The common bed bug, Cimex lectularius harbors the endosymbiotic microorganism, Wolbachia (wCle), in a gonad-associated bacteriome as an obligate nutritional mutualist. The obligatory nature of this association suggests that all individuals in C. lectularius populations would be infected with wCle. However, studies spanning the past several decades have reported variation in both infection frequency and relative abundance of wCle in field-collected samples of bed bugs. Since the growth kinetics of wCle is poorly understood, the objective of this study was to quantify wCle over the life cycle of two strains of C. lectularius. Our results highlight that wCle is dynamic during bed bug development, changing relative to life stage, intermolt stage, and blood-fed status. These results suggest new hypotheses about the coordination of Wolbachia growth and regression with its host’s physiology and endocrine events. The observed quantitative modulation of wCle during the bed bug life cycle and during periods of starvation may explain the disparities in wCle infections reported in field-collected C. lectularius.
Insect bodies are inhabited by diverse bacterial communities, ranging from parasitic, commensal, to facultative or obligatory mutualistic associations with their host[
Bed bugs are obligatory hematophagous insects with hemimetablous development from egg to adult through five nymphal stages (instars), each of which requires a blood-meal to molt to the next stage. The common bed bug, Cimex lectularius (Cimicidae) harbors a hereditary, Gram (-), intracellular α-proteobacterium, Wolbachia that is primarily concentrated in a gonad-associated bacteriome. Wolbachia’s relationship with C. lectularius presumably evolved from a facultative association to obligate mutualism where the bacteria garner protection and nutrients within their host in exchange for supplementing the host’s nutritional needs[
The obligate association of wCle with its host, transovarial transmission, and poor performance of bed bugs cured of wCle infection, all would suggest that all C. lectularius populations worldwide should be infected with wCle. Yet, early research[
The growth kinetics of wCle in C. lectularius is poorly understood, but endosymbiont titers have been quantified in other organisms in relation to host development. For example, Wolbachia fluctuates over the course of the life cycle of its host, the nematode Brugia malayi. The endosymbiont resides intracellularly inside host-derived vacuoles in the hypodermal cord cells[
Nonetheless, to our knowledge, no reports have quantified the endosymbiont titer within the intermolt stage and in relation to feeding in bed bugs. We were particularly interested in this relationship as well as the wCle titer before and after the nymphal and adult molts. Moreover, we sought to understand whether the reported highly variable relative abundance of wCle in field-collected C. lectularius could be attributed to variation in bed bug developmental and blood-fed status. In this study, we quantified the amount of wCle during nymphal and adult development in two strains of C. lectularius using droplet digital (ddPCR), which has greater accuracy of quantification at low target concentrations than qPCR, and absolute quantification does not require reference genes or the generation of a standard curve[
We designed a ddPCR duplex assay for the absolute quantification of target amplicon copy number in bed bugs and their associated Wolbachia. The ddPCR optimization resulted in little variation in samples, where coefficients of variation of estimated DNA copy numbers of bed bug and Wolbachia in the five control samples were 2.1% and 2.4% respectively, with a mean (±SE) of 632,480 ± 3,565 for bed bug DNA (RPL18) per individual, and 385,920 ± 4,473 for Wolbachia DNA (16S rRNA) per individual (Fig. 1A). The Wolbachia-free control bed bugs removed from antibiotics 90 d post-antibiotic treatment contained 532,480 ± 7,634 DNA copies of RPL18 and no detectable Wolbachia DNA (Fig. 1B), and no DNA was detected in the no-template controls (Fig. 1C). However, one Harold Harlan (HH) bed bug and five Jersey City (JC) bed bugs were excluded from further analysis because they contained <15 copies/µl of the bed bug RPL18 or Wolbachia 16S rRNA genes.Droplet digital PCR optimization results. Copy number/µl of DNA for Wolbachia and Cimex lectularius (A), Wolbachia-free Cimex lectularius removed from antibiotics for 90 d maintained on blood fortified with vitamins only (B), and no-template control (C). Wolbachia (wCle) droplet spectrum (blue), Cimex lectularius droplet spectrum (green), droplets with both targets (orange), and droplets with neither target (gray).
