Reproductive fitness and survival are enhanced by adaptive behaviors that are modulated by internal physiological states and external social contexts. The common bed bug, Cimex lectularius, is an obligate hematophagous ectoparasite that requires host blood for growth, development, and reproduction. We investigated how mating, starvation and social interactions affect host-seeking, blood feeding, oviposition, and survival of female bed bugs. The percentage of females that fed and the amount of blood they ingested were greater in mated females (90–100%) than in unmated females (48–60%). Mating state also modulated the female's orientation towards human skin odor in an olfactometer; more mated (69%) than unmated (23%) females responded to human odors. The response rate of unmated females (60%) to skin odor increased with longer starvation period, while the opposite pattern was observed in mated females (20%). Although fecundity after a single blood meal was unaffected by long or short residence and interaction with males, females subjected to frequent copulation attempts had lower survivorship and lifespan than females housed with males for only 24 h. Taken together, these results indicate that by adaptively and coordinately expressing behaviors based on the internal physiological state, females maximize their survival and reproductive fitness.
Nutritional and mating states can profoundly affect the behavior of animals, including insects. The prioritization of various behaviors based on external sensory information and internal states, and their coordinated expression promote reproductive fitness and survival in a given evolutionary and ecological context[
The common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae) is an obligate blood-feeding human ectoparasite and a public health pest worldwide[
We recently showed that whereas mated females responded robustly to human odors 7–10 days after ingesting a blood meal, unmated females did not respond in these bioassays[
To explore the relationship between mating status and nutritional state, we fed virgin C. lectularius females, retained only fully engorged females, set them up into treatment groups of virgin (hereafter 'unmated') and mated females, and starved them for 8, 20, 30, and 40 days. For each experimental group, we examined the percentage that blood-fed and the size of their blood meals using an artificial feeder system. Whereas a high percentage of mated females fed (90–100%), independent of the length of starvation and the size of their blood meals, significantly fewer unmated females blood-fed (48–60%) after 8 and 20-days of starvation (Fisher's exact test (two-tailed): P < 0.0001 and P = 0.0001, respectively; Fig. 1a). The percentage of unmated females that blood-fed increased over time (75–97%) so that after 30 and 40-days of starvation there were no significant differences between mated and unmated females (Fisher's exact test (two-tailed): P = 0.1120 and P = 0.6139, respectively; Fig. 1a).
Graph: Figure 1 Effects of mating and starvation on the feeding parameters of Cimex lectularius females. (a) Percent feeding represents the percentage of tested females that ingested blood, determined visually by presence of blood in the digestive system. Unmated and mated female body mass before (b) and after (c) blood-feeding. Blood meal mass (d) was derived for each bed bug by subtracting its unfed body mass before feeding from its body mass after feeding. Females were allowed to fully engorge in a single blood meal within 8–10 days of eclosion and divided into three treatments. Unmated-Reference: not exposed to males; Unmated-Long: each female was housed with a sterile male (intromittent organ surgically ablated, denoted by an X within the male sign) until she died; and Mated-Long: each female was housed with a fertile male (denoted by a male sign) until she died. Both Unmated-Long and Mated-Long females were exposed to four starvation periods: 8, 20, 30 or 40 days. Numbers within bars represent the total number of replications for that treatment. The percent feeding differences between unmated and mated females for 8, and 20-days starvation periods are significant at ****P < 0.0001 and ***P = 0.0001, according Fisher's exact test (two-tailed). Bars with different lower-case letters indicate significant difference (P < 0.05) according to Kruskal–Wallis, Dunn's post hoc test.
