Exposure to opposing temperature extremes causes comparable effects on Cardinium density but contrasting effects on Cardinium-induced cytoplasmic incompatibility

Terrestrial arthropods, including insects, commonly harbor maternally inherited intracellular symbionts that confer benefits to the host or manipulate host reproduction to favor infected female progeny. These symbionts may be especially vulnerable to thermal stress, potentially leading to destabilization of the symbiosis and imposing costs to the host. For example, increased temperatures can reduce the density of a common reproductive manipulator, Wolbachia, and the strength of its crossing incompatibility (cytoplasmic incompatibility, or CI) phenotype. Another manipulative symbiont, Cardinium hertigii, infects ~ 6–10% of Arthropods, and also can induce CI, but there is little homology between the molecular mechanisms of CI induced by Cardinium and Wolbachia. Here we investigated whether temperature disrupts the CI phenotype of Cardinium in a parasitic wasp host, Encarsia suzannae. We examined the effects of both warm (32°C day/ 29°C night) and cool (20°C day/ 17°C night) temperatures on Cardinium CI and found that both types of temperature stress modified aspects of this symbiosis. Warm temperatures reduced symbiont density, pupal developmental time, vertical transmission rate, and the strength of both CI modification and rescue. Cool temperatures also reduced symbiont density, however this resulted in stronger CI, likely due to cool temperatures prolonging the host pupal stage. The opposing effects of cool and warm-mediated reductions in symbiont density on the resulting CI phenotype indicates that CI strength may be independent of density in this system. Temperature stress also modified the CI phenotype only if it occurred during the pupal stage, highlighting the likely importance of this stage for CI induction in this symbiosis.


Introduction
Temperature has profound effects on the biology of ectothermic organisms as well as on the microbiota inhabiting these organisms [1][2][3][4]. The heritable symbionts of arthropods, which are primarily vertically transmitted from mother to offspring, are closely integrated with host biology [5], and may be particularly vulnerable to temperature stress [4,6]. These symbionts rely on strategies, ranging from mutualistic to parasitic, to modify host phenotype in ways that increase the likelihood of their maternal transmission [5]. While transmission of these symbionts and their phenotypic effects are usually stable under benign conditions, both aspects may be threatened as the environment becomes less hospitable [4]. Extreme temperatures can reduce symbiont density [7,8,9], induce phenotypic failure [7][8][9][10], and/or reduce rates of vertical transmission, resulting in uninfected offspring [8,9]. From the host's perspective, the limited thermotolerance of the symbiont compared to the host is a vulnerability, since long-term associations are likely to make hosts metabolically or reproductively dependent on these symbionts, even those that originally acted as reproductive parasites [11][12][13][14]. In these cases, temperature-induced disruption of these symbioses can be disastrous for the host, reducing host fitness by rendering hosts vulnerable to natural enemies [10,15] or causing reproductive failure [13,16,17].
Temperature extremes thus pose a clear challenge to heritable symbioses, including those involving one of the best-studied symbionts, Wolbachia, a diverse genus of widespread bacteria (Phylum Alphaproteobacteria). Wolbachia typically acts as a reproductive parasite of hosts using one of four major strategies, the most common of which is cytoplasmic incompatibility (CI) [18]. CI is a mating incompatibility between symbiont-infected males and uninfected females, resulting in inviable offspring. Offspring viability is restored (or "rescued") in crosses with infected females [18]. CI increases infected female fitness at the cost of uninfected female fitness, thus driving the symbiont infection through a population [19][20][21]. Some Wolbachia strains also improve host resistance to viruses and macroparasites, including several important human pathogens, instigating the use of this symbiont as a means of controlling important human diseases, including dengue fever, zika, and chikungunya [22][23][24].
Multiple studies using different host species and Wolbachia strains have found that Wolbachia's CI phenotype can be modified by temperature [7,8,25]. These changes to CI appear to be generally mediated by changes in Wolbachia density, which in turn influences CI strength [26]. Temperature stress reduces Wolbachia density, thereby weakening CI [8,25].
