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Developmental stage affects survival of the ectoparasitoid Tamarixia triozae exposed to the fungus Beauveria bassiana
Accepted Manuscript Developmental stage affects survival of the ectoparasitoid Tamarixia triozae exposed to the fungus Beauveria bassiana Fernando Tamayo-Mejía, Patricia Tamez-Guerra, Ariel W. Guzmán-Franco, Ricardo Gomez-Flores PII: DOI: Reference:
S1049-9644(15)30050-5 http://dx.doi.org/10.1016/j.biocontrol.2015.11.006 YBCON 3348
To appear in:
Biological Control
Received Date: Revised Date: Accepted Date:
20 January 2015 9 November 2015 15 November 2015
Please cite this article as: Tamayo-Mejía, F., Tamez-Guerra, P., Guzmán-Franco, A.W., Gomez-Flores, R., Developmental stage affects survival of the ectoparasitoid Tamarixia triozae exposed to the fungus Beauveria bassiana, Biological Control (2015), doi: http://dx.doi.org/10.1016/j.biocontrol.2015.11.006
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Running title: Tamarixia triozae survival following B. bassiana exposure
Developmental stage affects survival of the ectoparasitoid Tamarixia triozae exposed to the fungus Beauveria bassiana
Fernando Tamayo-Mejíaa,‡, Patricia Tamez-Guerraa,*, Ariel W. Guzmán-Francob and Ricardo Gomez-Floresa
a
Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, LIV-DEMI. AP 46-F. San Nicolás
de los Garza, NL. México. 66450. b
Postgrado en Fitosanidad-Entomología y Acarología, Colegio de Postgraduados, Km. 36.5 Carretera
México-Texcoco, Montecillo, Texcoco, Estado de Mexico, Mexico. 56230.
‡Current address: Secretaría de Desarrollo Agroalimentario y Rural. Av. Irrigación 102-Int. 2A, Col. Monte Camargo, Celaya, Guanajuato. México. 38010.
* Corresponding author: Patricia Tamez-Guerra, Río Guadalquivir 401 Ote. Col. Del Valle, San Pedro Garza García, Nuevo León, México. 66220. [email protected]; [email protected]
1
ABSTRACT: Biological control of Bactericera cockerelli using a combination of parasitoids and pathogens has potential. However, their combined use could result in complex multitrophic interactions, the outcomes of which are uncertain. In this study we evaluated the effects of applications of two Beauveria bassiana isolates (pathogen) on the parasitoid Tamarixia triozae during its development on the host, B. cockerelli. Three concentrations (LC20, LC50 and LC90 values against B. cockerelli) of the fungal isolates BB40 and GHA were applied to 4th instar B. cockerelli nymphs that had been successfully parasitized by T. triozae 3, 5, 8 or 12 day earlier at 25 °C. The greatest infection of T. triozae was observed when the calculated LC90 was inoculated, regardless of isolate, and when the parasitoids were 3-5 days old. The lowest infection of T. triozae was observed when they were 8 and 12 days old regardless of isolate or conidial concentration. The highest parasitoid emergence was achieved in the untreated control compared with all other treatments and combinations tested. Within treatments, the highest parasitoid emergence was obtained at the lowest B. bassiana concentration tested and when the pathogen was applied to B. cockerelli supporting 12 day-old parasitoids. Longevity of adult parasitoids that had emerged from B. cockerelli nymphs was greatest in the control treatment, followed by those from nymphs treated with GHA when applied to B. cockerelli supporting 12 dayold parasitoids. In conclusion, biological control of B. cockerelli may require the synchronized use of high B. bassiana concentrations, applied only on T. triozae late pupal stage (8-12 days of development), in order to allow the parasitoid to survive and also achieve good overall pest control. Key words: Multitrophic interactions; Bactericera cockerelli entomopathogenic fungi; ectoparasitoid, potato/tomato psyllid biocontrol agents 2
1. Introduction
Interspecific competition amongst natural enemy species utilizing the same host is considered one of the most important factors determining the structure and dynamics of biological communities (Hochberg, 1991). When two or more species of natural enemies interact in the same habitat, the outcome can be synergistic, additive or antagonistic with respect to host mortality (Roy and Pell, 2000). Regardless of the ultimate effect on host mortality, interactions can influence the overall fitness of the two competing species. For example, transmission and dispersal of species-specific entomopathogenic fungi can be increased by the activity of insect natural enemies (Rännbäck et al., 2015); however, some predators also consume infected hosts, potentially reducing transmission (Agboton et al., 2013). Furthermore, if environmental conditions are particularly favourable for fungi, then insect natural enemies may be directly susceptible to infection by fungal species with broader host ranges (Roy and Pell, 2000; Vega et al., 1995); if parasitoids survive infection, these may have reduced fecundity and/ or longevity and conidia production from infected hosts that are also parasitized may be reduced (Tamayo-Mejía et al., 2015).
The outcomes of interactions between entomopathogenic fungi and beneficial parasitoids depend on different factors including the dose of the fungus inoculated (Nielsen et al., 2005) and the parasitoid developmental stage when the fungus is applied (FuentesContreras et al., 1998; Hamdi et al., 2011; Kim et al., 2005). Prior residence time has been demonstrated to have a significant effect on the outcomes of interactions on other study systems (de Roode et al., 2005; Lohr et al., 2010; Sandoval-Aguilar et al., 2015), where it is 3
normally assumed that a parasite attempting to colonize a host that is already colonized by another parasite could be at a disadvantage (Lohr et al. 2010); very little research has been done on the effect of prior residence on the interactions between parasitoids and entomopathogenic fungi (Furlong and Pell, 2000). Ultimately, all such interactions could modify the effectiveness of the two interacting biological control agents (Furlong and Pell, 2005).
Bactericera cockerelli (Sulc.) (Hemiptera: Triozidae) is a major pest of solanaceous crops and causes severe damage, despite the intensive use of chemicals for its control (Butler and Trumble, 2012; Gharalari et al., 2009; Yang and Liu, 2009). The damage caused when nymphs and adults of this species feed on the phloem of infected plants is manifested as yellowing, curling, growth retardation, aerial tuber formation and purple top in potato (Gharalari et al., 2009). Bactericera cockerelli is the main vector of the disease Zebra Chip in potato, which is caused by the bacterium Candidatus Liberibacter psyllaurus, also known as Candidatus Liberibacter solanacearum. This disease results in severe economic losses (Butler and Trumble, 2012; Gharalari et al., 2009; Yang and Liu, 2009; Secor et al., 2009).
