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Shifting the Balance: Heat Stress Challenges the Symbiotic Interactions of the Asian Citrus Psyllid, Diaphorina citri (Hemiptera, Liviidae).

Global warming may impact biodiversity by disrupting biological interactions, including long-term insect-microbe mutualistic associations. Symbiont-mediated insect tolerance to high temperatures is an ecologically important trait that significantly influences an insect's life history. Disruption of microbial symbionts that are required by insects would substantially impact their pest status. Diaphorina citri, a worldwide citrus pest, is associated with the mutualistic symbionts Candidatus Carsonella ruddii and Candidatus Profftella armatura. Wolbachia is also associated with D. citri, but its contribution to the host is unknown. Symbiont density is dependent on a range of factors, including the thermosensitivity of the host and/or symbiont to heat stress. Here, we predicted that short-term heat stress of D. citri would disrupt the host-symbiont phenological synchrony and differentially affect the growth and density of symbionts. We investigated the effects of exposing D. citri eggs to different temperatures for different periods of time on the growth dynamics of symbionts during the nymphal development of D. citri (first instar to fifth instar) by real-time polymerase chain reaction. Symbiont densities were assessed as the number of gene copies, using specific molecular markers: 16S rRNA for Carsonella and Profftella and ftsZ for Wolbachia. Statistical modeling of the copy numbers of symbionts revealed differences in their growth patterns, particularly in the early instars of heat-shocked insects. Wolbachia was the only symbiont to benefit from heat-shock treatment. Although the symbionts responded differently to heat stress, the lack of differences in symbiont densities between treated and control late nymphs suggests the existence of an adaptive genetic process to restore phenological synchrony during the development of immatures in preparation for adult life. Our findings contribute to the understanding of the potential deleterious effects of high temperatures on host-symbiont interactions. Our data also suggest that the effects of host exposure to high temperatures in symbiont growth are highly variable and dependent on the interactions among members of the community of symbionts harbored by a host. Such dependence points to unpredictable consequences for agroecosystems worldwide due to climate change-related effects on the ecological traits of symbiont-dependent insect pests.


Global warming impacts biodiversity by disrupting the interactions among associated partners at different levels in nature (Robinet and Roques, 2010), including beneficial interactions between insects and their microbial symbionts (Kikuchi et al., 2016). Insects maintain a myriad of symbiotic interactions with beneficial microbes that provide a wealth of metabolic benefits to their hosts. The ecological innovations provided by symbionts allow the hosts to withstand environmental stressors, including tolerance to heat stress (Baumann, 2005; Feldhaar, 2011; Hussa and Goodrich-Blair, 2013). These intimate and often long-term associations led to the development of specialized structures, for example, bacteriocytes or bacteriome, for harboring (Baumann, 2005) and even for controlling beneficial symbionts (Login and Heddi, 2013). The events during the formation of these symbiont-dedicated structures and the maintenance of the close association involve complex mechanisms for host-cell invasion, transmission, and control of symbiont growth within the host (Braendle et al., 2003; Koga et al., 2012; Sloan and Moran, 2012; Matsuura et al., 2015). Endo-symbionts can engage in a nutrition-based and highly interdependent obligate association (i.e., primary symbionts) with insect hosts. Primary symbionts (P-symbionts) are harbored inside bacteriocytes, which in some host species can construct a complex organ, the bacteriome (Baumann, 2005: Simonet et al., 2018). Because of the nature of the interaction, P-symbionts are critical to the nutritional ecology of their hosts, providing essential nutrients and vitamins that are lacking in their host's diet (Baumann et al., 2006; Feldhaar, 2011). Alternatively, facultative symbionts (i.e., secondary symbionts) have quite variable metabolic integrations with their hosts, influencing the host's ecophysiology by modifying its plasticity to biotic and abiotic stressors (Werren et al., 2008; Feldhaar, 2011; Sloan and Moran, 2012).