Likewise, 8, 20, and 30-days starved unmated females ingested significantly smaller blood meals than mated females (mean ± SEM: 7.96 ± 0.30, 8.92 ± 0.38, and 9.54 ± 0.36 mg, respectively; Kruskal–Wallis (Dunn's multiple comparison) test: P < 0.05; Fig. 1b), but their blood intake increased with longer starvation, with no difference between the two groups after 40 days of starvation. This difference is because unmated females retained most of the blood from their first blood meal, as indicated by their significantly greater unfed body mass than in mated females in all four starvation periods (Kruskal–Wallis (Dunn's multiple comparison) test: P ≤ 0.0001; Fig. 1c). In mated females that were starved for 8 days, the blood meal mass was only twofold their unfed body mass of 5.07 mg (mean ± SEM: 2.06 ± 0.08 fold; Figs. 1b, 2), likely because these females retained some blood from their previous blood meal and were still maturing oocytes (oviposition lasts for ~ 10 days after each blood-feeding). However, after 20 days of starvation the blood meal mass of mated females increased to 3.4-fold their unfed body mass of 3.28 mg (mean ± SEM: 3.46 ± 0.15 fold; Figs. 1b, 2), as these females digested all their previously ingested blood and oviposited all their eggs. Blood meal mass continued to increase in mated females through 40 days of starvation (Fig. 1b).
Graph: Figure 2 Effects of mating and starvation on the size of the blood meal of Cimex lectularius females. The ratio of blood meal mass to the unfed body mass of three treatment groups. Unmated- Reference (not exposed to males); Unmated-Long: each female was housed with a sterile male (denoted by an X within the male sign) until she died; and Mated-Long: each female was housed with a fertile male (denoted by a male sign) until she died. Both Unmated-Long and Mated-Long females were exposed to four starvation periods: 8, 20, 30 or 40 days. Numbers within bars represent the total number of replications for that treatment. Bars show mean ± SEM (n = 30–50) and all replicates are displayed. Bars with different lower-case letters indicate significant difference (P < 0.05) according to Kruskal–Wallis, Dunn's post hoc test.
In contrast, the blood meal mass of unmated females that were starved for 8 days was significantly smaller than in mated females (mean ± SEM: 7.96 ± 0.32 mg; Kruskal–Wallis (Dunn's multiple comparison) test: P = 0.0100; Fig. 1b), likely because of a larger unfed body mass of 7.96 ± 0.20 mg (SEM), related to their slow digestion of the previous blood meal and retention of eggs (Fig. 1c). In these females, a smaller blood meal was sufficient to attain a large fed body mass of 15.92 ± 0.35 mg (Fig. 1d). The ratio of blood meal mass to unfed body mass increased to only twofold and was not significantly different among the 20, 30, and 40-day starvation groups (Kruskal–Wallis (Dunn's multiple comparison) test: P > 0.9900; Fig. 2). This pattern represented larger blood meals and increasingly smaller body mass as the previous blood meal was slowly digested (Fig. 2).
For reference, we also assessed the same parameters in a group of virgin females that took their first adult blood meal within 8–10 days after eclosion, and were never exposed to males after the eclosion. These females behaved more like mated starved females than unmated starved females. The percentage of females that fed (Fig. 1a), their blood meal mass (Fig. 1a–c), and the ratio of blood meal mass to unfed body mass (Fig. 2) were similar to the respective parameters of mated females, and much higher than for unmated 8-day starved females.
To investigate the effects of mating and starvation on orientation to human odor, we deemed it important to control for recurrent copulations and the presence of males in the mated group. Therefore, we conducted olfactometer assays to determine the effects on starved females of being housed with fertile males (i.e., mated females) vs. sterile males (male with a surgically ablated intromittent organ) that could not copulate (i.e., unmated females); females in both groups experienced social interactions. When these conditions persisted throughout the female's adult life (i.e., 'Long' treatments), significantly more mated than unmated females responded when starved for 8- or 20-days (Fisher's exact test (two-tailed): P < 0.0003 for both; Fig. 3a). Strikingly, responses of 40-day starved females were reversed, with significantly more unmated than mated females showing robust upwind orientation by walking towards the human skin swab (Fisher's exact test (two-tailed): P < 0.0086; Fig. 3a). Overall, the response rate of unmated females towards a skin swab increased with longer starvation, whereas the response rate of mated females declined with longer starvation.