Biological control of B. cockerelli is an important strategy that requires further research before it can be implemented in the field. Tamarixia triozae Burks (Hymenoptera: Eulophidae) is one of the most important parasitoids of B. cockerelli (Jensen, 1957). It is considered a solitary, idiobiont and synovigenic ectoparasitoid (Jervis and Kidd 1986; Rojas et al., 2015). However, some authors are not convinced that T. triozae alone can 4
effectively reduce B. cockerelli populations (either nymphs or adults) (Butler and Trumble, 2012), although others have reported high parasitism rates (up to 85%) in the field (LomelíFlores and Bueno-Partida, 2002), and particularly under greenhouse conditions (Weber, 2013; McGregor, 2013). For this reason, the use of entomopathogenic fungi in combination with T. triozae has been suggested as a useful strategy for biological control of this pest (Lacey et al., 2009; Lomelí-Flores and Bueno-Partida, 2002; Tamayo-Mejía et al., 2014). The nymphs of B. cockerelli are highly susceptible to B. bassiana s.l. isolates under laboratory and field conditions (Tamayo-Mejía et al., 2014, 2015). The use of combinations of control agents to reduce B. cockerelli populations is based on the philosophy of integrated pest management (Gharalari et al., 2009) and must be carefully evaluated to avoid antagonistic effects that may reduce overall pest control (Roy and Pell, 2000).
Information regarding the outcomes of interactions between B. bassiana s.l. and the parasitoid T. triozae in populations of B. cockerelli nymphs is scarce. However, recently Tamayo-Mejía et al. (2015) reported that T. triozae was unable to recognize B. cockerelli nymphs previously infected with B. bassiana, resulting in a significant proportion of parasitoids becoming infected after attempting to parasitize infected B. cockerelli nymphs; this suggests the potential for long term antagonism between the two species. The present study provides further information to better understand the interactions between B. bassiana and T. triozae in B. cockerelli populations. Rather than considering the implications for parasitoids attacking B. cockerelli nymphs already infected by B. bassiana, here we consider the implications of applying B. bassiana to B. cockerelli nymphs already parasitized by T. triozae. 5
2. Material and Methods
2.1 Beauveria bassiana isolates
The isolates of B. bassiana used were BB40 from the culture collection of the Laboratory for Production of Beneficial Organisms (Guanajuato State Committee for Plant Health, Irapuato, Guanajuato, Mexico), and GHA (ARSEF 6444) (Mycotrol, Laverlam Int. Corp.), which is available as a commercial control product. Both isolates were grown as monosporic cultures in 90-mm diameter Petri dishes containing 20 mL of Sabouraud dextrose agar supplemented with 1% yeast extract (Bioxon®) at 25°C in total darkness for 10 days. Prior to experimentation, viability of conidia was estimated using the methods described by Inglis et al. (2012). Germination was always above 95%.
Conidial suspensions were prepared by scraping mycelia and conidia, using a sterile metal spatula, from Petri dish cultures in to 50 ml centrifuge tubes each containing 50 ml of sterile 0.03% Tween 80 (Sigma Aldrich Química, SA de CV, Toluca, Estado de México). Each tube was vortexed (Vortex-Genie® 2 Model G560, Scientific Industries, Inc. USA) for 5 min; the suspension was filtered into another clean centrifuge tube through a double layer of sterile cloth placed in a 5 cm diameter glass funnel. The concentration of conidia per milliliter of each suspension was determined using a Neubauer haemocytometer and adjusted to the concentrations required.
6
2.2 Rearing B. cockerelli and T. triozae colonies
The B. cockerelli colony was maintained by repeated subculture on chilli pepper Capsicum annum (L.) var. Rebelde (Seminis®). The T. triozae colony was maintained on fourth instar nymphs of B. cockerelli, also on chilli pepper plants. Both colonies were reared using the same methods and environmental conditions as described by TamayoMejia et al. (2015). Tamarixia triozae is a parasitoid for which the egg stage lasts approximately 1.5 d, larval stages last approximately 3.5 d and the pupal stage lasts approximately 5 d. at 26 °C (Rojas et al., 2015) Therefore, the average time from egg to adult emergence is 12 d; female longevity was 20 d under the same conditions (Rojas et al., 2015).
2.3 Effect of B. bassiana inoculation of T. triozae juvenile stages
A population of approximately 150 4th instar B. cockerelli nymphs feeding on chilli pepper plants, was introduced in to a clean gauze-covered cage (50 x 50 x100 cm in length, width and height respectively) with 50 10-day-old T. triozae adults and incubated at 25 ± 2°C, 60 ± 10% relative humidity in 16:8 h light:dark regime, for 24 hours. After this time the parasitized B. cockerelli nymphs were transferred to a new cage without parasitoids and incubated under the same conditions. The process was repeated on subsequent days and in this way we obtained, on the same day, cohorts of B. cockerelli nymphs containing the following developmental stages of the parasitoid: larvae (3 and 5 days after parasitism), pupae (8 days after parasitism), and adults that had formed but not emerged (12 days after 7
parasitism). From each parasitism category, groups of 12 nymphs per leaf, were confined inside sterile 9 cm wide x 2 cm high Petri dishes, thus our experimental unit was a Petri dish containing a chilli pepper leaf with 12 fourth instar nymphs of B. cockerelli that had each been parasitized by T. triozae. The petiole of each leaf was inserted through a hole in the lid of an Eppendorf tube containing 1 mL of sterilized water to allow the leaf to remain turgid. Nine Petri dishes were prepared for nymphs containing parasitoids from each of the development times (36 dishes in total). Previously estimated LC20, LC50 and LC90 concentrations (against B. cockerelli) for each B. bassiana isolate (six Petri dishes for the two isolates) (Tamayo-Mejía et al., 2015; Table 1) were applied to Petri dishes of B. cockerelli nymphs parasitized by T. triazae for either 3, 5, 8 or 12 days (24 Petri dishes). For each developmental stage a further three dishes (12 Petri dishes) were inoculated with 0.03% Tween (control treatments). Fungal inoculations were made as described by Tamayo-Mejía et al. (2015) using 1 mL of conidial suspension (or 0.03% Tween in controls) for each dish of nymphs. Each group of treated nymphs on leaves were then transferred to new clean 9 cm Petri dishes with a filter paper (Whatman No. 1) disk in the base onto which 1 mL of sterile distilled water was applied to maintain humidity. This amount of distilled water produced a damp filter paper but it was not saturated. Each dish was sealed with parafilm and incubated at 25 °C in a 16:8 light: dark regime.