Wolbachia titer per bed bug ranged from 45,428 ± 15,349 in 1
Wolbachia titer per bed bug in the HH strain remained low (X¯ = 60,449) and steady in 1
In both bed bug strains there was a large and significant increase in Wolbachia titer between the end of the 1
Another large, but not statistically significant, increase in Wolbachia per bed bug occurred within one day between the last instar and the adult molt (~2-fold increase in HH bugs, ~3.3-fold increase in JC bugs) (Fig. 2A). However, this increase disappeared when Wolbachia DNA was normalized to the amount of bed bug DNA (Fig. 2B), suggesting that increases occurred in both Wolbachia and body mass. Subsequently, in the adult stage in both strains, Wolbachia titer declined significantly with prolonged starvation even when Wolbachia DNA was normalized relative to bed bug DNA. Wolbachia titer per HH bed bug declined by 95.5% 40 d after feeding, and in JC this decline was ~93.0% (Fig. 2A).
We generated a normalization within each life stage, relative to unfed 1
Our results highlight that Wolbachia of C. lectularius (wCle) is dynamic during host development, changing relative to life stage, intermolt stage, and blood-fed status. Overall, we observed that (a) Neonate unfed, lab-reared, bed bugs (HH strain) had 4.3-fold more wCle per bug than field-collected bugs (JC strain), and when normalized to the amount of bed bug DNA a 1.7-fold difference remained; (b) wCle DNA titer per bed bug increased ~30-fold (HH) and ~168-fold (JC) between the 1
Endosymbiotic bacteria, such as Wolbachia, are broadly associated with many insect species in a variety of parasitic, commensal and mutualistic associations[
A central feature of the association of Wolbachia with C. lectularius is their obligate mutualism, where wCle provisions the bed bug with B vitamins in exchange for being hosted and transmitted as a “hereditary” component of the oocyte. wCle are therefore essential for nutrient synthesis and embryonic development. wCle-cured bed bugs grow more slowly and experience lower fecundity, but these effects can be reversed with biotin (vitamin B7)[
The blood-meal is the only external source of nutrients for bed bugs, and indirectly for wCle. In contrast to other hematophagous insects such as adult fleas or lice that live exclusively on their host, bed bugs take larger and infrequent blood-meals, and substantial degradation of erythrocytes is delayed up to 12 hours[
In adults, the combined effects of starvation and reproduction also affect wCle populations. As in 5
The fine coordination of wCle titer with bed bug feeding needs further investigation. wCle appears to respond to nutritional conditions in its host much faster than the time resolution of our study. It is possible, for example, that we missed peaks in wCle populations immediately after the blood meal that damped out within 2 d after the blood meal was ingested. Highlighting this response is wCle’s rapid increase (2-fold in HH, 3.3-fold in JC) in one day between the late 5
Precise mechanisms of coordination of insect-bacterial symbiosis are not well known. Buchner[
One could postulate that wCle has relinquished control over replication and cell division to its bed bug host through lateral gene transfer or genome reduction, since this symbiosis is a highly specialized obligate mutualism. Interestingly, this does not appear the case. In contrast to other Wolbachia genomes, wCle has not undergone extensive genome reduction, or experienced significant loss of genes that control cell division, replication, or are responsible for other essential functions[
The coordination of wCle with host development and physiology may be driven by nutrition or the bed bug’s endocrine cycle. Wolbachia, like other intracellular endosymbionts such as Spiroplasma, require macronutrients from the host for replication and proliferation[
Competition between Wolbachia infections and the host for nutrients has also recently been suggested. Wolbachia had a direct impact on cholesterol availability in Aedes aegypti mosquitoes[
In the related hemipteran R. prolixus, a blood-meal stimulates a molt cycle through humoral factors and neuronal signals generated by stretch receptors in the gut[
The results presented in this study suggest new hypotheses about the coordination of Wolbachia growth and regression with its host’s physiology and endocrine events. Future experiments could include quantitative measurements of Wolbachia’s rapid response to feeding, molting, mating, and oviposition, as well as to manipulations of juvenile hormone and ecdysone titers in the bed bug. As well, the broad-scale changes in wCle in various life stages of C. lectularius bear on recent failures to detect wCle in some field-collected bed bugs. This appears to be in conflict with the ostensible obligate symbiosis of wCle and C. lectularius, suggesting (a) that wCle titers in some field-collected bugs were below the detection limit of the assays, or (b) that C. lectularius lineages may vary in their degree of dependence on wCle. Regarding the latter, the field-collected JC strain had much higher titers of wCle than the lab-adapted HH strain, suggesting that the lab bed bugs might have adapted to frequent and possibly higher quality blood meals by depending less on wCle. Consistent with this suggestion, multiple recent surveys indicate that sizeable human populations in developed countries fail to consume the minimum recommended quantity of B-vitamins[
Two Cimex lectularius bed bug strains were used in these experiments. The Harold Harlan strain (HH) was collected in 1973 in Ft. Dix, NJ, USA. It is an insecticide-susceptible strain used as a standard in many laboratories working on bed bugs. This strain has been maintained at NC State University since 2008 on defibrinated rabbit blood (Hemostat Laboratories, Dixon, CA). The Jersey City (JC) strain of C. lectularius was collected in 2008, also in NJ, and maintained on defibrinated rabbit blood at NC State University since then. It is known to be highly resistant to pyrethroid insecticides[
Our objective was to determine if the relative abundance of Wolbachia fluctuated during the developmental cycle and in relation with the blood-fed status of C. lectularius for both HH and JC strains.