Temperature can affect the density of symbionts by altering important symbiont-dependent metabolic pathways, which can ultimately disrupt the biological performance of host insects (Anbutsu et al., 2008; Shan et al., 2014). The reduced density or complete elimination of symbionts following exposure to heat stress is reported to affect host development in many species, including aphids (Oliver et al., 2010), white-flies (Brumin et al., 2011), bedbugs (Zhao and Jones, 2012), and stinkbugs (Kikuchi et al., 2016). Most of the information regarding the effects of acute or prolonged heat shock is associated with the exposure of immatures or adults, stages in which insects can actively search for favorable micro-environments and regulate their physiological mechanisms for thermoregulation (Howe, 1967; Feder and Hofmann, 1999; Hamdoun and Epel, 2007). In addition to the behavioral mechanisms for avoiding heat stress, insects can deal with deleterious effects of heat stress by using sophisticated mechanisms to minimize water loss (Prange, 1996; Neven, 2000), such as evaporative cooling (Prange, 1996), respiration and oxidative-defense mechanisms (Neven, 2000; Ju et al., 2014), metabolism of thermoprotectant polyols (e.g., mannitol and sorbitol) (Hendrix and Salvucci, 1998; Salvucci, 2000; Guo et al., 2012), and synthesis of heat-shock proteins (HSPs) (reviewed by King and MacRae, 2015). Insects may also benefit from the production of molecular chaperones (HSPs) by their symbionts (Montllor et al., 2002; Brumin et al., 2011; Kupper et al., 2014; Xiao et al., 2016). Chaperones are critical components of the protein post-translational mechanism for protecting cells under stress conditions, by preventing protein misfolding and the consequent loss of function (Zhao and Jones, 2012; Kupper et al., 2014; King and MacRae, 2015).

Heat stress can also indirectly affect insects by altering symbiont density and, consequently, the host-symbiont interactions (Burke et al., 2010), for example, by impairing the efficiency of symbiont transmission to the host's offspring (Sacchi et al., 1993; Dunbar et al., 2007). Several studies have examined the thermosensitivity of symbionts associated with insects (Chang, 1974; Montllor et al., 2002; Wemergreen, 2012). Thermosensitivity of symbionts exposed to high temperatures can lead to a reduction in the minimum number of symbiotic cells that must be transferred to the progeny to secure the host-symbiont association (Mira and Moran, 2002; Miura et al., 2003; Wilkinson et al., 2003; Feldhaar, 2011).

Diaphorina citri Kuwayama, 1908 (Hemiptera, Liviidae), the Asian citrus psyllid (Ouvrard, 2017), threatens citriculture worldwide as the vector of the causative bacterium of huanglongbing (HLB), Candidatus Liberibacter spp. The distribution of D. citri can be explained at least in part by its ability to develop in a wide range of temperatures, but the optimum biological fitness is attained at 28 [degrees]C (Nava et al., 2007). Additionally, immatures reared under laboratory conditions at 30 [degrees]C or higher suffer total mortality (Nava et al., 2007). Diaphorina citri exists in close association with mutualistic bacteria harbored in the cytoplasm of bacteriocytes or in the syncytium of the bacteriome (Nakabachi et al., 2013). The endosymbiotic association is based on nutritional and defensive benefits to the host insect, and the association is perpetuated through transovarial transmission (Nakabachi et al., 2013). Eggs of D. citri are laid on the tips of growing shoots on and between unfurling leaves, and they can use the leaf heat balance to reduce or prevent the deleterious effects of heat stress. The thermoregulation ability of leaves may vary even between leaves on the same shoot, and leaves can provide different microhabitats to maintain temperatures within a safe range for tiny eggs (Potter et al., 2009: Pincebourde and Woods, 2012). Thus, endosymbiont density may be influenced by several factors, including temperature (Clancy and Hoffmann, 1998; Chen et al., 2009), and may directly or indirectly affect host fitness. Under optimal rearing conditions (Nava et al., 2007), bacteriome-associated symbionts and facultative Wolbachia in D. citri have been shown to reach their maximum growth rates and population densities at different host development stages, although males have higher symbiont densities than females (Dossi et al., 2014).

In view of the extraordinary complementarity of the association between mutualistic symbionts and psyllids (Sloan and Moran, 2012), the sensitivity of D. citri to high temperatures (Nava et al., 2007; Hall and Hentz, 2014), and the expected thermosensitivity of their associated symbionts, we investigated the effects of heat shock on the growth pattern of the bacteriome-associated symbionts Candidatus Carsonella ruddii, Candidatus Profftella armatura, and Candidatus Wolbachia spp., an endosymbiont of unknown function that occurs in insect somatic and germline tissues but that is fixed in D. citri populations (Guidolin and Consoli, 2013). We assessed endo-symbiont densities by using real-time quantitative polymerase chain reaction (qPCR) during the development of nymphs of D. citri hatched from eggs exposed to heat treatment at high temperatures (32 [degrees]C and 34 [degrees]C) that are considered unsuitable for full development of D. citri (Tsai and Liu, 2000; Nava et al., 2007). Because symbionts can influence the host's ecophysiology and population dynamics, our findings will be useful for understanding the potential for success of D. citri across different environments, serving as the basis for predictive modeling of D. citri distribution and management under a global warming scenario.