The number of parasitoids infected was recorded every day for 15 days in each of the isolate × concentration × parasitoid development time (3, 5, 8 and 12 days) treatment combinations and the control treatment. To determine whether parasitoids had become infected, all treated and control nymphs of B. cockerelli were observed individually under a 8
stereomicroscope every day to look for infected parasitoids. Using a fine brush, each nymph was carefully lifted leaving its abdominal section exposed. This allowed us to observe the presence of parasitoid larvae on host surfaces; parasitoid larvae developed a red colour when initial infection was taking place, which was later confirmed when sporulation began. Although in the results section we focus our description on infection of parasitoids, we want to emphasize that these infection rates were the same for the B. cockerelli nymphs; if the parasitoid became infected then so did the B. cockerelli nymph. The number of adult parasitoids that emerged from the B. cockerelli nymphs was recorded every day for 15 days for treatments with 3 and 5 day old parasitoid developmental stages, and for 6 days for treatments with 8 and 12 day old of parasitoid developmental stages. Each emerged adult parasitoid was placed individually in a Petri dish with filter paper. Drops of 99% corn syrup (Vita Real®, Alimentos Naturistas Siglo XXI, México, D.F.) as a food source, and a piece of moistened cotton as a water source were added. The longevity (days) of the parasitoids was determined by daily observation until all the parasitoids died. Dead parasitoids were placed into clean 9 cm diameter Petri dishes with moistened filter paper (1 ml of distilled water was added) to determine whether mortality was due to fungal infection, as evidenced by characteristic external sporulation.
The experiment was conducted under a completely randomized design where all treatments were made on the same day (36 dishes), and the complete experiment was repeated on four separate occasions (for a total of 144 dishes) where each occasion represented an independent repetition of the experiment.
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2.3.2 Statistical analyses
Data on infection (representing the final cumulative infection values at the end of the incubation time) of T. triozae were analyzed using logistic regression assuming a binomial distribution. Infection was estimated as the proportion of the total number of parasitoids observed. Where necessary, the presence of more variability in the data than expected under the binomial assumptions (over-dispersion) was allowed. The main effects and interactions for the factorial set of treatments defined by isolates (combining conidial concentration and developmental stage of the parasitoid), conidial concentration (combining isolates and developmental stage of the parasitoid) and developmental stages of the parasitoid (combining isolates and conidial concentrations) were estimated and tested. Control data were excluded from the analysis as no mortality was recorded in the controls. The number of emerged parasitoids was analyzed using ANOVA with a similar treatment structure as described above; in addition, the effect of developmental stage at the time of inoculation, on the emergence of adult parasitoids, was also assessed in the control treatments. The longevity of the emerged adult parasitoids was analyzed with the same treatment structure using ANOVA, but because different numbers of parasitoids emerged for each treatment and replicate, parasite longevity was analyzed using an unbalanced design. GenStat statistical package V. 8.0 was used for all analyses (Payne et al., 2005).
3. Results
3.1 Infection of T. triozae by B. bassiana 10
Overall, infection rates of the parasitoids by the two B. bassiana isolates were not significantly different (F1,
72
= 0.07, P = 0.796). When infection of parasitoids was
compared amongst the three different conidia concentrations, significant differences were observed (F2, 72 = 27.48, P <0.001); the highest proportion of parasitoids became infected in the LC90 treatment, regardless of isolate (F2, 72 = 1.36, P = 0.264) (Fig. 1).
The highest proportion of parasitoids that became infected was observed in treatments where B. bassiana was inoculated after only three days of parasitoid development (F3,
72
= 100.33, P <0.001), (Fig. 1). There was no significant interaction
between the fungal isolate used and the parasitoid developmental stage (F3, 72 = 2.10, P = 0.108) or with the conidial concentration (F2, 72 = 1.36, P = 0.264). Overall, higher levels of infection were observed in early-stage parasitoid larvae (three and five days old) compared with later stages, and this effect was greater effect for isolate BB40 (Fig 1A) than the GHA isolate (Fig 1B); however, the greatest level of infection was observed for isolate GHA but only in the LC90 treatment (Fig. 1B). Both isolates caused least infection when the parasitoids had developed for 8 or 12 days (Fig. 1).
3.2 T. triozae adult emergence from B. bassiana-treated and untreated B. cockerelli nymphs
Significantly more parasitoids emerged from the control treatment than all the other treatments combined (F1,
113
= 50.64, P <0.001; Fig. 2A). Within the fungal treatments,
there were no significant differences between the two isolates tested (F1,
113
= 1.47, P = 11
0.229). There was a significant difference in parasitoid emergence amongst the different fungal concentrations (F2,
113
= 11.59, P = <0.001), where the highest number emerging
occurred when the LC20 concentration was applied, followed by the LC50 and LC90 concentrations (Fig. 2 B, C). This result was the same for the two fungal isolates tested (F2, 113 =
1.4, P = 0.250). There was a significant effect of parasitoid developmental stage at the
time of inoculation on the number of adult parasitoids that emerged (F3, 113 = 70.12, P = <0.001); as the developmental time at inoculation increased, so the number of parasitoids emerging increased with the greatest number of parasitoids emerging when the fungal treatments were made after 12 days of development, and the smallest number emerging occurred when the fungal treatments were made after only 3 days of development (Fig. 2B, C), and these results was regardless of the fungal isolate used (F3, 113 = 1.96, P = 0.124).
Comparing amongst control treatments there was also a significant difference in the number of parasitoids emerging in relation to the developmental times at which the Tween 80 was applied, although the number of adults emerging was not directly related to the developmental stage of the parasitoids (F3,
113
= 4.15, P = 0.008). The greatest mean
number of parasitoids emerging was observed after 12 days of development (10.83±0.568, d.f. 113), followed by five days (9.92±0.568, d.f. 113), eight days (8.83±0.568, d.f. 113) and three days of development (8.42±0.568, d.f. 113).
3.3 Longevity of T. triozae adults emerging from B. bassiana-treated and untreated B. cockerelli nymphs treated with B. bassiana
12
Overall, the longevity of adult parasitoids emerging from control treatments was significantly longer than for parasitoids emerging from the fungal treatments (F1,
943
=
23.35, P <0.001; Fig. 3A). Parasitoid longevity was not significantly different amongst treatments with different parasitoid developmental times, either for the control treatments (F3, 943 = 2.27, P = 0.079) or the fungus treatments (F3, 943 = 2.52, P = 0.057) (Fig. 3B, C). Within the fungus-inoculated treatments, the longevity of the adult parasitoids was not significantly different for the two isolates used (F1, concentrations of conidia used (F2,
943
943
= 0.28, P = 0.600) and the three
= 1.34, P = 0.262) (Fig. 3B, C). There was a
significant interaction between developmental time and isolate that affected longevity (F3, 943
= 2.78, P = 0.040), where the greatest longevity was observed after 8 days of
development for isolate BB40 (Fig 3B) and 12 days of development for isolate GHA (Fig 3C). When all the dead adult parasitoids from the fungal treatments were incubated, we generally observed sporulation on only a few. The percentage of sporulation increased according to the LCs with 4, 7 and 26% for the LC20, LC50 and LC90 respectively. For the different developmental stage of the parasitoid, we observed a decrease in the proportion sporulating as the developmental stage at the time of inoculation increased: 21, 14, 9 and 8% sporulated in the 3, 5, 8 and 12 days of development treatments respectively.