Temperature-induced Wolbachia reduction also leads to partial failure of the rescue phenotype and reduced efficacy of vertical transmission [8,27]. From these studies, it seems that temperature stress in some habitats could limit the ability of some Wolbachia strains to maintain high levels of infection or spread through novel populations, limiting both the natural spread of this symbiont as well as efforts to use this symbiont in disease vector control [8].
Although Cardinium symbioses present an alternative study system for the study of CI, research concerning the impact of environmental factors upon this manipulative symbiont is limited to a handful of studies investigating Cardinium presence or absence at various temperatures. One such study found that Cardinium prevalence was reduced in natural populations of Culicoides biting midges (Diptera: Ceratopogidae) occupying arid regions, offering evidence for a destabilizing effect on the symbiosis attributed to extreme temperature fluctuations in those regions [43]. More recently, an analysis of global Cardinium infection frequencies found increased Cardinium prevalence in insect host populations occupying warmer regions (mean temperature of~25˚C) compared to cooler ones [44]. Other studies using laboratory cultures of the spider mite, Metaseiulus occidentalis, found that mite cultures held at 34˚C for >1 year were either uninfected with Cardinium or had a reduced amount of the symbiont compared to cultures maintained at room temperature [45,46]. Together, these studies provide some evidence for a general destabilizing effect of extreme temperatures on Cardinium-host symbioses; yet we still do not know if temperature modifies other aspects of Cardinium symbioses, particularly the CI phenotype.
Here we examined how temperature stress modifies this manipulative symbiosis. We tested the effects of prolonged exposure, including one or more host stages, to either high or low temperatures on both Cardinium CI modification (in males) and rescue (in females; Fig 1). We next tested whether observed temperature-induced changes to Cardinium's CI modification machinery and rescue capabilities were due to changes in symbiont density. We also tested the effect of temperature stress on the duration of host pupal development, a host factor that appeared to correlate with Cardinium's CI strength in a previous study [47]. We next used temperature shock treatments during specific life stages to test whether temperature's influence on CI was life-stage dependent. Finally, we tested the effect of temperature stress on vertical transmission rates of this heritable symbiont. day/17˚C night; 16:8 h) temperatures caused dramatically different levels of CI-induced offspring mortality compared to males reared at 27˚C (Fig 2). Prolonged exposure to warm temperatures, beginning either when the host was a larva or pupa, resulted in a significant decrease in CI-induced offspring mortality (Fig 2; S1 Table; Logistic regression F 1,32 larva = 7.15, p = 0.01; F 1,31 pupa = 7.49, p = 0.01). Interestingly, exposure to cool temperatures beginning in the pupal stage had an opposite effect on CI strength and resulted in an increase in CI-induced mortality (F 1,38 = 9.98, p = 0.003). In this treatment, the CI phenotype became not only stronger but also less variable than CI typical of this Cardinium strain (Fig 2). However, we found that neither warm nor cool temperatures affected CI strength when the exposure period only included the adult stage (Fig 2;  We found no effect of temperature on offspring viability in control ("N") crosses, in which no CI-induced mortality is expected, showing that changes in offspring mortality in warm and cool treatments are not due to a general effect on male viability (Fig 2;

Warm and cool temperatures modify Cardinium's ability to rescue CI
When we tested the effectiveness of the ability of Cardinium in females to rescue Cardiniummodified sperm, the 27˚C control treatment showed comparable offspring survival between the rescue cross ("R"; infected mother x infected father) and the control cross ("N"; infected mother x uninfected father), indicating that cEper1 reliably rescues CI when mating The heat-exposed control (N) crosses showed lower offspring survival rates than the 27˚C controls, which suggests that heat reduced the viability of the experimental females independent of the CI phenotype. To account for this general reduction in viability, we subsequently compared offspring survival between the rescue (R) cross and its corresponding control (N) cross for each heat treatment. We then found an additional significant reduction in offspring survival in the R cross compared with its corresponding N cross when females were exposed to heat starting in the larval stage (Fig 3; F 1,28 = 20.13, p = 0.0001). These results suggest a partial failure of the rescue phenotype with heat, although pupal and adult exposure to heat did not significantly influence rescue beyond the general reduction in viability (F 1,28 pupa = 2.85, p = 0. For this reason, we compared cold R rescue crosses to the R cross of females kept at the 27˚C control temperature (Fig 3). Like the heat treatment, the longest cold exposure resulted in reduced offspring survival in R crosses, indicative of partial rescue failure (

Warm and cool temperatures reduce Cardinium density in male and female hosts
We tested the effect of temperature stress on Cardinium density in male and female hosts. We found that Cardinium reached variable densities in adult males at the control rearing temperature of 27˚C, with densities ranging from 0.25 Cardinium: host cells to about 2:1 Cardinium:host cell (Fig 4a). Both warm and cool temperatures reduced Cardinium density in the male larval and pupal treatments (Fig 4a, S2 Table; Mann-Whitney U-test p warm-larva = 0.009; p warm-pupa = 0.04; p cool-larva = 0.004; p cool-pupa = 0.04), although we found no significant changes to Cardinium density in either of the male adult treatments, which also did not show evidence of temperature-modified CI strength (Figs 2 and 4a). While warm and cool treatments both suppressed Cardinium density in male hosts, these temperature treatments showed contrasting effects on CI strength. Furthermore, the Cardinium density of individuals across treatments did not correlate with CI strength (Spearmen's rho = 0.05, p = 0.72).
