell cytoplasm of its hosts (in excess of 20% of insect species (Jiggins et al. 2001), and over 80% of Lepidoptera (Ahmed et al. 2015)), and therefore can be transmitted vertically to offspring, but also likely via horizontal transfer to new host groups. The mechanisms and rates of horizontal transfer are not well known. There does not seem to be any significant phylogenic signal between closely related Lepidopteran groups for incidence or prevalence of Wolbachia, further suggesting that vertical transfer is not the only means of transfer.
Phylogenies were constructed to compare speciation between the host and parasite. The molecular markers chosen were a mitochondrial cytochrome C oxidase 1 (COI) gene in the host, and the Wolbachia surface gene (wsp) in the parasite. Mitochondrial DNA is transmitted maternally, and has an effective population size a quarter of the size of nuclear markers, resulting in greater drift. There is also low recombination, so the recent genealogical history of the whole MtDNA molecule is likely to be similar to the chosen gene. Regular COI sequence divergences at COI allow closely allied species to be distinguished, in nearly all animal phyla, including Arthropods. The wsp sequence is present only in the Wolbachia genome. Surface proteins often function as antigens in pathogenic bacteria, and to avoid the host’s immune response, recombination and rapid sequence evolution of these surface proteins confer a selective advantage (Jiggins et al. 2002). However, Wolbachia lineages are subject to genetic recombination, and so a phylogeny based on one gene may not be reflective of the strain histories. Another limitation is that Wolbachia can influence the mtDNA in the host, and may confound the evolutionary history that mtDNA suggests (Hurst and Jiggins, 2005). Both gene sequences are likely to have undergone recent modification, so genealogies of the host COI and parasite wsp should be congruent if the transmission of Wolbachia is entirely vertical. If there is a large amount of horizontal transfer, then the phylogenies will not necessarily resemble each other. These mtDNA changes are detectable because a strong positive selection acts on particular symbiont genes (Jiggins et al. 2002), whereas mitochondrial genes are usually acted on by purifying selection.
The bacterium has been implicated in many phenomena, involving speciation, the evolution of sex determination mechanisms and the synthesis of essential vitamins (Jiggins, 2016). Wolbachia are typically “reproductive parasites” in arthropods, and this diverse range of functions can have a significant influence on host fitness. Any feminization or male-killing could potentially reduce the population size, which is relevant information to direct conservation efforts within the Cambridge University Botanic Garden, where the moths were collected from. The aim of this study is to investigate whether horizontal transfer is a significant mode of transmission between species. This information could lead to further investigation as to which strains are most likely to host-switch, and whether these negatively impact the host.
In September and October of 2018, a range of non-endangered moths were obtained from the Cambridge University Botanic Garden using overnight moth traps. The moths were then identified using field guides.
Diagnostic PCR and DNA sequencing
Using small abdominal sections (3mm) from the insects, DNA was extracted, and amplified using PCR. The DNA was prepared using DNeasy, following the manufacturer’s instructions. From one individual, there were multiple DNA preparations with different primers, for COI, and for wsp. A negative control with water instead of DNA was prepared to see if there was any contamination, and a positive control from a moth that is known to be infected with Wolbachia was also run.
The Wolbachia wsp primers were 81F (5’-TGGTCCAATAAGTGATGAAGAAAC-3’) and 691R (5’-AAAAATTAAACGCTACTCCA-3’). The PCR cycling conditions were 5 min at 95°C, 35 cycles (60s at 94˚C, 60s at 55˚C, 60s at 72˚C), and 7 min at 72˚C.
The Lepidoptera COI primers were HC02198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) and LC01490 (5’-GGTCAACAAATCATAAAGATATTGG-3’). The PCR cycling conditions were 5 min at 95°C, 35 cycles (30s at 94°C, 30s at 50°C, 60s at 72°C), and 7 min at 72°C.
Gel electrophoresis confirmed that the extraction and amplification of DNA was successful. DNA was cleaned using the Exo-Sap protocol, before Sanger sequencing at an external facility.
Phylogenetic analysis of host mtDNA and parasite DNA
Sequences obtained from Lepidoptera infected with Wolbachia were used to reconstruct the phylogenies. Lepidoptera species were identified using nucleotide sequences run through the basic alignment search tool (BLAST), and the closest matches were used in the analyses. Additional sequences from both were obtained from the NCBI database for phylogenetic analyses. Sequences were trimmed to contain regions that could be robustly aligned using CodonCode. Phylogenies were inferred using the F84 Markov model of molecular evolution, with a neighbour-joining algorithm.
A) Lepidoptera phylogeny displayed on the left. The phylogeny was constructed using concatenated COI sequences.
B) Wolbachia phylogeny, displayed on the right. The phylogeny constructed using concatenated wsp sequences.
The trees were constructed using the F84 Markov model of molecular evolution, and a neighbour joining algorithm. The trees are mid-point rooted.