4. Discussion
In a previous study BB40, was shown to be more virulent against 4th instar B. cockerelli nymphs than the GHA isolate, but less virulent than GHA against adult T. triozae (TamayoMejía et al., 2015). In the current study we found that the two B. bassiana isolates were 13
similar to each other in terms of infection rates against parasitoids; Tamayo-Mejía et al. (2015) reported differences in virulence against the adult parasitoid using the same isolates. However, we consider that a direct comparison between the results from these two studies is not possible because there were two fundamental differences; each study used different developmental stages of the parasitoid (adult vs larvae), and secondly, the first study made dose-response assays while in the current study we tested the effect of only three conidial concentrations. However, it is important to note that while the overall infection rates observed in parasitoids were statistically similar, we did find a significant effect of conidial concentration, with the LC90 concentration, estimated against B. cockerelli by TamayoMejía et al. (2015), causing the greatest parasitoid mortality.
Due to their speed of kill, entomopathogenic fungi generally have a competitive advantage over parasitoids when they attack the host at the same time (Furlong and Pell, 2005). Our study showed that, even when parasitoids had the advantage of prior residency on the host, the fungus still infected them resulting in reduced emergence of adult parasitoids, especially when treated with high concentrations of conidia. However, when the parasitoid was very close to emerging as an adult (after 12 days of development) when the inoculum was applied, there was no evident effect of B. bassiana and the emergence rate was similar to the control treatment (Fig. 2). Overall we observed that the length of time that the parasitoid had been developing on B. cockerelli nymphs strongly influenced its susceptibility to infection; the longer the developmental time of the parasitoid, the less likely for it to become infected (Fig. 1). Although this result is consistent with other host/ fungus/ parasitoid studies (Fransen and van Lenteren 1994; Fuentes-Contreras et al., 1998; 14
Hamdi et al., 2011; Kim et al., 2005), it is difficult for us to provide a specific reason for this effect. It has been reported that the production of phenoloxidase may increase during host development (González-Santoyo and Córdoba-Aguilar, 2012), which may contribute to why later stages of T. triozae were less susceptible. Furthermore, the parasitoid may be secreting immunosuppressive substances in response to the defense mechanisms of its host (Hartzer et al., 2005), since with increasing age the cuticle thickness also increases, which is the first barrier that the pathogen must breach to achieve infection (Hajek and St Leger, 1994).
The overall emergence of adult parasitoids was not affected when fungus was inoculated after 12 days of development (Fig 2B, C), as emergence seemed similar to the control treatment (Fig 2A). This result suggests that time is a key factor in the outcomes of the interactions between and parasitoids, and entomopathogenic fungi.
Parasitoid emergence from inoculated hosts is commonly used as the indicator of parasitoid survival in studies evaluating the susceptibility of natural enemies to entomopathogenic fungal infection (e.g. Avery et al., 2008; Ren et al., 2010), since infected parasitoids cannot emerge from their hosts. However, in our study, despite many parasitoids emerging as adults, their longevity was reduced compared to parasitoids that emerged from non-fungal-inoculated B. cockerelli nymphs (Fig 3A), so measuring emergence only could underestimate the negative effects of the fungus. We observed that only a relatively low percentage of parasitoids emerging from fungal-treated nymphs became infected, where the highest percentage was obtained in the highest conidial 15
concentrations (Fig 2b) and early stages of parasitoid development (Fig 2c). The reduction in the longevity of adults that emerged in fungus-inoculated treatments but did not sporulate after death (i.e. were not infected), could be related to the potentially lower quality of the infected prey they ingested compared with uninfected prey (Lee et al., 2004). When an entomopathogen temporally coincides with a parasitoid on the same host, interspecific competition for food can inevitably arises (Furlong and Pell, 2005). Our results are similar to those reported by Brodeur and Rosenheim (2000) and FuentesContreras et al., (1998), but differ from the results of Aiuchi et al., (2012), who reported that longevity of Aphidius colemani (Dalman) females emerging from Aphis gossypii Glover nymphs treated with Lecanicillium spp. were no different to the longevity of control parasitoids. This may be due to the specificity of the fungi used or the concentrations applied as, in our study, the longevity of emerging parasitoids was greater when the concentration of B. bassiana applied was low, and when the developmental stage of the parasitoid was more advanced at the time of fungal exposure (Fig 3B, C). This observation was also reported by Ren et al., (2010), who evaluated the use of L. muscarium and Eretmocerus sp. nr furuhashii Rose & Zolnerowich against Bemisia tabaci (Gennadius).
It has been reported that T. triozae could not recognize infected B. cockrelli nymphs, as parasitoids became infected when they manipulated fungal-inoculated nymphs (Tamayo et al., 2015). This suggests a potential negative interaction between these two biological control agents. However, if the nymphs were parasitized first, as we demonstrated in this study, the parasitoids did have a greater opportunity to survive, especially if the parasitoid had been developing for 12 days before any fungus was inoculated. It would be important 16
to follow up our results by studying other factors such as the parasitism rates achieved by adult parasitoids that had emerged from B. bassiana-infected B. cockerelli nymphs, and also fungal transmission from parasitized hosts. This would provide further information on the potential for using these two biological control agents together.
Overall, our results showed that the two B. bassiana isolates studied here, had negative effects on the survival of T. triozae by causing direct infection of larvae parasitizing B. cockerelli nymphs. However, if a time separation between applications of the two agents could be allowed, we consider that a greater reduction in the pest population could be achieved compared with when each agent is used separately. Our results also show that it is important to release the parasitoid first; doing the contrary results in greater negative impacts on the parasitoid because it cannot effectively distinguish between healthy and infected nymphs (Tamayo-Mejía et al., 2015).
5. Conclusion
It is possible to use the pathogen B. bassiana in combination with the parasitoid T. triozae to control B. cockerelli populations, but the parasitoid must be released first. In this scenario, it is only feasible to apply the selected B. bassiana between 8 to 11 days after release of T. triozae to ensure parasitoid survival is not compromised. The use of this time synchronization may limit competition between these two control agents; however, field evaluations are required to confirm this.