In female wasps reared at 27˚C, Cardinium density was less variable than in males, with females harboring about 2.5 Cardinium: 1 host cell (Fig 4b). Temperature treatments all significantly reduced Cardinium density in females relative to those kept at 27˚C, regardless of temperature range or exposure period (Fig 4b, S3 Table; Mann-Whitney U-test p = 0.012 for all comparisons to control). Because cool temperatures simultaneously reduced Cardinium density yet resulted in significantly stronger CI, we looked for another factor that could be responsible for the cold-modified CI phenotype. We found that cool temperatures significantly prolonged the duration of the pupal stage from~6 days at 27˚C to~14 days (Fig 5, S4 Table; Mann-Whitney U-test p = <0.0001). Additionally, we found that warm temperatures significantly reduced pupal developmental time (from~6 days to~4 days) when those exposures began in the larval or pupal stage (Fig 5, S4 Table; p = <0.01). The adult treatments underwent their temperature exposure after the pupal stage, and we found no difference in pupal duration between adultexposed wasps and control wasps kept at 27˚C (Fig 5). As in [47], we also found that the duration of the pupal stage positively correlated with CI strength (Fig 6; Spearman's rho = 0.57, p = <0.0001).

Heat-shock during the male pupal, but not adult, stage weakens CI
We designed a second experiment using temperature-shocks of equal duration to test whether temperature effects on Cardinium's CI were dependent on host life stage. We found that heat shock (40˚C for 2 hours) during the pupal, but not the adult stage, reduced offspring mortality in CI crosses (Fig 7, S1 (Fig 2), we found no difference in CI-induced mortality between the control and either the pupal or adult cold-shock (4˚C for 2 hours) treatments (Fig 7, S1

High temperatures reduce vertical transmission efficiency
Prolonged exposure to high and low temperatures resulted in a reduction of the ability of Cardinium to rescue its CI (Fig 3) and a reduction of the density in female hosts (Fig 4b). This rescue failure could have resulted from vertical transmission failure, which would generate

Discussion
We tested the effect of temperature stress on a symbiosis between a minute parasitoid wasp, E. suzannae, and its CI-inducing heritable symbiont, Cardinium. Male hosts exposed to warm temperatures during the larval or pupal life stages showed faster pupal development, as well as reduced Cardinium density and reduced expression of the CI phenotype relative to hosts kept at a benign temperature. On the other hand, exposure to cool temperatures beginning during  the pupal stage strengthened CI expression, despite also reducing Cardinium density. This stronger CI likely resulted from the prolonged pupal stage of cold-treated males, as cold shock treatments that did not prolong the pupal stage or modify Cardinium density also did not result in stronger CI. We also found that lifetime exposure to both warm and cool temperatures reduced Cardinium density in female hosts. Although both temperature ranges reduced Cardinium density in female hosts, heat exposure weakened the CI rescue phenotype more than cold. Why heat exposure caused more rescue failure is unclear, although the decreased vertical transmission rates we observed from heat-treated females to their offspring could account for additional mortality in these rescue crosses. The symbiosis between Cardinium and E. suzannae, like other heritable symbioses, is vulnerable to environmental stress [4]. Specifically, warm temperatures can weaken the CI phenotype of this manipulative symbiont, with lifetime exposure to higher temperatures almost completely nullifying the phenotype. Like most symbionts, Cardinium infection is expected to carry a fitness cost to infected females under neutral conditions, and, like other symbionts, Cardinium is expected to rely on its CI phenotype to compensate for maintenance costs, allowing it to successfully invade new host populations when above the unstable equilibrium threshold [19-21, 48, 49]. With its CI phenotype weakened, the cost of harboring Cardinium could prevent its spread into regions experiencing prolonged bouts of warmer temperatures.
