Indirect Effects of Temperature on Stink Bug Fitness, via Maintenance of Gut-Associated Symbionts

Adults of A. hilare and egg masses of M. histrionica were acquired from researchers at University of California Berkeley (Kent Daane) and Riverside (Jocelyn Millar) to establish laboratory colonies. Insects were maintained using a diet that consisted of green beans (Phaseolus vulgaris), broccoli (Brassica oleracea var. italica), and peanuts (Arachis hypogaea) to rear A. hilare and of broccoli and cauliflower (Brassica oleracea var. botrytis) for M. histrionica (26). We supplied the colonies with fresh food twice weekly. Both species were reared in a walk-in insectary room at UC Berkeley kept at 24 ± 2°C with a photoperiod of 16/8 (light/dark [L/D]). We collected the egg masses from these colonies to initiate all experiments.

We tested whether high temperature affects symbiont maintenance in A. hilare, using 30 egg masses housed individually in plastic containers with mesh screen covers (26). Fifteen replicate containers were placed in a controlled temperature chamber at 25°C, while the other 15 were kept in a chamber at 30°C; both had a 16/8 L/D photoperiod. We added fresh green beans to the containers every other day and censused the insects every 7 days to estimate development and mortality. Censuses continued until all the insects died or reached adulthood. As adults developed, we transferred them to a separate cage and allowed them to breed under the same conditions. While the insects were in the adult stage, we evaluated insect mortality and fecundity. To establish the second generation, we collected 15 egg masses per temperature, laid by first-generation adults. Fifteen egg masses were placed in their respective temperature-controlled chambers (25 and 30°C) with the same measurements taken as for the first generation. We counted the number of dead insects and the number of egg masses produced during the females' lifetimes at each temperature. A smaller experiment was conducted following the same protocol but analyzing only the role of temperature on symbiont maintenance for one generation (see the supplemental material).

A similar experiment was conducted with M. histrionica, using 40 egg masses, housed in the same plastic containers. We placed 20 replicate containers in each of two chambers, set to 25 or 30°C with a 16/8 L/D photoperiod. We initiated a second generation as before, with 45 replicate egg masses at 25°C and 20 egg masses held at 30°C. We replaced the insects' diet every other day and censused all replicate cages every three days for both generations until the insects' death.

For A. hilare, two nymphs of second, third, fourth, and fifth stages and one adult male and female were sampled from each replicate for detection of the symbiont by PCR as described previously (26). For M. histrionica, we sampled one individual of each of the same stages from each replicate. Sampling of more individuals per replicate could negatively affect our capacity to estimate stink bug demographical parameters. First instars were not used due to their small size and potential symbiont detection problems. We compared the proportion of insects that tested positive for symbionts via PCR using a two-way general linear model with temperature and generation as fixed effects and binomial errors (6). Separate tests were conducted for each of the development stages, with adjustment of α to account for multiple tests.

We estimated a suite of demographic parameters for each of the stink bug species: net reproductive rate (R₀), mean generation time (T), intrinsic rate of increase (r), finite rate of increase (λ), doubling time (DT), and gross reproductive rate (GRR), as described by Carey (4). We also determined adult emergence (day first adult appeared) and oviposition parameters (preoviposition period, oviposition period, number of egg masses, and total number of eggs per egg masses) as described by Carey (4). Effects on development rate were analyzed using the median development time (MDT) method described by Peterson and Painting (23).

All statistical analyses were performed on the two species, separately, using the software R, version 2.6.1 (30). We analyzed mortality using a two-way analysis of variance (ANOVA) with temperature and generation as fixed factors and cumulative mortality as the response variable. Comparisons of MDT were performed using a linear mixed-effects model with development stage as a random, repeated measure (6). The statistical analyses of the biological parameters for M. histrionica were calculated using a separate one-way ANOVA with three treatment levels (25°C for generation 1, 25°C for generation 2, and 30°C for generation 1). Pairwise t tests were used to compare means among the treatments, with α adjusted for multiple comparisons. We also used one-way ANOVA to compare the total number of eggs laid throughout the first-generation lifetime of A. hilare and M. histrionica.

To visually confirm our PCR-based detection of symbionts in stink bugs we also used FISH. We collected the cecum region of the midgut (or crypts, where symbionts are located) of five fifth instar nymphs of each insect species reared at 25 and 30°C (independently from experiments described above) and fixed them with 10% formalin in phosphate-buffered saline (PBS) for 4 h at 4°C. We resuspended the samples in ice-cold PBS-96% ethanol (vol/vol) and stored them at −20°C. A drop of 20 μl of the stored sample was spotted to a glass slide, dehydrated through ethanol series, air-dried, and submitted to in situ hybridization (7, 34). We used the probe SPAHFinv (5′-CGAAGCGTATTAAGCCTC-3′) to specifically detect the gut-associated symbiont of A. hilare and SPMHFinv (5′-GGTTGTGAAACATTATGCG-3′) to specifically detect the gut-associated symbiont of M. histrionica. These probes were designed using the 16S rRNA gene sequences of the bacteria previously identified as the gut symbionts of these pentatomid species (25). Both probes were linked with the fluorescent cyanine dye Cy3 at the 5′ end. For both insect species we also used the universal bacterial probe EUB338 linked to the dye ALEXA 488 at the 5′end as an internal control (7). We applied 100 μl of the hybridization buffer (5 M NaCl, 1 M Tris-HCl, 10% sodium dodecyl sulfate, 40% formamide) containing 30 ng/μl of each probe onto a glass slide. Samples covered with a coverslip were incubated in a humidified and dark environment at 56°C for about 2 h. We washed the slides with preheated buffer (5 M NaCl, 1 M Tris-HCl, 0.5 M EDTA) and incubated them in the same buffer for 15 min at 52°C to eliminate nonspecifically bound probes. Finally, we added mounting medium containing DAPI (4′,6-diamidino-2-phenylindole) to the slides and stored them in the dark at −20°C if they were not immediately used (7). We used a Zeiss Axiophot epifluorescence microscope equipped with a 5MPix QImaging Micropublisher low-light, cooled charge-coupled device (CCD) color digital camera to obtain pictures. All probes used in this work were also tested without the fluorescent label as a negative control for sample fluorescence; the samples were not fluorescent.