17
Acknowledgements
This study was supported by Laboratorio de Inmunología y Virología, DEMI, FCB-UANL, by Fundación Guanajuato Produce A.C. (grant 542/09 to PT); and by CONACYT (grant 155771 to PTG and scholarship 79390 to FTM). We thank Suzanne Clark for her valuable help on the statistical analyses and Robert W. Behle and Julissa Ek-Ramos for manuscript draft review.
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Mauchline, N. A., Stannard, K. A., Zydenbos, S. M. 2013. Evaluation of selected entomopathogenic fungi and bio-insecticides against Bactericera cockerelli (Hemiptera). N. Z. Plant Protection. 66, 324-332. http://www.nzpps.org/journal/66/nzpp_663240.pdf McGregor, R. R. 2013. Bactericera cockerelli (Sulc), Tomato/Potato Psyllid (Hemiptera: Triozidae). in: Mason, P. G., Gillespie, D. R. (Eds.). Biological Control Programmes in Canada 2001-2012. CAB International. Oxfordshire, UK. pp: 107-112. Morales, A. S. I., Martínez, A. M., Figueroa, J. I., Espino, A. M., Chavarrieta, Y. J. M., Ortíz, R. R., Rodríguez, E. ChL., Pineda, S. 2013. Life parameters of the synovigenic parasitoid Tamarixia triozae (Hymenoptera: Eulophidae). Rev. Colomb. Entomol. 39, 243-249. Nielsen, C., Skovgård, H., Steenberg, T. 2005. Effect of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) on survival and reproduction of the filth fly parasitoid, Spalangia cameroni (Hymenoptera: Pteromalidae). Environ. Entomol. 34, 133-139. Payne, R., Murray, D., Harding, S., Baird, D., Soutar, D. 2005. GenStat for Windows, 8th Edition, Introduction. VSN International, Hemel Hempstead. Rännbäck, L.M., Cotes, B., Anderson, P., Rämert, B., Meyling, N.V. 2015. Mortality risk from entomopathogenic fungi affects oviposition behaviour in the parasitoid wasp Trybliographa rapae. J. Invertebr. Pathol., 124, 78-86. Ren, S. X., Ali, S., Huang, Z., Wu, J. H. 2010. Lecanicillium muscarium as microbial insecticide against whitefly and its interaction with other natural enemies. in: MéndezVilas, A. (Ed.). Current Research, Technology and Education Topics in Applied 22
Microbiology and Microbial Biotechnology. FORMATEX. Microbiology Series No. 2. Vol. 1. Spain. pp: 339-348. Rojas, P., Rodríguez-Leyva, E., Lomeli-Flores, J. R., Liu, T. X. 2015. Biology and life history of Tamarixia triozae, a parasitoid of the potato psyllid Bactericera cockerelli. BioControl, 60, 27-35. DOI 10.1007/s10526-014-9625-4. Roy, H. E., Pell, J. K. 2000. Interactions between entomopathogenic fungi and other natural enemies: implications for biological control. Biocontrol Sci. Technol., 10, 737-752. DOI: 10.1080/09583150020011708. Secor, G. A., Rivera, V. V., Abad, J. A., Lee, I. M., Clover, G. R. G., Liefting, L. W., Li, X., De Boer, S. H. 2009. Association of “Candidatus Liberibacter solanacearum” with zebra chip disease of potato established by graft and psyllid transmission, electron microscopy and PCR. Plant Dis. 39, 574-583. DOI: http://dx.doi.org/10.1094/PDIS-936-0574. Tamayo-Mejía, F., Tamez-Guerra P., Guzmán-Franco, A. W., Gomez-Flores, R. 2015. Can Beauveria bassiana Bals. (Vuill) (Ascomycetes: Hypocreales) and Tamarixia triozae (Burks) (Hymenoptera: Eulophidae) be used together for improved biological control of Bactericera cockerelli (Hemiptera: Triozidae)? Biological Control, 90: 42-48. DOI:10.1016/J.BIOCONTROL.2015.05.014. Tamayo-Mejía, F., Tamez-Guerra, P., Guzmán-Franco, A. W., Gomez-Flores, R., CruzCota, L. R. 2014. Efficacy of entomopathogenic fungi (Hypocreales) for Bactericera cockerelli (Sulc.) (Hemiptera: Triozidae) control in the laboratory and field. Southwestern Entomol. 39, 271-283. DOI: http://dx.doi.org/10.3958/059.039.0205.
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Vega, F. E., Dowd, P. F., Bartelt, R. J. 1995. Dissemination of microbial agents using an autoinoculating device and several insect species as vectors. Biol Control 5, 545-552. DOI: 10.1006/bcon.1995.1064. Weber, D. C. 2013. Biological control of potato insect pests. in: Giordanengo, P., Vincent, C. H., Alyokhin, A. (Eds.). Insect Pests of Potato. Global Perspectives on Biology and Management. Academic Press. Oxford, U. K. pp: 399-437. Yang, X. B., Liu, T. X. 2009. Life history and life tables of potato psyllid Bactericera cockerelli (Homoptera: Psyllidae) on eggplant and bell pepper. Environ. Entomol. 38, 1661-1667. DOI: http://dx.doi.org/10.1603/022.038.0619.
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Tables
Table 1. LC20, LC50 and LC90 values estimated previously for isolates BB40 and GHA against B. cockerelli (Tamayo-Mejía et al., 2015), which were used in this study to inoculate B. cockerelli nymphs parasitized with T. triozae. LC=Lethal concentration.
BB40
GHA
LC20 (conidia mL-1)
3.61 x 104
7.46x105
LC50 (conidia mL-1)
9.54 x 105
1.97x107
LC90 (conidia mL-1)
1.39 x 108
2.88x109
25
Figure legends
Figure 1. Proportion of Tamarixia triozae infected by the two Beauveria bassiana isolates BB40 (A) and GHA (B). Four experimental units (Petri dishes) were used to produce each bar. The LC concentrations applied were those determined previously against B. cockerelli.Error bars represent 95% confidence intervals back-transformed from the logistic scale.
Figure 2. Average number of Tamarixia triozae adults emerging from 4th instar Bactericera cockerelli nymphs. (A) Control treatment vs B. bassiana-treated nymphs (combining results from the two isolates). (B) Nymphs treated with isolate BB40 at three different concentrations and carrying parasitoids at different developmental stages. (C) Nymphs treated with isolate GHA at three different concentrations and carrying parasitoids at different developmental stages. For Figure A, the number of experimental units (Petri dishes) used is showed at the bottom of each bar, for Figures B and C, four experimental units (Petri dishes) were used to produce each bar. The LC concentrations applied were those determined previously against B. cockerelli. Error bars represent SEM.