Warm temperatures also caused Cardinium transmission rates to drop from essentially perfect (100% of offspring infected) to a 90% infection rate. Such a decrease, coupled with modification and rescue failure, may be enough to destabilize the Cardinium symbiosis and cause a drop in Cardinium frequency in an infected population. A similar scenario occurs with Wolbachia that infect Trichogramma wasps, in which the Wolbachia falls in frequency during warm periods and re-invades populations in cooler months [50]. However, given that symbiont strains can show substantial variation in their susceptibility to temperature [8,10], we caution against applying our results concerning cEper1 (the Cardinium strain used in this study) to other strains of Cardinium. If a strain of Cardinium is thermally sensitive, like cEper1, it may have dynamic infection frequencies that vary seasonally, although this should be investigated further using population-level studies and seasonal field collections.
Our study highlights the importance of the male pupal stage for this Cardinium strain's CI modification step, which has mechanistic implications for Cardinium CI modification. Temperature exposure only affected CI strength if it occurred throughout the pupal stage, suggesting that this symbiont is actively modifying male sperm as the testes develop during the pupal stage. Furthermore, E. suzannae adult males emerge with more than enough mature sperm for 14 successive matings in a laboratory setting (MRD, personal observation). During spermatogenesis the cytological contents, including any heritable symbionts, are dumped, leaving mature sperm bacteria-free [51,52]. Given that male E. suzannae emerge with most of the sperm they will use in their lifetime, and that these sperm likely don't contain Cardinium, we suspect that the majority of Cardinium's active modification of male chromatin occurs in the pupal stage. Indeed, adult males of many parasitoid species emerge with a majority of their lifetime sperm, and the pupal stage is potentially of prime importance for the modification step of CI symbionts in these hosts [7,53]. Future gene expression studies on Encarsia-Cardinium symbioses should focus on the pupal stage to catch expression of all possible bacterial genes involved in CI modification.
The strength of Wolbachia CI is generally dependent on its within-host density [8,21,26,50,52]. Reduction of Wolbachia density, whether by temperature or some other means, results in loss or weakening of the CI phenotype [8,25,54]. Wolbachia also harbors a bacteriophage, WO, which may further modify Wolbachia density and CI strength in response to extreme temperatures [7]. While we found that Cardinium density is also reduced by warm and cold temperature stress, this density reduction did not result in consistent changes to the CI phenotype. Instead, the duration of the pupal stage of male hosts seems to have a larger impact on this symbiont's phenotype. This can be seen most clearly in the case of cold exposure, which prolonged the pupal stage and increased CI mortality. A similar scenario occurs in Wolbachiainfected Nasonia vitripennis, a species of parasitic wasp, that also shows prolonged pupal development and stronger CI after long-term exposure to cool temperatures. However, this Wolbachia strain harbors the WO phage and it's possible that increased phage densities may also be responsible for the stronger CI phenotype [7]. It's not known whether Cardinium must be present within a spermatid in order to modify it or whether the CI factors employed by Cardinium diffuse across membranes of developing sperm, as has been shown for Wolbachia [55]; either case is compatible with a time-limited mechanism for modifying sperm during the pupal stage. Extension of the pupal stage may grant time for Cardinium to modify a greater proportion of sperm and generate stronger CI. This contrasts with what is observed in Drosophila melanogaster-Wolbachia symbioses, in which slower developing males exhibit weaker CI through an unknown mechanism that is also independent of density [56]. Time could be a limiting factor for modification steps in CI symbioses which occur in host organisms that produce most of their lifetime sperm prior to adult emergence, like many parasitic wasps [7,53,57], but not for hosts that continuously mature sperm, like Drosophila flies [56,58].