The prevalence of symbionts in the two insects varied greatly among temperatures and generations (Table 1, Fig. 1). For A. hilare there were significant differences in prevalence between temperatures for all stages, significant differences between generations for the second nymphal instar, and significant temperature-by-generation interactions for the later nymphal instars. Prevalence was generally high across all stages at 25°C for both generations. Conversely, at 30°C prevalence dropped off dramatically after the second instar, particularly in the second generation (Fig. 1A). A similar trend was observed in the small trial performed for only one generation, although in that experiment all PCR-tested fifth instars reared at 30°C were symbiont negative (see supplemental material).

For M. histrionica there were significant differences in prevalence between temperatures for all but the third nymphal instar, significant differences between generations for all but the egg and fifth instars, and significant temperature-by-generation interactions for the early nymphal instars (Table 1). Symbiont prevalence was slightly lower than in A. hilare at 25°C, especially in the second generation. Moreover, prevalence in M. histrionica showed large differences between generations at 30°C, with no symbionts detected in any of the second-generation insects (Fig. 1B).

The Acrosternum hilare mortality rate showed significant effects of temperature (t = −4.43; df = 305; P < 0.0001), generation (t = −3.46; df = 305; P = 0.0006), and the interaction between temperature and generation (t = −3.23; df = 305; P = 0.0014). Mortality in the first generation was substantially higher at 30°C than at 25°C (Fig. 2A). During the first generation, egg viability was low, and first (26.42 ± 8.8) and fifth nymphal stage (36.67 ± 13.2) mortality rates were higher. The first (40.71 ± 10.5) and second (22.60 ± 5.6) nymphal stages of the second generation also had high mortality rates. The M. histrionica mortality rate was not affected by temperature (t = 1.02; df = 547; P = 0.3079; but see Fig. 2B for first-generation data) but was slightly affected by generation (t = 2.04; df = 547; P = 0.0417), with a significant interaction between temperature and generation (t = −2.31; df = 547; P = 0.0212). Mortality rates were greater for the first (19.27 ± 4.8), second (20.41 ± 5.6), and fourth (20.24 ± 5.8) nymphal stages at 30°C of the second generation compared with the first generation (5.61 ± 2.8, 8.92 ± 2.4, and 3.59 ± 2.1, respectively, for first, second, and fourth instars).

The median development time for A. hilare was not significantly affected by generation (t = 0.60; df = 337; P = 0.55), temperature (t = −0.13; df = 337; P = 0.90), or the interaction between temperature and generations (t = −0.59; df = 337; P = 0.56). Similarly, the MDT for M. histrionica was not significantly affected by generation (t = 1.02; df = 374; P = 0.31), temperature (t = −0.55; df = 374; P = 0.59), or the interaction between temperature and generations (t = −1.16; df = 374; P = 0.25). Though not significant, nymphal development times of both species were shorter at the higher temperature (see Table S1 in the supplemental material).

The demographic parameters calculated for A. hilare and M. histrionica are shown in Tables S2 and S3 in the supplemental material. For A. hilare, high temperature slightly reduced the mean generation time, T. However, the higher temperature had a net negative impact on the insect's population performance. Insect rearing at 30°C resulted in R₀ and λ values of less than one and a negative value of r—all indicating a population in decline. As such, we could not estimate the population doubling time at 30°C. Moreover, GRR at 30°C was approximately half that at 25°C. Acrosternum hilare reproduction was similarly affected by temperature (see Table S3 in the supplemental material). The preoviposition period was reduced at the higher temperatures, but so were the number and size of egg masses (Fig. 3).

Murgantia histrionica showed trends toward lower population performance at higher temperature and in the later generation (see Table S2 in the supplemental material). However, the only significant difference was for the gross reproductive rate, GRR, at 25°C between the first and second generations. Murgantia histrionica reproduction showed trends toward reduced oviposition period and number of eggs (Fig. 3) at higher temperature, but the only significant difference was for adult emergence between generations at 30°C (Table S3).

Insects of both species reared at 25°C were positive for in situ hybridization with the universal (green signal) probe. Moreover, insects at 25°C were positive for their respective symbiont-specific probes (Fig. 4). Acrosternum hilare's symbiont was a rod-shaped bacterium. Murgantia histrionica's bacterial symbiont was an elongated rod up to 18 μm in length. Other insect symbionts have been found to be unusually large (30 μm) in length (18). No cells were observed in gut samples of either stink bug species reared at 30°C. A small proportion of individuals were symbiont positive in the life history experiments in the first generation at 30°C (Fig. 1). The absence of detectable symbionts in the 30°C FISH assays might be due to the small sample size used (n = 5/treatment) or low symbiont populations in the individuals tested.