Figure 3. Longevity (days) of Tamarixia triozae adults emerging from Bactericera cockerelli nymphs. (A) Untreated vs B. bassiana-treated B. cockerelli nymphs. (B) Nymphs treated with isolate BB40 at three different concentrations and carrying parasitoids at different developmental stages. (C) Nymphs treated with isolate GHA at three different concentrations and carrying parasitoids at different developmental stages. The number of 26
adults parasitoids used is showed at the bottom of each bar. The LC concentrations applied were those determined previously against B. cockerelli. Error bars represent SEM.
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28
29
31
Highlights • • • • •
B. cockerelli nymphs parasitized with T. triozae were treated with B. bassiana Fungus was applied after parasitoids had developed for 3, 5, 8 or 12 days Infection rate decreased with parasitoid age at the highest fungal dose More adult parasitoids emerged at the lower fungal doses Adult parasitoids from untreated hosts lived longer than those from treated hosts
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S1049-9644(15)30050-5 http://dx.doi.org/10.1016/j.biocontrol.2015.11.006 YBCON 3348
To appear in:
Biological Control
Received Date: Revised Date: Accepted Date:
20 January 2015 9 November 2015 15 November 2015
Please cite this article as: Tamayo-Mejía, F., Tamez-Guerra, P., Guzmán-Franco, A.W., Gomez-Flores, R., Developmental stage affects survival of the ectoparasitoid Tamarixia triozae exposed to the fungus Beauveria bassiana, Biological Control (2015), doi: http://dx.doi.org/10.1016/j.biocontrol.2015.11.006
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Running title: Tamarixia triozae survival following B. bassiana exposure
Developmental stage affects survival of the ectoparasitoid Tamarixia triozae exposed to the fungus Beauveria bassiana
Fernando Tamayo-Mejíaa,‡, Patricia Tamez-Guerraa,*, Ariel W. Guzmán-Francob and Ricardo Gomez-Floresa
a
Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, LIV-DEMI. AP 46-F. San Nicolás
de los Garza, NL. México. 66450. b
Postgrado en Fitosanidad-Entomología y Acarología, Colegio de Postgraduados, Km. 36.5 Carretera
México-Texcoco, Montecillo, Texcoco, Estado de Mexico, Mexico. 56230.
‡Current address: Secretaría de Desarrollo Agroalimentario y Rural. Av. Irrigación 102-Int. 2A, Col. Monte Camargo, Celaya, Guanajuato. México. 38010.
* Corresponding author: Patricia Tamez-Guerra, Río Guadalquivir 401 Ote. Col. Del Valle, San Pedro Garza García, Nuevo León, México. 66220. [email protected]; [email protected]
1
ABSTRACT: Biological control of Bactericera cockerelli using a combination of parasitoids and pathogens has potential. However, their combined use could result in complex multitrophic interactions, the outcomes of which are uncertain. In this study we evaluated the effects of applications of two Beauveria bassiana isolates (pathogen) on the parasitoid Tamarixia triozae during its development on the host, B. cockerelli. Three concentrations (LC20, LC50 and LC90 values against B. cockerelli) of the fungal isolates BB40 and GHA were applied to 4th instar B. cockerelli nymphs that had been successfully parasitized by T. triozae 3, 5, 8 or 12 day earlier at 25 °C. The greatest infection of T. triozae was observed when the calculated LC90 was inoculated, regardless of isolate, and when the parasitoids were 3-5 days old. The lowest infection of T. triozae was observed when they were 8 and 12 days old regardless of isolate or conidial concentration. The highest parasitoid emergence was achieved in the untreated control compared with all other treatments and combinations tested. Within treatments, the highest parasitoid emergence was obtained at the lowest B. bassiana concentration tested and when the pathogen was applied to B. cockerelli supporting 12 day-old parasitoids. Longevity of adult parasitoids that had emerged from B. cockerelli nymphs was greatest in the control treatment, followed by those from nymphs treated with GHA when applied to B. cockerelli supporting 12 dayold parasitoids. In conclusion, biological control of B. cockerelli may require the synchronized use of high B. bassiana concentrations, applied only on T. triozae late pupal stage (8-12 days of development), in order to allow the parasitoid to survive and also achieve good overall pest control. Key words: Multitrophic interactions; Bactericera cockerelli entomopathogenic fungi; ectoparasitoid, potato/tomato psyllid biocontrol agents 2
1. Introduction
Interspecific competition amongst natural enemy species utilizing the same host is considered one of the most important factors determining the structure and dynamics of biological communities (Hochberg, 1991). When two or more species of natural enemies interact in the same habitat, the outcome can be synergistic, additive or antagonistic with respect to host mortality (Roy and Pell, 2000). Regardless of the ultimate effect on host mortality, interactions can influence the overall fitness of the two competing species. For example, transmission and dispersal of species-specific entomopathogenic fungi can be increased by the activity of insect natural enemies (Rännbäck et al., 2015); however, some predators also consume infected hosts, potentially reducing transmission (Agboton et al., 2013). Furthermore, if environmental conditions are particularly favourable for fungi, then insect natural enemies may be directly susceptible to infection by fungal species with broader host ranges (Roy and Pell, 2000; Vega et al., 1995); if parasitoids survive infection, these may have reduced fecundity and/ or longevity and conidia production from infected hosts that are also parasitized may be reduced (Tamayo-Mejía et al., 2015).
The outcomes of interactions between entomopathogenic fungi and beneficial parasitoids depend on different factors including the dose of the fungus inoculated (Nielsen et al., 2005) and the parasitoid developmental stage when the fungus is applied (FuentesContreras et al., 1998; Hamdi et al., 2011; Kim et al., 2005). Prior residence time has been demonstrated to have a significant effect on the outcomes of interactions on other study systems (de Roode et al., 2005; Lohr et al., 2010; Sandoval-Aguilar et al., 2015), where it is 3
normally assumed that a parasite attempting to colonize a host that is already colonized by another parasite could be at a disadvantage (Lohr et al. 2010); very little research has been done on the effect of prior residence on the interactions between parasitoids and entomopathogenic fungi (Furlong and Pell, 2000). Ultimately, all such interactions could modify the effectiveness of the two interacting biological control agents (Furlong and Pell, 2005).
