Increased plasma selenium levels correlate with ... - naldc - USDA

Journal of Invertebrate Pathology 95 (2007) 77–83 www.elsevier.com/locate/yjipa

Increased plasma selenium levels correlate with elevated resistance of Heliothis virescens larvae against baculovirus infection Kent S. Shelby ¤, Holly J.R. Popham USDA, Agricultural Research Service, Biological Control of Insects Research Laboratory, Columbia, MO 65203, USA Received 14 December 2005; accepted 8 January 2007 Available online 19 January 2007

Abstract We reported that dietary selenium (Se) impacted the growth and development of Trichoplusia ni reared for many generations on diet containing extremely low levels of Se. Larvae had an elevated resistance to per os infection with a baculovirus. In this study, we examine how dietary Se (in the form of selenite) aVects the growth, development, and Se content of Heliothis virescens that have been laboratory reared for less than two years. Larvae fed a commercial tobacco budworm diet supplemented with greater than 20 ppm Se grew at a slower rate than insects fed lower levels of Se and had an increase in the amount of Se sequestered in pupae. Larvae fed diets containing from 10–60 ppm Se exhibited elevated plasma concentrations of the micronutrient and increased plasma virucidal activity against Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). Larvae reared on diet supplemented with 10 or 60 ppm Se until the onset of the penultimate instar were then infected per os or by injection with increasing concentrations of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Larvae fed dietary Se and infected with occluded virus per os displayed a signiWcantly lower mortality compared with infected larvae not fed Se. Our results suggest that dietary Se levels are directly correlated with plasma Se levels, and that plasma Se levels are in turn correlated with baculovirus resistance. © 2007 Elsevier Inc. All rights reserved. Keywords: Heliothis virescens; Selenium; Biological control; Resistance; Nutritional immunology; Autographa californica multiple nucleopolyhedrovirus; Helicoverpa zea single nucleopolyhedrovirus

1. Introduction The micronutrient selenium (Se) plays a vital role in the resistance of vertebrates against viral infection (Beck et al., 2004). Se is a cofactor required for the activity of a number of selenoenzymes involved in the stress response, and the maintenance of high tissue antioxidant levels, which may contribute to a more robust antimicrobial and antiviral defense (Beck et al., 2004). We documented the possibility that Se may impact the eYcacy of microbial biological control agents by tracking the mortality of Se-supplemented cabbage loopers, Trichoplusia ni, to the baculovirus Autographa californica multiple nucleopolyhedrovirus

*

Corresponding author. Fax: +1 573 875 4261. E-mail address: [email protected] (K.S. Shelby).

0022-2011/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2007.01.001

(AcMNPV) (Popham et al., 2005). We reported that the presence of Se in larval tissues during viral infection lowers the susceptibility of larvae, particularly in initial mortality. On the basis of this information we hypothesized that dietary Se supplementation would elevate tissue Se concentrations. We further hypothesized that elevated tissue Se concentrations would correlate with increased resistance against a baculovirus challenge. Here we report on experiments designed to test both hypotheses. 2. Materials and methods 2.1. Insects and insect diets Heliothis virescens eggs were received from the North Carolina State University Department of Entomology Insectary. The insectary colony was established from Weld

78

K.S. Shelby, H.J.R. Popham / Journal of Invertebrate Pathology 95 (2007) 77–83

insects in July of 2002. Larvae were reared individually on an artiWcial wheat germ based diet (Catalog # F9781B, BioServe, Frenchtown, NJ) under a photoperiod of 14 h:10 h (L:D) at 55% relative humidity at 28 °C (Popham et al., 2005; Shelby and Popham, 2006). Diets were supplemented with Se in the form of Na2SeO3 at 10, 20, 60, and 125 ppm. Diet without added Se was considered to be 0 ppm Se. Pupation, adult emergence and mortality data were collected daily on insects reared on increasing levels of Se. Midpoint pupation and emergence times were determined by the ViStat 2.1 program (Hughes et al., 1986).

(Popham et al., 2004). HzAM1 cells were seeded at 5 £ 104 cells ml¡1 in 96-well plates (BD Falcon, Franklin Lakes, NJ) and allowed to attach for 1 h. The cells were infected with dilutions of virus/plasma or virus/PBS at dilutions of 10¡1 to 10¡6 and plates were incubated for 1 week at 28 °C. The plate wells were then scored positive, if polyhedra were visible within two or more cells, or negative for viral infection, and the results were used to calculate the viral titer as the tissue culture infectious dose per ml (TCID50 ml¡1) of inoculum. When indicated, statistical comparisons were done using the SigmaStat program (SPSS Inc., Chicago, IL).