Figure 2: Null distribution of correlation between Wolbachia and Lepidoptera, assigned at random. There were 1000 random permutations in total. The red line indicates the observed correlation value (0.22).
25 moth species out of 48 gave positive results after PCR for infection with Wolbachia. Negatives, however, do not indicate a definite absence of Wolbachia in that species or individual (as samples were only from the abdomen). Related Lepidoptera species were more likely to be infected by similar Wolbachia strains than by chance, and so we can reject the hypothesis that the Wolbachia phylogeny is independent of the Lepidoptera phylogeny (Figure 2, Null distribution, p=0.002). This may be a statistical signature of vertical transmission, but it is possible that recent host switching could produce spurious congruence between the two trees, especially if the bacteria preferentially colonise related hosts. However, the trees (Figure 1) are not identical, and some of the Wolbachia sequences from moths in Cambridge were 100% sequences matches to bacteria found in beetles, spiders, and butterflies (when samples were compared with known databases).
The phylogenies constructed for Lepidoptera and Wolbachia are similar, reflecting vertical transfer, but not identical, reflecting horizontal transfer. However, congruence may also arise from host-switches followed by parasite speciation, or similarities may also reflect preferential host switching between closely related species. The latter is possible, as certain Wolbachia strains seem to be restricted to the same genus, from their host range distribution (Jiggins et al. 2002). The incongruence of the trees is likely to indicate host-switching events, as other evidence suggests similar strains can be found in phylogenetically divergent host taxa (Baldo et al. 2006b). This is additionally supported by sequence matches between our Wolbachia samples and those found infecting orders other than Lepidoptera, e.g. Araneae. As this sequence correlation cannot be from hybridisation, it either reflects convergent evolution, or horizontal transfer. Another possibility is that incongruence between the two phylogenies has arisen as a result of differential survival of multiple parasite lineages rather than host switching i.e. extinctions. If the bacteria diverged prior to the hosts diverging then the two bacterial lineages are paralogues, not orthologues. Information on relative coalescence times could help to discriminate between these possibilities.
From Figure 2, it can be seen that some distantly related species have Wolbachia with more similar sequences than closely related species. An example is that the two species of Coenonympha were infected with bacteria with more recent common ancestors in different genera than with each-other (a similar pattern is also seen in Phengaris, Carterocephalus). On the other hand, indistinguishable sequences represented by species on the same vertical line with no terminal branches also reflect vertical transmission in some cases, where individuals of the same species Agrotis puta, a European moth, are infected with Wolbachia strains that have indistinguishable wsp sequences. This same line also indicates horizontal transfer as these individuals are also grouped with e.g. Ideopsis similis, an Asian butterfly. 4 wsp sequences (out of 6) obtained from the Botanic Garden samples were indistinguishable, and this may reflect high rates of horizontal transfer due to geographic proximity of the species. However, it is not immediately obvious how some of these host switching events could have occurred, due to large distances separating species’ ranges. This could be investigated further, as to whether similarities are arising due to convergent evolution. The wsp sequence will be under selection, as it functions like an antigen, and motif exchanges are favoured (Baldo et al, 2006). As a result the wsp sequence may not reflect the history of the rest of the genome.
The Wolbachia strain infecting Agrotis puta shared 100% wsp sequence similarity with Wolbachia infecting Culex pipiens, mosquitoes in which the bacteria can induce cytoplasmic incompatibility, as sperm from Wolbachia-infected males produce inviable progeny. It is interesting to note the potential link between the strains observed and known strains with negative fitness implications, as the moths from the Botanic Gardens are part of a conservation project. Agrotis puta moths are common but horizontal spread of bacterial that potentially could cause cytoplasmic incompatibility would be a risk for rarer species. There are also species (e.g. Xestia cnigrum) in the Botanic Garden infected with Wolbachia that share 99.45% sequence similarity with strains infecting Hypolimnas bolina. This strain is known to have effects on the sex ratio, producing a female-bias, which again could reduce population size of infected species. The shared wsp sequence does not guarantee that the cytoplasmic incompatibility genes are present in the bacteria from the moths, or that they would function in a similar way. This would also be a point for further investigation, to study whether the rest of the Wolbachia genome matches the virulent strains, and whether similar effects are seen. This would allow management of the spread of fitness-reducing strains.
Using only one gene to construct a phylogeny does not necessarily give a ‘gene tree’ that reflects the ‘species tree’, particularly if introgression is common between species. Wolbachia genes do not evolve in a bifurcating manner due to horizontal gene transfer. Grouping of truly derived strains cannot be inferred from traditional cladistics methods. As a result of this recombination, a single gene such as wsp is insufficient for a high level of support for the clade produced.
Neighbour-joining joins a pair of taxa at each step to give the greatest decrease in the estimated tree length. This procedure does not always give the topology which is optimal by the balanced minimum evolution criterion, although it often does, it is not guaranteed to be correct.
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