Materials and Methods

Insect stock colony

A colony of Diaphorina citri was maintained under controlled laboratory conditions (28 [degrees]C [+ or -] 2 [degrees]C; 60% [+ or -] 10% relative humidity [RH]; 14-h photophase), using seedlings of Murraya paniculata (L.) Jack (Rutaceae) as the host plant (Nava et al., 2007). We used a 2-y-old laboratory colony that often received field-collected psyllids to maintain high genetic variability and to avoid insect fitness losses during continuous rearing. We believe this rearing procedure was successful in avoiding the accumulation of mutations in the laboratory-maintained colony that would make it not representative of the genetic diversity of a field population.

Egg collection and heat-shock treatment

Diaphorina citri adults (5 d after emergence) were placed (1 female : 1 male sex ratio) in plastic cages (65 cm x 65 cm x 40 cm) to feed and oviposit for 24 h on M. paniculata seedlings. Adults were removed, and seedlings containing eggs (0-24 h after oviposition) were subjected to heat-shock treatments. Since D. citri eggs are laid on leaves at the tips of growing shoots and because the pedicel of each egg is inserted just below the plant epithelial cell layer, eggs were kept on seedlings where they were laid to avoid any damage due to handling. Seedlings containing eggs were exposed to heat shock in climate-controlled, ventilated chambers with the temperature set at 28 [degrees]C (control), 32 [degrees]C, or 34 [degrees]C ([+ or -]1 [degrees]C), 60% [+ or -] 10% RH, and a light: dark photoperiod of 14 h : 10 h for 3, 6, 12, or 24 h. Heat-shocked seedlings with psyllid eggs were immediately transferred to a rearing room under climate-controlled conditions (28 [degrees]C [+ or -] 2 [degrees]C; 60% [+ or -] 10% RH; 14-h photophase) for egg hatching and nymph development. The development of nymphs was evaluated daily, and nymphs comprising each of the five instars were collected just after molting and were stored in absolute ethanol. Each treatment (= each heat-shock temperature and time of exposure) was replicated three times (biological replicate). Each replicate was composed of five pooled D. citri nymphs per temperature and exposure time. Samples were stored at 4 [degrees]C until genomic DNA (gDNA) extraction.

Genomic DNA extraction and quantitative polymerase chain reaction

Extraction of gDNA from nymphs of D. citri followed Gilbert et al. (2007). All other steps comprising the qPCR assay, including the yield and quality of gDNA samples, primer sets for each symbiont studied, plasmid preparation, serial dilutions for generating standard curves, qPCR conditions, and calculation of bacterial copy numbers per insect-equivalent, followed Dossi et al. (2014).

Statistical analysis

Data were tested for homoscedasticity (Levene test) and normality (Cramer-von Mises test) before transformation into the natural log of each observation, ln(x). Then data were subjected to analysis of covariance (P [less than or equal to] 0.05) to evaluate the effects of host age, temperature, exposure time, and the interactions between the factors temperature x time and symbiont x symbiont (symbionts as covariates). All analyses were conducted using the software SAS 9.1 (SAS Institute, Cary, NC).


Exposure of Diaphorina citri eggs to heat-shock treatments significantly affected (P < 0.05) the density of all three symbionts evaluated during nymph development. However, the intensity of the response of each symbiont to heat shock varied according to temperature and exposure time (Table 1). Short-term exposure (3 h) to heat shock led to an increase in symbiont densities, while extended exposure time, especially for 24 h of exposure, decreased Carsonella and Profftella densities (Fig. 1). Wolbachia was the only symbiont to be benefited by the exposure to heat-shock treatments, particularly at the longest period of exposure (Fig. 1). In addition to the influence of temperature and duration of exposure on symbiont density during nymph development (Fig. 1), the growth curve of one symbiont in response to heat-shock treatments was influenced by the densities reached by the other symbionts (Table 2). However, the degree to which each symbiont affected the others was dependent on each symbiont-symbiont interaction (Table 2). The density of Carsonella was more intensely affected by Profftella (P < 0.0001) than by Wolbachia (P < 0.0224) (Table 2). Despite the reciprocal effect between Carsonella (sum of squares of residues [SQR] = 9.022; P < 0.0001) and Profftella (SQR = 9.114; P < 0.0001), both showed a greater reduction in density immediately following heat shock. In this same period of time, the density of Wolbachia was increased. Wolbachia influenced the density of Profftella (SQR = 1.011; P < 0.0001) more strongly than that of Carsonella (SQR = 0.629; P < 0.0224) (Table 2).

