Our results highlight the importance of Wolbachia as a nutritional mutualist in bed bugs. The elimination of Wb significantly increased nymph development time and reduced adult size and survivorship, eggs laid per female, and hatch rate. When aposymbiotic bed bugs were fed blood supplemented with B-vitamins, nymph development time, egg hatch rate and adult survival were fully recovered to the levels of control bed bugs, but adult size and per capita egg production only partially recovered. This is the first study in bed bugs that fully eliminated Wb before and throughout experimentation, and that mitigated the side-effects of antibiotics by discontinuing the use of antibiotics for a generation before the start of experiments. It is also the first study that used a complete design that included normal Wb-present (Wb+) control bed bugs in all fitness-related assays. Our results support and extend the findings of previous studies that used a range of Wb-free (Wb–) and Wb-deficient bed bugs. Our results also add life history traits that are affected by Wb to previously documented traits.
What motivated this work?
Pioneering studies of the Wb-bed bug symbiosis confirmed that Wb is housed in a gonad-associated bacteriome and is vertically transmitted, and demonstrated that Wb produces biotin and riboflavin that benefit the bed bug host16,17,18. Hosokawa et al.16 examined several fitness-related traits, including the rates of egg and nymph development, and the percentage of nymphs that emerged into adults. Their results showed that Wb was an essential nutritional symbiont for normal growth and reproduction of C. lectularius. Four main reasons motivated us to re-examine these traits and consider additional traits. First, in the previous studies, “aposymbiotic” bed bugs obtained with rifampicin treatments were shown to be Wb-deficient, with titers reduced by 100 to 10,000 wsp gene copies; only 8 of 30 (26.7%) of the “aposymbiotic” bed bugs were shown by qPCR to be free of Wb16. Second, in previous work, rifampicin was used throughout the experiments in two treatment arms—“aposymbiotic” bed bugs fed blood and “aposymbiotic” bugs fed blood supplemented with B-vitamins16. Adverse effects of the antibiotic might therefore affect life-history traits throughout the experimental period. Third, in some experiments, control bed bugs harboring Wb were not used17,18, so it was difficult to determine if B-vitamins fully or partially recovered the traits that were examined. Finally, we felt that some incongruent results for numbers of eggs laid and adult emergence in Hosokawa et al.16 and Moriyama et al.18 required closer examination. For example, the number of eggs laid was unaffected by antibiotic treatment in the early study16, but the same treatment resulted in complete elimination of egg production in the later study18. Likewise, adult emergence was greatly reduced in the 2010 and 2014 reports16,17, but the 2015 study18 showed almost no adult emergence in the Wb-deficient colonies. It is important to emphasize, however, that although our results differ quantitatively from these previous studies, they broadly support the general conclusions of these groundbreaking studies.
To address these four issues, we developed procedures for absolute quantification of Wb titers in bed bugs and used these procedures to confirm that our rifampicin-treated bed bugs were indeed aposymbyotic. Compared to qPCR, droplet digital PCR (ddPCR) has higher sensitivity, specificity, and reproducibility; it can quantify low target numbers20 (our empirically derived lower detection level was 0.5 gene copies per µl) and it provides the absolute quantification of DNA in a sample21,22, making it an appropriate technology for confirming the absence of Wb in bed bugs. Using ddPCR, we showed that 100% of the third instars, fifth instars, and adult males, and 90% (9 of 10) of the adult females tested in our experiments were Wb-free; the one adult female that contained Wb was highly Wb-deficient with 1000-fold fewer Wb wsp gene copies than normal Wb+ females. These procedures ensured that the changes in life history traits attributed to the presence or absence of Wb and B-vitamin supplementation were not confounded by incomplete elimination of the endosymbiont.
Persistent antibiotic treatments have many unintended side effects. Yet, many studies on insect-microbe symbiosis administer large doses of antibiotics during the implementation of experiments. If available, chemicals with similar side effects but no antibiotic properties could be used in control treatments. However, this approach is complicated and we are not aware of its use in research on insect symbiosis. Instead, we treated a colony of bed bugs with rifampicin for multiple generations (experimental design in Fig. 1), confirmed that they were aposymbiotic by ddPCR, then reared one generation without rifampicin to clear the antibiotic from the colony, and again confirmed by ddPCR that they remained aposymbiotic throughout the experiments. We also included Wb+ control groups in all fitness tests to determine if vitamins fully or partially recovered the effect of Wb elimination.
Specific effects of aposymbiosis on bed bugs and B-vitamin supplementation
Table 1 summarizes the biological traits tested in our study, whether the elimination of Wb affected the trait and if blood meals fortified with B-vitamins could recover the trait to the levels observed in control bed bugs (Wb+Vit–).