2.2. Insect cells and virus 2.5. Bioassays Two Cell lines, an H. zea cell line (HzAM-1) and a Spodoptera frugiperda cell line (Sf21), were maintained as monolayers at 28 °C in Excel 401 medium (JRH Biosciences, Lenexa, KS) supplemented with 10% fetal bovine serum (Integen Co., Purchase, NY). Wild-type H. zea single nucleopolyhedrovirus (HzSNPV) isolate was used and ampliWed in HzAM1 cells for the virucidal assay (Popham et al., 2004). For larval bioassays, the L1 variant of AcMNPV was used and budded virus was ampliWed and titered in Sf21 for injection assays (O’Reilly et al., 1992). 2.3. Selenium content determination Individual pupae, plasma samples, or small portions of diet were frozen, placed in pre-tared vials, oven dried at 65 °C and the dry masses calculated. Se determinations were performed by the University of Missouri Research Reactor by instrumental neutron activation analysis using a modiWcation of the method described in (McKown and Morris, 1978). Se concentrations are expressed as ppm (g Se/gm dry mass) and are presented as standard Tukey box plots showing the mean, median, and the 5th/95th percentile ranges (SigmaPlot 8.0, SPSS, Inc. Chicago, IL). 2.4. Plasma in vitro virucidal assay Hemolymph was collected and virucidal activity in larval H. virescens plasma was quantitated by endpoint dilution assay as detailed (Popham et al., 2004). In short, hemolymph from early Wfth instar larvae was collected directly into a chilled 1.5 ml microcentrifuge tube containing ice cold, sterile phosphate buVered saline (PBS) (50 mM NaHPO4, pH 6.8). Hemolymph was adjusted to a Wnal dilution of 1:10 by addition of cold PBS after which hemocytes were removed by microcentrifugation at 8000 rpm for three minutes. The plasma supernatant was sterilized by centrifugation at 6000 rpm for three minutes through a 0.65 m Millipore Ultrafree™-MC centrifugal Wlter (Millipore, Inc., Bedford, MA). H. virescens plasma dilutions were combined with HzSNPV at a ratio of 3:1 (v/v), gently mixed and allowed to incubate at 20 °C for 1 h. PBS was used as a control in the absence of plasma. Viral titers of these incubations were determined by end-point dilution assay

Larvae fed diet with diVerent levels of Se were challenged either per os or by injection with varying concentrations of AcMNPV at the onset of the fourth instar. For per os bioassays, H. virescens larvae were fed polyhedra isolated and sucrose gradient puriWed. Larvae were infected by the droplet feeding method (Popham et al., 2005) with doses ranging from 1 £ 103 to 1 £ 107 polyhedra/ml of AcMNPV and placed in individual cups with diet. Injection bioassays were performed by injecting larvae in a proleg with 2 l of budded virus diluted to a treatment range of 0.0127 to 12.7 pfu/l (Lapointe et al., 2004). Injections were done manually using a 5 l Hamilton Syringe with a Wxed 26 gauge needle and a beveled noncoring needle point. Larvae were monitored two or three times daily for death for 10 days and the times the larvae were monitored was recorded. Per os and injection bioassays each contained Wve doses of virus with 30 larvae/dose. Per os assays were repeated four times and injection assays twice. 2.6. Statistical analysis of bioassays With multiple factors and the potential of interactions among these factors, something other than LC50 is needed for the analysis of bioassay data. Use of a Generalized Linear Model (Nelder and Wedderburn, 1972) allows us to deal with the complexity of a multi-factor experimental design with a binary response. In this paper, the probability of death was modeled with Generalized Estimating Equations (GEE) (Liang and Zeger, 1986), which allows an analysis that deals with the non-independence of observations through time. Graphic examination of the data showed there were few deaths before 120 h and the number of deaths remained fairly constant at more than 80% after 192 h. Because this consistency of no deaths or almost all deaths masked the eVects of viral dose, Se dose and time only data from 120 to 192 h were included in the analyses. In this study, there were two factorial experiments (per os and injected) with the factors: (1) Se dose level, (2) viral dose level, and (3) time, in each experiment. Analytical tools exist which can take into consideration that a factorial experiment was performed and also model the suspected