Graph: Figure 3 Effects of mating and starvation on orientation of female Cimex lectularius to human skin odor in an olfactometer. Percent response (a,b) represents the percentage of females that made a choice (skin swab or clean filter) of all females tested. (a) Unmated-Long: each female was housed with an infertile male (intromittent organ ablated, denoted by an X within the male sign) until she died; Mated-Long: each female was housed with a fertile male (denoted by a male sign) until she died. (b) Unmated-Short: each female was housed with an infertile male for 24 h and then with a female (denoted by an X within the male sign and then a female sign); Mated-Short: each female was housed with a fertile male for 24 h and then with a female (denoted by a male sign and then a female sign). (c,d) Olfactometer preference for human skin odor (left) vs. clean filter paper (right). Assays were conducted on four female treatment groups 8, 20, 30, and 40 days after they ingested a blood meal. Numbers within bars represent the total number of replications for that treatment. In (a,b), Control bioassays with clean filter papers of females of different starvation periods were pooled due to low response rates (< 15%, n = 40), and differences between unmated and mated females are significant at **P < 0.0096, ***P < 0.0003, according to Fisher's exact test.
A similar response pattern was apparent in females transiently exposed to males for only 24 h (i.e., 'Short' treatments). The orientation responses of unmated females gradually increased with longer starvation, whereas in mated females response rate increased and then declined; we found a significant difference between them only at 8 days of starvation (Fisher's exact test (two-tailed): P < 0.0096; Fig. 3b). However, we did not observe the sharp decrease in the response rate of mated females after 30- or 40-days of starvation that was apparent in the 'Long' experimental condition.
Females in all treatments were highly adept at detecting and preferably orienting toward human skin odor over the clean control filter paper, irrespective of their mating status, social conditions, nutritional state, and response rate in the olfactometer (Fig. 3c,d). Control assays with clean filter papers in both arms of the olfactometer resulted in low response rates (< 15%) and random orientation to the left and right arms of the olfactometer. Of note is that although we defined a "response" and "making a choice" as reaching 1.5 cm into either arm of the olfactometer, 100% of the females that responded reached the respective filter paper.
We hypothesized that long co-habitation with a fertile male would reduce female fecundity after a single blood-meal and lifetime survival because of constant harassment and physical damage to the female during repeated hemocoelic copulations. However, we found that the number of eggs oviposited during the first oviposition cycle (within 10 days after the blood-meal) was the same in both female groups (with a fertile male throughout vs. with a fertile male for only 24 h) and their cumulative mean numbers of eggs per female were similar (Fig. 4a,b). In both groups, females initiated egg laying on day-4 and ceased on day-10 post blood-feeding.
Graph: Figure 4 Egg production of female Cimex lectularius in the first oviposition cycle after a blood meal. (a) Mated-Long: each female was housed with a fertile male until she died (denoted by a male sign). (b) Mated-Short: each female was housed with a fertile male for only 24 h and the male was replaced with an antennaless female (denoted by a male sign and then a female sign). Females were monitored daily for the number of eggs they oviposited. Bars show mean daily eggs per female and cumulative mean number of eggs oviposited per female (± SEM, n = 40).
On the other hand, survival was significantly affected, with mated-short females (each with a fertile male for only 24 h) surviving for a median of 55.5 days, whereas median survival of mated-long females (each exposed to a fertile male throughout her lifetime) was only 35 days (Log-rank (Montel-Cox) test: df = 1; Hazard ratio (log-rank): 4.012; P < 0.0001; Fig. 5). The same pattern was evident in unmated females: The median survival times of unmated-short (24 h with a sterile male) and unmated-long females (lifetime with a sterile male) were 111 days and 73 days, respectively (Log-rank (Montel-Cox) test: df = 1; Hazard ratio (log-rank): 5.055; P < 0.0001). Overall, mated females died at a significantly higher rate than unmated females (Log-rank (Montel-Cox) test: df = 3; P < 0.0001; Fig. 5), suggesting that both matedness and long-term presence of either a fertile or infertile male reduced female survival.