One caveat to the apparent lack of dependence of CI strength on density in the current study is that qPCR, our method and the standard method for estimating symbiont density, does not give any indication of symbiont health. It's possible that while the warm and cool treatments equally depressed Cardinium density, the heat treatment may have damaged Cardinium or denatured proteins and other effector molecules involved in expression of CI, while the cold treatment only repressed Cardinium replication. Nevertheless, while density may be a reliable indicator of phenotypic strength for some symbionts, more examples are emerging in which phenotype strength and symbiont density are decoupled, and this rule may not be as general as originally thought [7,9,10,56,59]. In the case of low-density symbionts, like the CI-Cardinium strains infecting Encarsia wasps and citrus thrips (Pezothrips kellyanus) [37] or some CI-Wolbachia strains [59], other factors may be of equal or greater importance than symbiont density. These include host factors, like developmental time in this study, or the density of extrachromosomal elements harbored by many heritable bacteria [7]. These phages or plasmids can harbor genes responsible for the symbiont-conferred phenotype [60][61][62][63], replicate independently of the host bacterium [7,10], and may respond differently from their host bacterium to environmental stress [7,10]. In the case of Wolbachia, temperature stress inversely modifies the density of Wolbachia and its WO phage, which harbors the genes required for CI expression [7,62]. The cEper1 strain of Cardinium does not have a phage but does harbor a plasmid [39]. The role of the plasmid in Cardinium symbioses and temperature stress is not yet clear.
With a rapidly changing climate, the susceptibility of heritable symbionts to temperature stress confers similar vulnerability to the insect hosts that often depend on them, including hosts of economic or medical importance. Here, we show that a symbiosis involving Cardinium and its wasp host is modified by temperature. Whether this has cascading effects upon the host population remains to be seen. However, Encarsia spp. are used as widespread biological control agents for agricultural pests like whiteflies and armored scales [64][65][66][67][68]. Given the potential for temperature to disrupt these symbioses, further investigations into how environmentally susceptible Cardinium-Encarsia symbioses impact wasp and whitefly populations are warranted.

Insect and symbiont cultures
Encarsia suzannae (= E. pergandiella [38]) is a species of autoparasitic wasp (Hymenoptera: Aphelinidae). Like all Hymenoptera, these wasps are haplodiploid; females develop from fertilized, diploid eggs and males develop from haploid, unfertilized eggs. In autoparasitic wasps, females lay diploid eggs within whitefly nymphs, where they develop as solitary primary parasitoids. Male eggs are laid within developing aphelinid whitefly parasitoids enclosed within the remnant whitefly cuticle, and male E. suzannae develop obligately as hyperparasitoids [69]. Laboratory cultures of female E. suzannae were maintained on Bemisia tabaci (sweet potato whitefly) grown on Vigna unguiculata (cowpea); male wasps were maintained by exposing pupae of a second parasitoid species, Eretmocerus emiratus, to virgin E. suzannae females. Wasp cultures were kept at 27˚C and 16 D: 8 N photoperiod at ambient humidity unless otherwise noted.
The E. suzannae culture of this study was collected from the Rio Grande Valley of Texas, USA [33]. This wasp naturally harbors a CI-inducing strain of Cardinium hertigii, cEper1 [33,39,40]. Additionally, an uninfected wasp culture was established by feeding adults antibioticspiked honey, allowing for mating crosses between uninfected C(-) and cEper1 infected C(+) individuals [33]. Uninfected individuals used in this study were from a culture that had been antibiotically-cured at least 10 generations previously. The Er. emiratus used for generating male E. suzannae were not infected with Cardinium.