Table 1 Summary of the effects of Wolbachia (Wb) on life-history traits of C. lectularius and whether the addition of B-vitamins to blood meals partially or fully restored the trait to the level of control (Wolbachia-present, Wb+) insects.
Our results showed that the elimination of Wb did not affect feeding rate in bed bugs. It also did not affect survival of nymphs and the cumulative emergence of adults. The latter results were notably different from previous findings16,17,18. Consistent with earlier findings16, however, development of aposymbiotic nymphs was slower and their mean time to adult emergence was 1.8 weeks (22.2%) longer than control (Wb+Vit–) nymphs. Supplementation of the blood meal with B-vitamins restored the rate of development of aposymbiotic nymphs and their mean time to adult emergence was only 0.14 weeks (1.7%) slower than the control nymphs.
Adult size was significantly lower in aposymbiotic females and males than in normal Wb+ bed bugs. Interestingly, whereas normal females are significantly larger than normal males, aposymbiotic adults converged on the same smaller size, indicating that the absence of Wb affected female size more than male size. Smaller adult size was also observed in aposymbiotic stinkbugs, Megacopta punctatissma and Megacopta cribraria23, but the underlying mechanisms remain unknown. In our study, B-vitamin supplementation recovered the sexual dimorphism in adult size, but body size only partially recovered relative to normal adults. These findings suggest that the recovery may require multiple generations or that Wb provisions the bed bug with other factors, including other nutrients, in addition to B-vitamins.
Our results demonstrated dramatic effects of Wb on reproduction. Aposymbiotic females oviposited 85.8% fewer eggs than control females (Wb+Vit–), despite being fed every 5 days and remated once. Supplementation of the blood meal with B-vitamins significantly increased per capita oviposition by 5.5-fold, but it was only partially recovered to the oviposition rate of normal females. Of interest, however, was the observation that in B-vitamins-fed aposymbiotic females (Wb–Vit+) oviposition increased over time, reaching within 10.7% (2.15 eggs) of the oviposition rate of normal control females on days 41–45, but control females changed minimally. These observations suggest that the recovery of egg production with B-vitamin supplementation is a gradual process, and it may require more than 45 days for their full recovery.
In contrast, egg hatch rate in aposymbiotic females declined by 88.5% between days 5 and 45, whereas in normal control females it declined only 14.0% during the same period. B-vitamins fully recovered the effect of Wb elimination as early as day 5 (91.6% hatch), and the hatch rate of Wb–Vit+ eggs declined only 18% between days 5 and 45, similar to control females. These results are generally consistent with previous findings of low egg development in aposymbiotic C. lectularius16.
Effects of adding B-vitamins to Wb+ bed bugs
Bed bugs require a blood meal before every molt9 and have 5 instars. Therefore, we did not expect adults until week 6, and indeed 37.5% of the adult emergence in the Wb+Vit– control treatment occurred on week 6. The addition of B-vitamins to normal (Wb+) bed bugs sped up adult emergence, and the mean adult emergence of vitamin-supplemented Wb+Vit+ nymphs was a week earlier than that of normal bed bugs fed non-supplemented blood; two males (of 67 total, 3%) emerged after only 5 weeks. B-vitamin supplementation of normal bed bugs has not been reported previously, so this is the first report of faster nymph development on B-vitamin supplemented blood.
B-vitamin supplementation of normal nymphs did not affect their cumulative emergence to adults, nymph survival during the 4-month experiment, or adult size. However, it is interesting that the addition of B-vitamins to the blood meals of normal adult females reduced their per capita egg production and female survivorship over 45 days, but not the egg hatch rate. This was unexpected because B-vitamins are water soluble, animals excrete high levels of excess vitamins, and they store only moderate levels of B-vitamins8. It is possible that bed bugs are more susceptible to high levels of certain B-vitamins. For example, biotin (B-7), which is essential for processing of lipids, carbohydrates, and amino acids, is only required in minute doses for insects24 and dietary overdose of biotin has been shown to be an effective chemosterilant in the house fly, Musca domestica25 and it reduced fertility in the hide beetle, Dermestes maculatus, by suppressing embryogenesis26. Because Wb is known to produce biotin in bed bugs17 it is possible that the low concentration of biotin (10 ng/mL blood) fed to Wb+ bed bugs similarly affected female fecundity. The mechanisms underlying the differential effects of B-vitamin supplementation on development and reproduction are unknown and should be investigated.