Graph: Figure 5 Effects of mating frequency and starvation on the survival of female Cimex lectularius. The four treatments include, Unmated-Long: each female was housed with a sterile male (intromittent organ surgically ablated, denoted by an X within the male sign) until she died; Mated-Long: each female was housed with a fertile male until she died (denoted by a male sign); Unmated-Short: each female was housed with a sterile male for 24 h and the male was replaced with an antennaless female (denoted by an X within the male sign and then a female sign); and Mated-Short: each female was housed with a fertile male for 24 h and the male was replaced with an antennaless female (denoted by a male sign and then a female sign). Survival curves with different lower-case letters are significantly different (P < 0.0001) according to log-rank (Mantel-Cox) test.
Finally, we discovered a female "refusal" posture, whereby the female curled the ventral side of the abdomen, protecting the ectospermalege, a specialized cuticular region that guides the male paramere (intromittent organ), from males (Fig. 6a, Supplementary Video S1). This behavior was expressed only in the unmated-long and mated-long treatment females but not in the respective short treatment females (Fig. 6b), consistent with the idea that the "refusal" posture is expressed more in the presence of males.
Graph: Figure 6 Expression of a female refusal behavior in Cimex lectularius under various mating and starvation conditions on the survival of female Cimex lectularius. (a) Dorsal and ventral views of the refusal behavior, and a female in a normal position. (b) Refusal behavior frequency in four treatments, showing the percentage of females in each treatment that expressed the refusal behavior at least once in their lifetime. Unmated-Long: each female was housed with a sterile male (intromittent organ surgically ablated) until she died; Mated-Long: each female was housed with a fertile male until she died; Unmated-Short: each female was housed with a sterile male for 24 h and the male was replaced with an antennaless female; and Mated-Short: each female was housed with a fertile male for 24 h and the male was replaced with an antennaless female. Numbers within bars represent the total number of replications for that treatment.
The bed bug Cimex lectularius is an obligate ectoparasite that engages in recurrent blood-feeding events throughout its lifetime, punctuated by sheltering some distance from the host. This complex lifestyle requires coordination of diverse on-host and off-host behavioral repertoires, including host-seeking, blood-feeding, mating, oviposition, and aggregation to sustain development, reproduction, and survival[
Consistent with our previous results [
We speculated that the behavioral differences between unmated and mated females were related to the rate of processing of the blood meal, which would be affected in turn by the rate of oocyte maturation and oviposition. Specifically, we hypothesized that because unmated females resorb their eggs[
These results are consistent with the hypothesis that the differences between mated and unmated females are driven by the accelerated egg maturation and oviposition cycle of mated females. Mated females initiated egg laying 4 days after ingesting a blood meal, oviposited on average 15.3 eggs per female, and ceased oviposition 6 days later, 10 days after ingesting a blood meal (Fig. 4). Thus, 8 days after a blood meal, mated females digested most of the blood (Fig. 1b), oviposited most of their eggs, and became highly motivated to host-seek to support a second oviposition cycle. Indeed, both laboratory and field observations showed that, given the opportunity, mated females accept a second blood meal while the first blood meal is still being digested and females feed every 2.5 days on average[
The strategy of unmated females was to feed little when the host is available, but with longer starvation periods, they became more stimulated to host-seek and ingest increasingly larger blood meals. This strategy is likely driven by much-reduced nutritional demands related to resorption of oocytes, which allow unmated females to digest the blood meal more slowly and use it for somatic maintenance rather than egg maturation. As stated by Davis in[
Surprisingly, host-seeking declined in mated females that were starved for 30 or 40 days, and this was especially apparent in females housed with fertile males (mated-long treatment) (Fig. 3). Two factors might account for this observation. The first is female aging and senescence, as 40 days of starvation in these females was beyond the 35-day median survival of females in this treatment group, and 100% of these females died by day 49 (Fig. 5). Thus, the females that we bioassayed 40 days after they ingested a blood meal were likely weak and less responsive to olfactory stimuli. This reduction in the host-seeking and blood-feeding responses of older mated females might be associated with their higher metabolic rate, senescence, or aging that could negatively affect olfactory responses, as shown in the D. melanogaster[