79

The time required for larvae to progress to pupation and adult emergence increased with increasing concentrations of dietary Se (Fig. 1). Larvae fed basal diet attained 50% pupation after 12.2 § 0.1 days and 50% had emerged as adults by 25.1 § 0.2 days. At dietary Se levels of 10, 20, 60, and 125 ppm 50% pupation occurred at 12.2 § 0.1, 12.7 § 0.1, 15.1 § 0.3, and 17.5 § 0.2 days, while 50% emergence was measured at 25.0 § 0.2, 25.5 § 0.2, 27.5 § 0.4, and 31.7 § 0.3 days. Larvae reared on diet containing 60 and 125 ppm Se developed signiWcantly slower than larvae reared on lower levels of Se. In addition, mortality increased as dietary Se increased (Fig. 1). Pupae from larvae reared on diet containing 0, 10, 20, 60, and 125 ppm Se did not signiWcantly diVer in weight, either between groups

reared on diVerent levels of Se or between males and females within each group reared on diVerent levels of Se. The mean (§SE) amount of Se in 0, 10, 20, 60, and 125 ppm in Se-supplemented diet measured by instrumental neutron activation analysis was 0.27 § 0.01, 10.48 § 0.13, 21.4 § 0.37, 62.02 § 1.12, and 128.11 § 1.68 ppm, respectively (n D 9). The amount of sequestered Se in pupae from larvae reared on 0 ppm Se diets was higher than the amount available in the diet (compare 0.27 § 0.01 ppm Se in diet to 0.66 § 0.08 ppm in male pupae and 0.68 § 0.09 ppm in female pupae). However, as dietary Se increased, the amount of Se available in the diet far exceeded the amount sequestered in pupae (Fig. 2). Fig. 3a shows that the plasma level of Se increased with increasing dietary Se. Plasma collected from early 5th instar H. virescens larvae reared on Se-supplemented diet exhibited signiWcantly diVerent virucidal activities (Fig. 3b). As dietary Se increased, the virucidal activity increased. To rule out the possibility that Se added to the assay caused the changes in virucidal activity, Se was added to media at 10, 20, 60, and 125 ppm and the virucidal activity assayed. Virucidal activity in Se-supplemented media did not change (data not shown). In a second control experiment, Se was added to plasma from 5th instar larvae raised on 0 ppm Se diet to a Wnal concentration of 10, 20, 60, and 125 ppm Se. Again, virucidal activity was not diVerent between the 0 ppm plasma and the Se-augmented plasma samples (data not shown). When the statistical analyses of bioassays included Se dose, viral dose, hours post-infection, and the 2- and 3-way interactions between these parameters, viral injection showed only viral dose and hours (time) signiWcantly diVerent from controls (p 60.05). In the per os experiment, viral dose, hours and the Se dose by viral dose interaction were signiWcant (p 60.05).

Fig. 1. Increasing levels of dietary Se resulted in delayed pupation and adult emergence times of H. virescens larvae compared to those reared on control diet. Diets were supplemented with 10, 20, 60, and 125 ppm selenite. Larvae were placed on the diets as neonates (n D 80).

Fig. 2. Pupal sequestration of dietary Se increased as the amount of dietary selenium fed during larval development increased. Se levels were determined by instrumental neutron activation analysis. Data are presented in standard Tukey box plot format showing the 5th and 95th percentile range, mean (dashed line), and median (solid line) (n D 3). Values indicated with the same letters are not signiWcantly diVerent.

correlation between observations of a cohort over time. PROC GENMOD (SAS, 2004) was used to do that for each of the experiments. GEE were Wtted using various covariance structures to model the non-independence of the observations over time using methods found in Stokes et al. (2000). Initially the explanatory eVects used in the model were Se dose, viral dose, hours, with the interactions: Se dose by viral dose, Se dose by hours, viral dose by hours and Se dose by viral dose by hours. The covariance structures tried were Unstructured (UN), Compound-Symmetric (CS), and Autoregressive Order 1 [AR (1)]. From these analyses the best Wt was obtained with AR (1), with the logit transformation. For both experiments separate analyses were run for each time with viral dose, Se dose and viral dose by Se dose interaction included in the model. The probabilities (odds ratios) of death were computed for increasing viral and Se dosages. 3. Results

80


Fig. 3. Plasma Se levels correlate with elevated resistance of Heliothis virescens larvae against HzSNPV challenge. (a) Plasma Se levels following dietary supplementation with selenite were determined by instrumental neutron activation analysis (n D 2, mean § SE). (b) Virucidal activity in plasma is elevated as the level of dietary Se supplementation increases (n D 4, mean § SE).