Bactericera cockerelli (Sulc.) (Hemiptera: Triozidae) is a major pest of solanaceous crops and causes severe damage, despite the intensive use of chemicals for its control (Butler and Trumble, 2012; Gharalari et al., 2009; Yang and Liu, 2009). The damage caused when nymphs and adults of this species feed on the phloem of infected plants is manifested as yellowing, curling, growth retardation, aerial tuber formation and purple top in potato (Gharalari et al., 2009). Bactericera cockerelli is the main vector of the disease Zebra Chip in potato, which is caused by the bacterium Candidatus Liberibacter psyllaurus, also known as Candidatus Liberibacter solanacearum. This disease results in severe economic losses (Butler and Trumble, 2012; Gharalari et al., 2009; Yang and Liu, 2009; Secor et al., 2009).
Biological control of B. cockerelli is an important strategy that requires further research before it can be implemented in the field. Tamarixia triozae Burks (Hymenoptera: Eulophidae) is one of the most important parasitoids of B. cockerelli (Jensen, 1957). It is considered a solitary, idiobiont and synovigenic ectoparasitoid (Jervis and Kidd 1986; Rojas et al., 2015). However, some authors are not convinced that T. triozae alone can 4
effectively reduce B. cockerelli populations (either nymphs or adults) (Butler and Trumble, 2012), although others have reported high parasitism rates (up to 85%) in the field (LomelíFlores and Bueno-Partida, 2002), and particularly under greenhouse conditions (Weber, 2013; McGregor, 2013). For this reason, the use of entomopathogenic fungi in combination with T. triozae has been suggested as a useful strategy for biological control of this pest (Lacey et al., 2009; Lomelí-Flores and Bueno-Partida, 2002; Tamayo-Mejía et al., 2014). The nymphs of B. cockerelli are highly susceptible to B. bassiana s.l. isolates under laboratory and field conditions (Tamayo-Mejía et al., 2014, 2015). The use of combinations of control agents to reduce B. cockerelli populations is based on the philosophy of integrated pest management (Gharalari et al., 2009) and must be carefully evaluated to avoid antagonistic effects that may reduce overall pest control (Roy and Pell, 2000).
Information regarding the outcomes of interactions between B. bassiana s.l. and the parasitoid T. triozae in populations of B. cockerelli nymphs is scarce. However, recently Tamayo-Mejía et al. (2015) reported that T. triozae was unable to recognize B. cockerelli nymphs previously infected with B. bassiana, resulting in a significant proportion of parasitoids becoming infected after attempting to parasitize infected B. cockerelli nymphs; this suggests the potential for long term antagonism between the two species. The present study provides further information to better understand the interactions between B. bassiana and T. triozae in B. cockerelli populations. Rather than considering the implications for parasitoids attacking B. cockerelli nymphs already infected by B. bassiana, here we consider the implications of applying B. bassiana to B. cockerelli nymphs already parasitized by T. triozae. 5
2. Material and Methods
2.1 Beauveria bassiana isolates
The isolates of B. bassiana used were BB40 from the culture collection of the Laboratory for Production of Beneficial Organisms (Guanajuato State Committee for Plant Health, Irapuato, Guanajuato, Mexico), and GHA (ARSEF 6444) (Mycotrol, Laverlam Int. Corp.), which is available as a commercial control product. Both isolates were grown as monosporic cultures in 90-mm diameter Petri dishes containing 20 mL of Sabouraud dextrose agar supplemented with 1% yeast extract (Bioxon®) at 25°C in total darkness for 10 days. Prior to experimentation, viability of conidia was estimated using the methods described by Inglis et al. (2012). Germination was always above 95%.
Conidial suspensions were prepared by scraping mycelia and conidia, using a sterile metal spatula, from Petri dish cultures in to 50 ml centrifuge tubes each containing 50 ml of sterile 0.03% Tween 80 (Sigma Aldrich Química, SA de CV, Toluca, Estado de México). Each tube was vortexed (Vortex-Genie® 2 Model G560, Scientific Industries, Inc. USA) for 5 min; the suspension was filtered into another clean centrifuge tube through a double layer of sterile cloth placed in a 5 cm diameter glass funnel. The concentration of conidia per milliliter of each suspension was determined using a Neubauer haemocytometer and adjusted to the concentrations required.
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2.2 Rearing B. cockerelli and T. triozae colonies
The B. cockerelli colony was maintained by repeated subculture on chilli pepper Capsicum annum (L.) var. Rebelde (Seminis®). The T. triozae colony was maintained on fourth instar nymphs of B. cockerelli, also on chilli pepper plants. Both colonies were reared using the same methods and environmental conditions as described by TamayoMejia et al. (2015). Tamarixia triozae is a parasitoid for which the egg stage lasts approximately 1.5 d, larval stages last approximately 3.5 d and the pupal stage lasts approximately 5 d. at 26 °C (Rojas et al., 2015) Therefore, the average time from egg to adult emergence is 12 d; female longevity was 20 d under the same conditions (Rojas et al., 2015).
2.3 Effect of B. bassiana inoculation of T. triozae juvenile stages
A population of approximately 150 4th instar B. cockerelli nymphs feeding on chilli pepper plants, was introduced in to a clean gauze-covered cage (50 x 50 x100 cm in length, width and height respectively) with 50 10-day-old T. triozae adults and incubated at 25 ± 2°C, 60 ± 10% relative humidity in 16:8 h light:dark regime, for 24 hours. After this time the parasitized B. cockerelli nymphs were transferred to a new cage without parasitoids and incubated under the same conditions. The process was repeated on subsequent days and in this way we obtained, on the same day, cohorts of B. cockerelli nymphs containing the following developmental stages of the parasitoid: larvae (3 and 5 days after parasitism), pupae (8 days after parasitism), and adults that had formed but not emerged (12 days after 7
parasitism). From each parasitism category, groups of 12 nymphs per leaf, were confined inside sterile 9 cm wide x 2 cm high Petri dishes, thus our experimental unit was a Petri dish containing a chilli pepper leaf with 12 fourth instar nymphs of B. cockerelli that had each been parasitized by T. triozae. The petiole of each leaf was inserted through a hole in the lid of an Eppendorf tube containing 1 mL of sterilized water to allow the leaf to remain turgid. Nine Petri dishes were prepared for nymphs containing parasitoids from each of the development times (36 dishes in total). Previously estimated LC20, LC50 and LC90 concentrations (against B. cockerelli) for each B. bassiana isolate (six Petri dishes for the two isolates) (Tamayo-Mejía et al., 2015; Table 1) were applied to Petri dishes of B. cockerelli nymphs parasitized by T. triazae for either 3, 5, 8 or 12 days (24 Petri dishes). For each developmental stage a further three dishes (12 Petri dishes) were inoculated with 0.03% Tween (control treatments). Fungal inoculations were made as described by Tamayo-Mejía et al. (2015) using 1 mL of conidial suspension (or 0.03% Tween in controls) for each dish of nymphs. Each group of treated nymphs on leaves were then transferred to new clean 9 cm Petri dishes with a filter paper (Whatman No. 1) disk in the base onto which 1 mL of sterile distilled water was applied to maintain humidity. This amount of distilled water produced a damp filter paper but it was not saturated. Each dish was sealed with parafilm and incubated at 25 °C in a 16:8 light: dark regime.