Differences among studies and limitations of this study
In addition to differences in the Wb titers of aposymbiotic bed bugs in this and previous studies, other differences are notable. The bed bug strain used in previous studies16,17,18 to test the biological effects of Wb was the Japan Environmental Sanitation Center (JESC) from Kanagawa, Japan, which lacks a facultative γ-proteobacterial symbiont16,17,18. This symbiont is present in the Harold Harlan strain used in the current study, but we did not test whether it was eliminated by the rifampicin treatment. It is plausible that the γ-proteobacterial symbiont is also a nutritional mutualist and it too was eliminated by our extensive rifampicin treatments. This might explain the only partial recovery of several traits with B-vitamin supplementation. It is also important to note that in all studies using antibiotic treatments, the gut bacterial communities were likely dramatically affected or eliminated. Thus, any life history traits that could not be fully recovered in Wb-free bed bugs with B-vitamin supplementation might have been affected by a disrupted gut microbiome.
Moreover, as mentioned before, Nikoh et al.17 and Moriyama et al.18 reared one generation of rifampicin-treated bed bugs without blood meal supplementation with B-vitamins, to eliminate transgenerational carryover of B-vitamins. Insects do not have a large capacity to store B-vitamins8, but it is conceivable that this experimental design depleted certain B-vitamins in the developing nymphs. It is unclear if this difference in design contributed to the differences between our studies.
Finally, we used the Kao and Michayluk B-vitamin mix, as did Fisher et al.27,28, which differs substantially from previous studies with aposymbiotic bed bugs16,17,18. Nevertheless, direct comparisons of these vitamin mixes showed that both had no effect on egg production in Wb-present (Wb+) bed bugs and both similarly, but only partially, restored egg production in Wb-free (Wb–) females. Both vitamin mixes equally restored hatch rate of Wb– females to the high levels of Wb+ females.
Outstanding questions on the role of Wolbachia in bed bugs
There are numerous questions to be addressed about the nutritional fitness-related benefits of Wb in bed bugs. B-vitamins are co-enzymes in various metabolic reactions8, so it is not surprising that Wb would contribute to nymph growth and development, egg production and hatch rate. Our study shows the importance of B-vitamins in bed bug development and reproduction, but adult size and egg production results suggest that either our B-vitamin mix was not optimal or that Wb, or other symbionts that were eliminated by rifampicin, contribute nutrients to its C. lectularius host. Our observations also suggest that B-vitamins aid in the digestion of blood meals. We observed that aposymbiotic bed bugs that were not supplemented with B-vitamins retained blood in their gut for a longer duration than in all other treatments. B-vitamin supplementation in aposymbiotic bed bugs partially recovered faster blood meal digestion. In tsetse flies, the endosymbiont Wigglesworthia glossinidia was shown to produce vitamin B-6 (pyridoxine) and elimination of the symbiont showed that Wigglesworthia influences immunity, reproduction and digestion29. A closer look at how tsetse flies utilized vitamin B-6 showed that it was critical for proline homeostasis, which tsetse flies rely on to produce ATP30,31. These metabolic downstream effects of vitamin provisioning could be present in bed bugs, requiring further investigation.
Furthermore, it remains to be determined whether dramatic effects on fecundity and hatch rate due to Wb elimination, are due exclusively to an effect on females, or whether reproduction is also affected in aposymbiotic males. It has been shown that the blood source fed to males can affect sperm competition and female fecundity32 but the effect of Wb removal on male reproductive fitness has not been investigated. Both sexes house Wb in gonad-associated bacteriomes16, so the elimination of Wb might uniquely affect reproductive traits in females and males. To date, all experimental designs in bed bugs have allowed males and females to mate within each treatment (e.g., either Wb+ or Wb–). A cross-treatment design, involving reciprocal crosses of Wb+ × Wb–, should reveal sex-specific effects of Wb.
Finally, Wb has been investigated extensively for its ability to interfere with host reproduction through cytoplasmic incompatibility (CI)13 which is typically detected as a reduction in egg hatch rate33. CI expression in C. lectularius thus would result in unfertilized eggs, which would be resorbed, resulting in lower oviposition with minimal effects on egg hatch. It would be fascinating to discover whether Wb-free bed bugs experience fitness-related deficiencies in part due to CI.
In summary, we found ddPCR much more reliable than qPCR for detecting low copy numbers and confirming the absence of Wb in aposymbiotic bed bugs. We therefore urge a careful re-examination of other nutritional symbioses with ddPCR to confirm that the symbiont was fully eliminated. The results presented in this study not only highlight the nutritional importance of Wb in bed bugs but also provide a useful experimental design to examine other endosymbiont-host relationships and methods for confirming the elimination of a symbiont. It is possible that Wb is responsible for providing additional nutritional benefits that contribute to adult size and fecundity in bed bugs. The mechanisms of transfer of B-vitamins from Wb to its host and how B-vitamins influence development, fecundity and survival are still unknown and would be a valuable area for future investigations. In addition, elucidating male vs. female effects on fitness traits would be useful to understand how Wb influences bed bug success.