The effect of exposing eggs of D. citri to heat-shock treatments on the density of Carsonella was apparent as early as the initial instars, when greater fluctuations in density were detected after egg exposure to 32 [degrees]C for 6, 12, or 24 h. The density of Carsonella in subsequent instars varied with the exposure time: densities decreased in nymphs that hatched from eggs exposed to 32 [degrees]C for 24 h (Fig. 1 A). However, regardless of the duration of exposure at 32 [degrees]C, the density of Carsonella at the beginning of the fifth instar of D. citri was similar to that of the control (Fig. 1 A). Interestingly, exposure to 34 [degrees]C was less harmful to this symbiont compared to the effects observed at 32 [degrees]C (P < 0.05). But the longer the duration of exposure to heat shock, the greater the impact on the density of Carsonella during the development of D. citri immatures (Fig. 1B; Table 2). Furthermore, despite the changes in the density of Carsonella at early nymphal stages after heat shock (exposure to 34 [degrees]C for 3 h), the lack of a difference in density at the last instar compared to control nymphs (Fig. 1B) suggests the existence of a control mechanism that can regulate symbiont density in bacteriocytes to a maximum optimum density at the end of the development of immatures (fifth instar).

Profftella density was affected by the temperature and duration of exposure to heat shock in a similar manner to Carsonella (Fig. 1C, D; Table 2). However, no differences (P > 0.05) were observed between the mean densities in the two temperatures during the nymphal stages (Tables 1, 2). Heat shocks for 6, 12 and 24 h at 32 [degrees]C significantly reduced Profftella density in the first and second instars of D. citri. In contrast, the negative effects of heat shock at the egg stage were detected in late instars only after 24 h of exposure (Fig. 1C). Considering only the exposure time, the harmful effects of heat shock occurred in the longer exposure times (Table 2). The remaining exposure times led to an increase in Profftella density after the second instar. Symbiont density stabilized in the fifth instar, except in insects heat shocked for 3 h at 32 [degrees]C (Fig. 1C). The exposure of eggs for 3 or 6 h at 34 [degrees]C resulted in an increase in Profftella density until the fourth instar. However, the density of Profftella decreased in the fifth instar, compared to most of the other treatments (Fig. 1D).

Unlike the primary symbionts, Wolbachia density increased in all heat-shock treatments from the first instar to the fourth instar (Fig. IE, F), with the highest density observed at 32 [degrees]C (Table 2). In addition, the rapid increase in the density of Wolbachia produced a growth curve with a different trend from the other symbionts. The duration of exposure was the most important factor (Tables 1, 2) (P < 0.0001) among the variables tested. Significant growth was observed in response to all treatments, with a trend toward reaching a similar density in the last instar. Nevertheless, the density of Wolbachia was higher in the fifth instar of eggs exposed for 12 and 24 h at 32 [degrees]C or for 6 h at 34 [degrees]C (Fig. 1E, F).


Our results highlight the complexity of the interactions among the insect host, its symbionts, and the environment by describing the effects of short-term heat stress on eggs of Diaphorina citri on the growth responses and densities of their associated symbionts.

The effects of heat stress depend on multiple factors, including behavioral responses and adaptive genetic mechanisms to avoid heat stress. The recovery of symbionts and the phenological synchrony of symbiotic systems after stressful conditions can also have an impact on the negative effects that heat exposure may cause (Potter et al., 2009; Pincebourde and Woods, 2012). Although the densities of Carsonella, Prof-ftella, and Wolbachia were affected in the early developmental stages of D. citri immatures after egg exposure to heat, their densities in last-instar nymphs were similar to those in control nymphs. These results indicate that the effects of short-term heat shock on eggs appear to be much more complex than a simple linear response to an increase in temperature. Previous studies demonstrated that temperatures higher than 32 [degrees]C were unsuitable for the nymph development of D. citri (Nava et al., 2007); however, adults were shown to respond to short periods of exposure at 42 [degrees]C by stimulating the production of HSPs (Marutani-Hert et al., 2010; Hall and Hentz, 2014).