Testing the effect of temperature stress on Cardinium-induced CI
Male C(+) wasps were generated by placing several (~5-7) virgin C(+) E. suzannae in a 35mm Petri dish with 25 Er. emiratus pupae on moist filter paper along with a small amount of honey. We observed females parasitize for 3 hours, removing parasitized Er. emiratus with a fine paint brush to limit superparasitism (multiple eggs oviposited in a single host). Parasitized Er. emiratus (i.e. developing male E. suzannae) were placed into individual 1.2 ml vials. Then, using an experimental design modified from [7], we tested the effect of temperature stress on Cardinium's ability to induce CI in crosses between C(+) males and C(-) females by exposing these developing males to a variety of temperature treatments (Fig 1). One subset of males was kept at 27˚C in a Percival incubator throughout development to serve as a control group. Other males were kept in Percival incubators set to either a "warm" or "cool" temperature range of 32˚C day/ 29˚C night or 20˚C day/ 17˚C night, respectively. We monitored internal incubator temperatures with HOBO data loggers. These ranges, particularly the "warm" temperatures, reflect the temperature extremes at which E. suzannae will tolerate constant exposure in the laboratory (MRD, personal observation). Temperature treatments were further split into three groups that began at either the larval 1 st instar (4-day old wasps), pupal (7-day old), or adult (upon emergence) stage [70]. All temperature treatments continued until two days post-adult emergence. This resulted in a total of seven temperature treatments that differed in their temperature range as well as the host life stages that they included (Fig 1). As cEper1's CI phenotype was previously found to positively correlate with male pupal developmental time [47], we also recorded the duration of the pupal stage for each individual male.
Two days after male adult emergence, males were mated either with virgin C(+) females for the control cross ("N"), to test for temperature-induced effects on general male viability, or with virgin C(-) females for the inviable "CI" cross to test for the effect of temperature on the strength of Cardinium's CI phenotype. Mating was visually confirmed by observing male and female wasps confined in a 1.2 ml vial plugged with cotton for~5 minutes or until mating.
Mating typically occurred < 1 min after bringing the pair together, but in cases where mating had not yet occurred after 5 minutes, the female was removed, discarded, and replaced. After mating, males were stored at -80˚C and females were transferred to individual arenas to parasitize whiteflies. The arenas consisted of a 35mm Petri dish with a ventilated lid, containing a cowpea leaf disc resting on 1% agar. The cowpea leaf disc bore~50 2 nd -3 rd instar B. tabaci nymphs. Females were allowed to parasitize whiteflies in these arenas for 24hrs and the dish was then incubated at 27˚C. Ten days later, we counted the number of whiteflies that either contained a developing E. suzannae pupa (resulting from a successful cross) or were developmentally arrested (i.e. parasitized by E. suzannae but the wasp failed to develop-a reliable proxy for the dead wasp embryos indicative of CI [33]). We then compared the proportion of arrested whiteflies across treatments using logistic regression with a quasibinomial distribution. We compared male pupal developmental time using the non-parametric Kruskal-Wallis test and post hoc multiple pairwise Mann-Whitney U Tests with Benjamini-Hochberg-corrected p-values in R v 3.3.1 [71]. We also compared male pupal developmental time and CI strength using Spearmen's rho correlation.

Testing the effect of temperature stress on Cardinium's rescue phenotype
To test the effect of temperature stress of Cardinium's ability to rescue its CI phenotype, we first generated female C(+) E. suzannae by allowing mated C+ female E. suzannae to parasitize leaf dishes with 2 nd -3 rd instar whiteflies for 24hrs. Leaf discs with developing female wasps were then exposed to one of seven temperature treatments in Percival incubators as previously described. Two-days after adult emergence, experimental females were mated with either C(-) males (N; control cross) to test for temperature-induced effects on female viability, or C(+) males (R; rescue cross) that developed at 27˚C to test for effects on Cardinium's rescue phenotype. Here a decrease in offspring survival relative to the control would indicate a reduction in the rescue ability of Cardinium in the temperature treated female host. After mating observations, females were transferred to individual parasitism arenas and given 24hrs to parasitize whitefly hosts. Females were then removed and stored at -80˚C. Ten days later, we counted the number of surviving wasp pupae and arrested whiteflies and analyzed rescue efficiency across temperature treatments using logistic regression with a quasibinomial distribution, as in the CI experiment.