Neonate unfed 1
Three individuals from each vial (n = 9) were randomly collected for testing at the following time intervals: unfed 1
Sixty newly molted unfed 5
Total genomic DNA was extracted using the DNeasy Blood and Tissue kit (QIAGEN, Germantown, MD, USA) using a modified protocol for purification of total DNA from animal tissues (spin-column). Heads were removed from 5
To obtain absolute quantification of Wolbachia in each individual bed bug, we used a droplet digital PCR (ddPCR
The bed bug/Wolbachia ddPCR assay comprised 22 µl of 1 × Droplet Supermix (Bio-Rad), 5 µl of bed bug template DNA, 2 U of MseI restriction enzyme (New England Biolabs, Ipswich, MA, USA), 500 nM each of forward and reverse primers and 250 nM each of FAM- or HEX-labeled TaqMan probes for bed bug and Wolbachia strains, respectively. The 22 µl of PCR mixture was partitioned into an emulsion of ~20,000 droplets using the ddPCR system. PCR was performed on a T100 Thermal Cycler using the following protocol: 95 °C for 10 min and (94 °C for 30 s, 56 °C for 2 min) x 40 cycles, and 98 °C for 10 min. Post PCR, droplets were analyzed on the QX200 Droplet Reader. Absolute DNA amounts of bed bug and Wolbachia sequences in a sample were calculated on the Poisson distribution using the Quantasoft software version 1.7.4 (Bio-Rad). Data are reported as Wolbachia DNA (16S) copy number per individual bed bug and as Wolbachia DNA copy number per C. lectularius DNA (RPL18) copy number. Bed bug DNA samples containing Wolbachia (+control) and without Wolbachia (- control) were included in each experiment as checks on the ddPCR results. A no-template control was also included in each experiment to control for non-specific amplifications. To estimate the limit of detection of the ddPCR assay, serial dilutions of a DNA sample were prepared in water (
We used a General Linear Model (GLM) Univariate Analysis of Variance (ANOVA) and Tukey’s HSD (α = 0.05) in SAS 9.4[
The data supporting the findings in this study are available as Supplementary Information.
We would also like to thank Lauren Jurczak for her dedicated assistance with DNA extractions and Rick Santangelo for maintaining the bed bug colonies. This study was supported in part by the Blanton J. Whitmire Endowment, US Department of Housing and Urban Development Healthy Homes program (NCHHU0017-13), Alfred P. Sloan Foundation (2013-5-35 MBE), W.M. Keck Center for Behavioral Biology, and a National Institute of Environmental Health Sciences grant (P30ES025128) to the Center for Human Health and the Environment.
M.L.F., D.W.W. and C.S. designed the study. M.L.F. performed the experiments. H.M. and M.B. contributed to the acquisition and interpretation of ddPCR data. J.A.O. contributed to statistical analysis. M.L.F. and C.S. wrote the manuscript, and all authors contributed to revisions and approved the final manuscript.
The authors declare no competing interests.
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Supplementary information accompanies this paper at 10.1038/s41598-018-29682-2.
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By Michael L. Fisher; David W. Watson; Jason A. Osborne; Hiroyuki Mochizuki; Matthew Breen and Coby Schal