The second factor that likely underlies their early senescence is the unusual extra-genitalic, hemocoelic (traumatic) insemination in C. lectularius. Females housed with fertile males would receive multiple copulations that represent constant harassment, stress, and physical damage. These interactions with sexually aggressive males reduced their median adult lifespan by 63% (mated-long treatment) relative to females that were housed with fertile males for only 24 h and then with another female (mated-short treatment) (Fig. 5). These findings are consistent with previous studies in bed bugs on the adverse effects of multiple copulations on female lifespan[
We found no significant difference in female fecundity in the first oviposition cycle (~ 10 days post blood meal) between two treatment groups—females with long- and short-term presence of fertile males that represented high and low mating rates, respectively (Fig. 4). It is important to emphasize, however, that this experiment was limited to a single feeding and only one oviposition cycle. The lifespan of mated females was dramatically reduced by both the high and low mating rates, but significantly more so by high mating rate (Fig. 5), consistent with previous observations[
Remarkably, we also detected a significant effect on females of non-copulatory harassment by males. Females housed with a male that could not copulate (intromittent organ ablated) for only 24 h and then with another female (unmated-short treatment) lived to a median age of 111 days (100% dead by day 142), whereas females housed continuously with an infertile male (unmated-long treatment) lived to a median age of only 73 days (100% died by day 104). This 34% decline in expected lifespan, independent of copulation and egg production, can be attributed to male-specific harassment (Fig. 5). Males engage in a stereotyped behavior where the male repeatedly mounts the female's dorsum, bends his abdomen to her ventral surface, and probes the female's sternites with the paramere. We observed that 50% of the unmated-long and 62.5% of mated-long females exhibited a 'refusal' posture at least once in their lifetime thereby making the ectospermalege inaccessible to males, while none of the unmated-short or mated-short females displayed this behavior (Fig. 6b). This behavior may be similar to a behavior noted by N. Davis [in[
Mating in female bed bugs strongly influences their host-seeking and blood-feeding behaviors, and the size of their ingested blood meal. Whereas mated females were highly stimulated by host odors to host-seek, and they ingested large and frequent blood meals, unmated females were less driven to seek a host and they ingested smaller and less frequent blood meals. The length of starvation also affected mated and unmated females differently. Host-seeking declined over starvation time in mated females, likely due to mating-induced physiological changes, whereas host-seeking responses in unmated females gradually increased. To our knowledge, this is the first report showing that multiple matings impair host-seeking in older starved female bed bugs, and the first to show that non-copulatory male harassment dramatically reduces female lifespan. Finally, our results show that female bed bugs that respond in the olfactometer orient towards human odor regardless of their mating and nutritional status.
Cimex lectularius used for this study was the Harold Harlan (Harlan) population, originally collected in 1973 at Fort Dix, NJ, USA, and maintained in the laboratory. Colonies were maintained in an incubator at 27 °C, approximately 30–50% relative humidity (RH) and under a 12 h light:12 h dark cycle (lights off at 0800). Bed bugs were fed defibrinated rabbit blood (HemoStat Laboratories, Dixon, CA) using an artificial feeder system. A heated water bath (B. Braun Biotech Inc., Allentown, PA) circulated warm (34 °C) water through a custom-made water-jacketed glass feeder. Blood in each glass feeder was retained with a stretched plant grafting tape (A.M. Leonard Horticultural Tool and Supply Co., Piqua, OH) on the bottom and ~ 4 mL of blood was introduced through the opening at the top. Bed bugs were housed in a polystyrene wide-mouth threaded colony container (Consolidated Plastics Company, Inc., Stow, OH) constructed by replacing the bottom with a nylon screen (0.3 mm mesh opening; BioQuip Products Inc., Rancho Dominguez, CA) and a piece of folded manila paper was provided so bed bugs could walk up to the screen and feed. Colonies were fed weekly by placing each container screen side up under a feeder for 15–20 min.