With the injection experiment, when each time was analyzed separately, the viral dose was signiWcant for all times but Se dose was only signiWcant for 96 and 120 h (4 and 5 days) (Table 1). The odds of death were slightly greater than 1:1 for an increase in Se dose of 10 U for both times where Se dose was signiWcant. The odds of death for an increase of 1 U in the viral dose ranged from 2:1 to 10:1 with the higher values occurring at 168 and 192 h (7 and 8 days). When the data from the per os infection experiment was analyzed separately at each time the interaction of viral dose with Se dose was signiWcant at 120, 144, 168, 192, and 210 h (5, 6, 7, 8, and 9 days) (Fig. 4; Table 1). Viral dose was signiWcant at all times and Se dose was signiWcant at 96 h (4 days) in addition to the times when the interaction was signiWcant. The odds of death for an increase of 100,000 U viral dose was 1.03:1 for all times from 96 to 240 h and the odds ratio for Se dose was always less than 1:1 (range of 0.81:1 to 0.95:1) for an increase of 10 U Se dose. 4. Discussion Increased susceptibility of H. zea larvae to baculovirus infection under a deWciency of speciWc nutrients such as

sterols (Mac Donald and Ritter, 1988), or of micronutrients such as Se on T. ni larvae (Popham et al., 2005), as well as the stimulatory eVect of Zn on Manduca sexta hemocytes (Willot and Tran, 2002), and of Fe on Galleria mellonella hemocytes (Dunphy et al., 2002), suggests that the immunocompetence of herbivorous insects may be aVected by a range of foliar nutrients and phytochemicals. In support of this hypothesis, plant derived inhibitors of phenoloxidase have been documented (Dowd, 1988, 1999; Kubo et al., 2003) leading to speculation that insects feeding upon these compounds would exhibit suppressed melanization capabilities, and consequently be immunosuppressed (Popham et al., 2004; Ourth, 2004; Shelby and Popham, 2006). Soil Se levels vary by geographic region and this variation correlates with the Se status of large herbivores such as beef cattle (Baghour et al., 2002; Hintze et al., 2002). Dietary Se was assimilated from selenate irrigated alfalfa by the larval beet armyworm, Spodoptera exigua, to selenite and to selenomethionine (Vickerman et al., 2004). Dietary Se is further converted to a nucleophilic redox cofactor, selenocysteine (Sec), which is co-translationally incorporated into oxidoreductases as the “21st amino acid”. These enzymes primarily are involved in the stress response and maintenance of high tissue antioxidant levels, which may contribute to a more robust antiviral defense. Paradoxically, however, most insect orthologs of these selenoproteins utilize the catalytically inferior Cys in their active sites (Bauer et al., 2003; Gromer et al., 2003; Castellano et al., 2005), and Drosophila deletion mutants incapable of incorporating Sec into selenoproteins appear to suVer no deWciencies of defense against oxidative damage by reactive oxygen species (Hirosawa-Takamori et al., 2004). The impact of larval S. exigua Se accumulation on higher trophic levels was demonstrated by a two day delay in development of the parasitoid Cotesia margeniventris, and similar delays in development of the generalist pentatomid predator Podisus maculiventris (Vickerman and Trumble, 2003; Vickerman et al., 2004). High levels of Se provided systemically may actually act as feeding deterrents for some herbivores (Hanson et al., 2003, 2004), and an intriguing case of a Se-resistant strain of Plutella xylostella has been recently reported (Freeman et al., 2006). Foliar fertilizer application is an eVective means for Se biofortiWcation of wheat and other crops intended for human consumption (Genc et al., 2005; Lyons et al., 2005). These results suggest that widespread adoption of this agronomic biofortiWcation method might actually elevate pest insect resistance to biological control agents such as baculoviruses, while adversely aVecting populations of predators and parasitoids. Natural baculovirus infections progress from infected midgut epithelial foci to tracheoblasts, the hemocoel, hemocytes, and other tissues (Engelhard et al., 1994; Trudeau et al., 2001; Haas-Stapleton et al., 2003). Interactions of occluded baculovirus particles with enzymes and foliar compounds within the highly alkaline midgut lumen, passage through the peritrophic membrane, and interactions


81

Table 1 EVect of Se and viral dose by per os infection and injection of HzSNPV into H. virescens larvae Interval

Per os infection

Injection

2a

p>2

4 days (96 h) Viral_Dose Se_Dose Viral_Dose ¤ Se_Dose

111.20 5.42 1.59

2

1.03 (1.02–1.03) 0.81 (0.67–0.98)

231.49 16.97