We observed a reduction in Carsonella and Profftella densities (10%--14%) in the bacteriome of D. citri even after 24 h of exposure of eggs to heat shock. These consequences of heat stress agree with the detrimental effects of higher temperatures on psyllid development reported by Nava et al. (2007) or on differential tolerances of immatures and adults of D. citri to high temperature in laboratory conditions (Hall and Hentz, 2014). The variation in the densities of symbionts is suggested to result from disruption of the cellular and molecular processes that coordinate the bacteriome-endosymbiont homeostasis, and not simply a direct negative consequence of exposure to high temperatures on the symbionts. However, further studies at the cellular and molecular levels are required to elucidate their interactions. Thus, differences in the density of Carsonella and Profftella early in the development of immatures tend to decrease as the nymphs develop, until the symbionts reach cell densities in the last instar similar to those in the control nymphs. Rio et al. (2006) also found alterations in the profiles of mutualistic symbionts of tsetse flies during the development of hosts subjected to environmental stresses, with re-establishment of the symbiont densities in the late stages of the host fly.

Wolbachia benefited, with an increase in density, when host eggs were subjected to heat stress. The higher densities reached by Wolbachia in D. citri are not clearly related to the decrease in the densities of Carsonella and Profftella. Wolbachia densities were reduced in field-collected D. citri populations from China and Pakistan when exposed to 40 [degrees]C for 24 h, but density was recovered 3 d after heat exposure (Hussain et al., 2017). Interestingly, Carsonella density that was initially less affected by heat treatment (24 h) declined rapidly after 3 d of exposure compared to Wolbachia (Hussain et al., 2017). The increase in density of Wolbachia in D. citri nymphs eclosed from heat-shocked eggs differs from the expected response of a symbiont known for its thermo-sensitivity (Serbus et al., 2008; Werren et al., 2008; Bor-denstein and Bordenstein, 2011). Unexpectedly, Wolbachia benefited the most from host egg exposure to 32 [degrees]C, developing higher densities in these nymphs than in control nymphs (28 [degrees]C). Therefore, we suggest that the greater and more rapid increase of Wolbachia density, accompanied by the reduction in the densities of primary symbionts, particularly at the initial stages of nymph development, may partially explain the limited development of D. citri at high temperatures (Nava et al., 2007). This is especially expected when D. citri is continuously exposed to high temperatures (Nava et al., 2007), because 32 [degrees]C is close to the upper temperature threshold for many insects and is unsuitable for their optimal growth and development (Hoffmann et al., 2013). The detrimental effects of exposure to high temperatures may be even more evident in species infected with Wolbachia, due to fitness costs for the hosts (Serbus et al., 2008; Werren et al., 2008).

The increase in symbiont density after exposure of D. citri to heat shock differs from what is commonly reported for other host-symbiont systems, including the aphid-Buchnera system (Dunbar et al., 2007; Chen et al., 2009). Aphids harbor one primary symbiont but may also carry secondary symbionts that play different roles in their host's physiology, such as tolerance to high temperature (Russell and Moran, 2006; Heyworth and Ferrari, 2015). However, this beneficial contribution of symbionts to their hosts is dependent on microbe-microbe and host-microbe interactions, as demonstrated for Serratia symbiotiea and the X-type symbiont in the protection of aphids against heat shock (Montllor et al., 2002; Russell and Moran, 2006; Heyworth and Ferrari, 2015, 2016).