Quantifying Cardinium relative density post-temperature exposure After the crossing experiments, we next tested whether changes in Cardinium's CI modification and rescue phenotypes were due to changes in the within-host density of this symbiont. Experimental wasps (both male and female) had been stored at -80˚C for DNA extraction. Extractions consisted of homogenizing a single wasp in 3 μL of 20 mg ml -1 proteinase k, then adding the homogenate to 50 μL of 5-10% w/v Chelex [72]. Samples were incubated at 37˚C for 1 hour with periodic vortexing, followed by incubation at 97˚C for 8 min and storage at -20˚C. We estimated Cardinium density relative to host cells by performing quantitative PCR (qPCR) using Maxima SYBR Green/ROX qPCR Master Mix (2×) (ThermoFisher Scientific) with primers for the single-copy Cardinium gyrB gene [47] and the host Ef1a gene on a Bio-Rad CFX Connect Real-Time cycler [73]. We also created standards via serial dilutions of PCR products from both primer sets. PCR products were diluted to a concentration of 1.0 ng/μL DNA, which was confirmed with a Qubit 4.0 fluorometer prior to serial dilution. Samples were run in triplicate and each qPCR plate included standards for both primer sets to correct for between-plate differences in reaction efficiency. Raw Cq values were averaged and corrected before conversion to relative density [47].
We then compared Cardinium density across temperature treatments for male and female wasps using the Kruskal-Wallis non-parametric test with post hoc multiple pairwise Mann-Whitney U Tests with Benjamini-Hochberg-corrected p-values. We also compared Cardinium density and CI strength using Spearmen's rho correlation. All statistical analyses were performed in R v. 3.3.1 [71].

Using temperature-shocks to standardize exposure period
The experiments described in the current study tested the effect of prolonged temperature stress on the Cardinium CI phenotype and thus included one or more host life stages and different durations of temperature exposure. To test for life stage-specific effects of temperature stress on the CI phenotype, we performed temperature "shocks" of uniform duration on male C(+) E. suzannae, during either their pupal stage or upon adult emergence. Temperature shocks consisted of a two-hour ramping step, where temperatures increased from 27˚C to 40˚C (heat shock) or decreased from 27˚C to 4˚C (cold shock), followed by a two-hour period at 40˚C or 4˚C, and then a two-hour ramp back to 27˚C in a Percival incubator. After the temperature shock, males were maintained at 27˚C until mating two days after emergence. As in the previous CI experiment, males were kept in individual 1.2 ml vials with a small amount of honey throughout development. Mating and parasitism were otherwise performed and analyzed as in the first experiments, and males were kept at -80˚C for DNA extractions and Cardinium density estimates using qPCR.

Testing the effects of temperature on vertical transmission efficiency
In addition to modifying symbiont-induced phenotype and/or density, temperature stress can destabilize symbiont vertical transmission [8]. Inefficient or "leaky" vertical transmission can slow the spread or cause the loss of symbionts within a population. We tested the effect of temperature stress on Cardinium vertical transmission by exposing C(+) female E. suzannae to one of three temperature treatments: a 27˚C control, 32˚Cday/29˚Cnight (warm), or 20˚Cday/ 17˚Cnight (cool). Female wasps were generated as previously described and kept at 27˚C until they were 4-days old (1 st instar larvae), when they were moved to their respective temperature treatments. Exposure continued through their development until two-days post adult emergence, when the females were mated to C(-) males, a control cross that should generate viable progeny regardless of female infection status. After mating, females were placed on arenas with whitefly nymphs and given 24hrs to parasitize hosts. These offspring were kept at 27˚C until adult emergence, at which point they were frozen at -80˚C. Offspring DNA was extracted and tested for the presence of Cardinium DNA using Cardinium-specific 16S rRNA primers [49]. PCRs included a positive and negative control and were run on a 1% agarose gel with SYBR Safe to visualize the product. Negative samples were re-run with the Cardinium primers. We next confirmed the presence of wasp DNA in negative samples using general CO1 primers [74]. Negative samples that failed to show DNA bands with the CO1 primers were discarded from the final analysis. We then tested whether the vertical transmission rates were significantly lower than 1.0 (perfect transmission) using the Exact Binomial test with 95% confidence intervals.