To obtain unmated females, freshly fed 5th instars were individually placed into glass vials (7.5 mL), each with a paper strip. Adult unmated females were collected on the day of eclosion and up to two days after eclosion and grouped in a polystyrene wide-mouth threaded container with a screened bottom, through which bed bugs could feed as described above. The females were group-fed to satiation within 8–10 days and each placed in a clean glass vial (7.5 mL) with a paper substrate. The following day (24 h after feeding) fully engorged individual females (9–11 days post eclosion) were subjected to one of the following four experimental treatments: (
We visually recorded female 'refusal' behavior as a result of starvation and male mating harassment at least once daily until the death of the female, in all four experimental treatments: Mated-Long, Unmated-Long, Mated-Short, and Unmated-Short. We scored 'presence' of refusal behavior if a female curled the ventral side of the abdomen, protecting the ectospermalege from males as illustrated in Fig. 6a and Supplementary Video S1.
We assayed the effects of mating and starvation on the female's propensity to blood-feed and the size of her blood meal using the unmated-long and mated-long treatments (n = 30–40 per treatment). All the feeding experiments were performed between 3 and 6 h after onset of the scotophase (under red-light) and at 26 ± 1 °C. At the beginning of the scotophase (1 h before testing), each female was weighed (MP8-1, Sartorius, Goettingen, Germany) and placed in a glass vial (7.5 mL) containing a folded paper strip leading to a nylon screened lid (0.3 mm mesh opening), so she could walk up to the screen and feed. Then, two artificial glass feeders (described above) were prepared side by side, and four vials were placed randomly under each feeder for 10 min—two vials were of unmated-long females and two mated-long females of one of four starvation periods (
We used a filter paper to obtain human skin odors to test bed bug attraction to human odors, as previously described by DeVries et al.[
We used a vertical Y-tube custom-made glass olfactometer (14 mm inner diameter) to test the effect of mating and starvation on the orientation of female bed bugs to skin odors, as previously described[
Fisher's exact test (two-tailed) was performed to compare the effects of mating and starvation on the feeding propensity of females between treatments of the same starvation period. 'Blood meal mass' was calculated as (Fed body mass – Unfed body mass). The ratio of blood meal mass to unfed body mass was calculated as (Fed body mass – Unfed body mass)/Unfed body mass. The non-parametric Kruskal–Wallis test followed by Dunn's post hoc test were used to compare female blood meal mass, unfed body mass, fed body mass, and the ratio of blood meal mass to unfed body mass among treatments. In all statistical analyses, the significance level (α) was set to 0.05. 'Unmated-Reference' females were not included in the analysis. We used Fisher's exact test (two-tailed) to compare olfactometer response rates of unmated and mated females on the same starvation day. Because control filter papers in both sides of the olfactometer resulted in low response rates (< 15%), we pooled the controls of the four starvation periods of unmated and mated females. The Kaplan–Meier method was employed to create survival curves from the raw data and a log-rank (Mantel–Cox) test was used to compare the survival curves. Statistical analysis was performed in GraphPad prism v.5.0a.
Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the position or the policy of the U.S. Government and no official endorsement should be inferred.
A.M.S., Z.C.D., R.S., and C.S. designed the study; A.M.S. conducted the experiments, analyzed the data, and prepared the figures; A.M.S., Z.C.D., and C.S. wrote the manuscript. All authors reviewed and approved the final version of the manuscript.
This study was supported by the Blanton J. Whitmire Endowment at North Carolina State University, and grants from the US Department of Housing and Urban Development Healthy Homes program (NCHHU0053-19), the Alfred P. Sloan Foundation (2013-5-35 MBE), the US National Science Foundation (DEB-1754190) and the Department of the Army, U.S. Army Contracting Command, Aberdeen Proving Ground, Natick Contracting Division, Ft Detrick MD.
The authors declare no competing interests.
Graph: Supplementary Legend.
Graph: Supplementary Video S1.
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By Ahmed M. Saveer; Zachary C. DeVries; Richard G. Santangelo and Coby Schal
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