The growth curves of all three symbionts that we analyzed were affected by the other symbionts, and the different trends observed for D. citri symbionts in response to heat shock may arise from specific interactions among these symbionts. As reported elsewhere, the specificity at which symbionts regulate each other is linked to the long coevolutionary history between partners (e.g., Rio et al., 2006; Russell et al., 2013; Heyworth and Ferrari, 2015). The high metabolic complementarity suggested between Carsonella and Prqfftella (Sloan and Moran, 2012) could explain their reciprocity and their similar response to heat exposure. Hence, mutualistic symbionts of D. citri may suffer from alterations in the intracellular environment (e.g., reactive oxygen species [ROS] metabolism) of the bacteriome and from disturbances in the functional dependence of Carsonella and Profftella on host metabolism (Naka-bachi, 2009; Sloan and Moran, 2012). Wolbachia responses seemed to be opportunistic (Kondo et al., 2005; Goto et al., 2006), taking advantage of the higher sensitivity of Carsonella and Profftella to heat stress. Nevertheless, others have suggested that the higher sensitivity of Carsonella and Profftella to heat stress may be attributed to their location (e.g., sharing the bacteriome) or to the existence of an obligate complementarity (Hussain et al., 2017). The idea that Wolbachia density responded to changes in the density of the primary symbionts of D. citri is based on the opposite growth trends between Wolbachia and the two mutualistic symbionts after heat shock. Likewise, heat stress is known to influence Wolbachia infection by attenuating host phenotypes (Hurst et al., 2000; Saridaki and Bourtzis, 2010). For example, a brief exposure of eggs of Drosophila melanogaster Meigen, 1830 (Dip-tera, Drosophilidae) to heat shock during early embryogenesis causes morpho-functional changes in Wolbachia, influencing secretory mechanisms and interfering with protein synthesis (Zhukova et al., 2008). Metabolic alterations can include activation of intense lysosomal proteolytic activity and alteration in gene expression, such as stress-related chaperone-encoding genes (Feder and Hofmann, 1999; Nishikori et al., 2009). Furthermore, such conditions may lead to symbiont degradation by lysosomal activity in several tissues and stages of development, including embryonic development (Hinde, 1971; Nishikori et al., 2009). Increased lysosomal degradation in heat-exposed eggs could explain the reduced abundance of symbionts in early nymphs of D. citri. Moreover, the degradation products produced by the lysis of symbionts could serve as a source of nutrients to surviving symbionts and the insect host (Burke et al., 2010; Vigneron et al., 2014). Hence, the availability of nutrients after the recycling of microbial symbionts and the reduced competition for the intracellular environment as a consequence of the lower density of primary symbionts would explain the increase in Wolbachia densities in heat-shocked D. citri. The survival and faster growth of Wolbachia during D. citri nymph development may also result from the ability of Wolbachia to deal with ROS experienced by the host after heat shock (Brennan et al., 2008).

Our data demonstrated both the influence of heat stress on the growth pattern of associated symbionts during the development of the host nymphs and the symbionts' recovery of densities at the last instar similar to those of control nymphs. At this point, we are unable to fully understand the implications of Wolbachia for the physiology of the host or for the mutualist bacteria carried by D. citri. We hypothesize that D. citri has an adaptive response to heat-shock exposure to recover the homeostasis of its associated symbionts. Further studies on D. citri stress physiology, including ROS production and the metabolism of host antioxidant proteins related to heat shock during bacteriome morphogenesis and nymph development, are necessary to clarify the intricate microbe-microbe and microbe-host interactions. Our findings illustrate the potential effects of high temperatures on host-symbiont interactions, and they highlight the importance of better understanding the biology of animal-symbiont associations and their implications for predicting the persistence and distributional shifts of insect species in a changing climate.


This study was supported by the National Council of Technological and Scientific Development (CNPq, scholarship provided to FCAD) and the Sao Paulo Research Foundation (FAPESP grant 2011/50877-0).

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(1) Insect Interactions Laboratory, Department of Entomology and Acarology, Luiz de Queiroz College of Agriculture, University of Sao Paulo, Avenida Padua Dias 11, 13418-900 Piracicaba, Sao Paulo, Brazil; and(2) Avian Science Laboratory, Department of Animal Science, College of Agricultural and Veterinarian Sciences, Sao Paulo State University, Via de Acesso Professor Paulo Donato Castellane, S/N, 14884-900 Jaboticabal, Sao Paulo, Brazil

Received 19 August 2017; Accepted 16 July 2018; Published online 1 October 2018.

(*) To whom correspondence should be addressed. E-mail: ORCID; 0000-0002-2287-0782.

Abbreviations: gDNA. genomic DNA; HSPs. heat-shock proteins; P-symbionts, primary symbionts; qPCR, quantitative polymerase chain reaction; RH, relative humidity; ROS, reactive oxygen species; SQR; sum of squares of residues.
Table 1
Analysis of covariance of the densities of endosymbionts during the
five nymph instars of Diaphorina citri following exposure of the egg
stage to two high temperatures for different periods of time

Dependent variable
and covariables       Type III SQR  F      P        CV (%)  F

Carsonella                                          1.80    156.33
Age                    1.898         4.03   0.0041
Temperature            0.633         5.39   0.0219
Time                   1.405         2.99   0.0214
Profftella             9.113        77.42   0.0001
Wolbachia              0.629         5.35   0.0224
Temperature vs. time   0.931         1.98   0.1018
Profftella                                          1.56    159.58
Age                    1.990         4.27   0.0028
Temperature            0.054         0.47   0.4957
Time                   2.167         4.65   0.0015
Carsonella             9.021        77.42  <0.0001
Wolbachia              1.010         8.67   0.0001
Temperature vs. time   3.954         8.48   0.0001
Wolbachia                                           3.37    126.83
Age                   18.632        15.14  <0.0001
Temperature            3.124        10.15   0.0018
Time                  44.961        36.53  <0.0001
Profftella             1.645         5.35   0.0224
Carsonella             2.669         8.67   0.0038
Temperature vs. time   5.314         4.32   0.0026

Dependent variable
and covariables       P        [R.sup.2]

Carsonella            <0.0001  0.948
Temperature vs. time
Profftella            <0.0001  0.949
Temperature vs. time
Wolbachia             0.0001   0.937
Temperature vs. time

The dependent variables were the number of copies of the molecular
marker for each symbiont investigated per insect-equivalent. The
covariable for age includes first, second, third, fourth, and fifth
instars of D. citri; that for temperature includes 32 [degrees]C
and34[degrees]C; that for time includes the time of exposure of eggs
to heat treatment: 0 (control condition), 3, 6, 12 and 24 h. CV,
coefficient of variation; R'. sum of squares of the model/total sum
of squares; SQR, sum of squares of residues.

Table 2
Analysis of covariance of the copy number of each symbiont per
insect-equivalent (Aeq) as a function of the duration (h) of exposure
to heat shock compared to control (28 [degrees]C)

                   In copy number/[degrees]eq ([+ or -]SD)
Symbiont and heat
shock (h)          32 [degrees]C              34 [degrees]C

0                  19.21 [+ or -] l.41 (aA)   19.21 [+ or -] 1.41 (aA)
3                  18.98 [+ or -] 1.26 (abB)  19.55 [+ or -] 1.26 (aA)
6                  18.95 [+ or -] 1.65 (abA)  19.03 [+ or -] 1.64 (aA)
12                 18.87 [+ or -] 1.93 (bA)   18.84 [+ or -] 1.46 (bA)
24                 18.79 [+ or -] 2.19 (bA)   18.82 [+ or -] 1.12 (bA)
0                  21.75 [+ or -] 1.44 (abA)  21.52 [+ or -] 1.44 (aA)
3                  21.96 [+ or -] 1.45 (aA)   22.31 [+ or -] 1.45 (aA)
6                  21.78 [+ or -] 1.77 (abA)  21.75 [+ or -] 1.16 (aA)
12                 21.78 [+ or -] 1.91 (abA)  21.56 [+ or -] 1.45 (aA)
24                 21.53 [+ or -] 1.84 (bB)   21.65 [+ or -] 1.51 (aA)
0                  15.09 [+ or -] 2.48 (cA)   15.09 [+ or -] 2.48 (bA)
3                  16.37 [+ or -] 2.19 (bA)   16.74 [+ or -] 1.91 (aA)
6                  16.40 [+ or -] 2.21 (bA)   16.37 [+ or -] 2.69 (aA)
12                 17.06 [+ or -] 2.45 (aA)   16.53 [+ or -] 2.05 (aA)
24                 17.27 [+ or -] 2.09 (aA)   16.50 [+ or -] 1.75 (aB)

Symbiont and heat
shock (h)          CV (%)  F       P       [R.sup.2]

Carsonella         1.80    156.33  0.0001  0.948
Profftella         1.56    159.58  0.0001  0.949
Wolbachia          3.37    126.83  0.0001  0.937

For heat shock (h). control conditions (28 [degrees]C) are represented
by a 0-h heat-shock treatment. Means followed by the same lowercase
letters within a column or by the same capital letters within a row do
not differ by the Tukey-Kramer test (P > 0.05).
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Author:Dossi, Fabio Cleisto Alda; Silva, Edney Pereira Da; Consoli, Fernando Luis
Publication:The Biological Bulletin
Article Type:Report
Date:Dec 1, 2018
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