The number of parasitoids infected was recorded every day for 15 days in each of the isolate × concentration × parasitoid development time (3, 5, 8 and 12 days) treatment combinations and the control treatment. To determine whether parasitoids had become infected, all treated and control nymphs of B. cockerelli were observed individually under a 8
stereomicroscope every day to look for infected parasitoids. Using a fine brush, each nymph was carefully lifted leaving its abdominal section exposed. This allowed us to observe the presence of parasitoid larvae on host surfaces; parasitoid larvae developed a red colour when initial infection was taking place, which was later confirmed when sporulation began. Although in the results section we focus our description on infection of parasitoids, we want to emphasize that these infection rates were the same for the B. cockerelli nymphs; if the parasitoid became infected then so did the B. cockerelli nymph. The number of adult parasitoids that emerged from the B. cockerelli nymphs was recorded every day for 15 days for treatments with 3 and 5 day old parasitoid developmental stages, and for 6 days for treatments with 8 and 12 day old of parasitoid developmental stages. Each emerged adult parasitoid was placed individually in a Petri dish with filter paper. Drops of 99% corn syrup (Vita Real®, Alimentos Naturistas Siglo XXI, México, D.F.) as a food source, and a piece of moistened cotton as a water source were added. The longevity (days) of the parasitoids was determined by daily observation until all the parasitoids died. Dead parasitoids were placed into clean 9 cm diameter Petri dishes with moistened filter paper (1 ml of distilled water was added) to determine whether mortality was due to fungal infection, as evidenced by characteristic external sporulation.
The experiment was conducted under a completely randomized design where all treatments were made on the same day (36 dishes), and the complete experiment was repeated on four separate occasions (for a total of 144 dishes) where each occasion represented an independent repetition of the experiment.
9
2.3.2 Statistical analyses
Data on infection (representing the final cumulative infection values at the end of the incubation time) of T. triozae were analyzed using logistic regression assuming a binomial distribution. Infection was estimated as the proportion of the total number of parasitoids observed. Where necessary, the presence of more variability in the data than expected under the binomial assumptions (over-dispersion) was allowed. The main effects and interactions for the factorial set of treatments defined by isolates (combining conidial concentration and developmental stage of the parasitoid), conidial concentration (combining isolates and developmental stage of the parasitoid) and developmental stages of the parasitoid (combining isolates and conidial concentrations) were estimated and tested. Control data were excluded from the analysis as no mortality was recorded in the controls. The number of emerged parasitoids was analyzed using ANOVA with a similar treatment structure as described above; in addition, the effect of developmental stage at the time of inoculation, on the emergence of adult parasitoids, was also assessed in the control treatments. The longevity of the emerged adult parasitoids was analyzed with the same treatment structure using ANOVA, but because different numbers of parasitoids emerged for each treatment and replicate, parasite longevity was analyzed using an unbalanced design. GenStat statistical package V. 8.0 was used for all analyses (Payne et al., 2005).
3. Results
3.1 Infection of T. triozae by B. bassiana 10
Overall, infection rates of the parasitoids by the two B. bassiana isolates were not significantly different (F1,
72
= 0.07, P = 0.796). When infection of parasitoids was
compared amongst the three different conidia concentrations, significant differences were observed (F2, 72 = 27.48, P <0.001); the highest proportion of parasitoids became infected in the LC90 treatment, regardless of isolate (F2, 72 = 1.36, P = 0.264) (Fig. 1).
The highest proportion of parasitoids that became infected was observed in treatments where B. bassiana was inoculated after only three days of parasitoid development (F3,
72
= 100.33, P <0.001), (Fig. 1). There was no significant interaction
between the fungal isolate used and the parasitoid developmental stage (F3, 72 = 2.10, P = 0.108) or with the conidial concentration (F2, 72 = 1.36, P = 0.264). Overall, higher levels of infection were observed in early-stage parasitoid larvae (three and five days old) compared with later stages, and this effect was greater effect for isolate BB40 (Fig 1A) than the GHA isolate (Fig 1B); however, the greatest level of infection was observed for isolate GHA but only in the LC90 treatment (Fig. 1B). Both isolates caused least infection when the parasitoids had developed for 8 or 12 days (Fig. 1).
3.2 T. triozae adult emergence from B. bassiana-treated and untreated B. cockerelli nymphs
Significantly more parasitoids emerged from the control treatment than all the other treatments combined (F1,
113
= 50.64, P <0.001; Fig. 2A). Within the fungal treatments,
there were no significant differences between the two isolates tested (F1,
113
= 1.47, P = 11
0.229). There was a significant difference in parasitoid emergence amongst the different fungal concentrations (F2,
113
= 11.59, P = <0.001), where the highest number emerging
occurred when the LC20 concentration was applied, followed by the LC50 and LC90 concentrations (Fig. 2 B, C). This result was the same for the two fungal isolates tested (F2, 113 =
1.4, P = 0.250). There was a significant effect of parasitoid developmental stage at the
time of inoculation on the number of adult parasitoids that emerged (F3, 113 = 70.12, P = <0.001); as the developmental time at inoculation increased, so the number of parasitoids emerging increased with the greatest number of parasitoids emerging when the fungal treatments were made after 12 days of development, and the smallest number emerging occurred when the fungal treatments were made after only 3 days of development (Fig. 2B, C), and these results was regardless of the fungal isolate used (F3, 113 = 1.96, P = 0.124).
Comparing amongst control treatments there was also a significant difference in the number of parasitoids emerging in relation to the developmental times at which the Tween 80 was applied, although the number of adults emerging was not directly related to the developmental stage of the parasitoids (F3,
113
= 4.15, P = 0.008). The greatest mean
number of parasitoids emerging was observed after 12 days of development (10.83±0.568, d.f. 113), followed by five days (9.92±0.568, d.f. 113), eight days (8.83±0.568, d.f. 113) and three days of development (8.42±0.568, d.f. 113).
3.3 Longevity of T. triozae adults emerging from B. bassiana-treated and untreated B. cockerelli nymphs treated with B. bassiana
12
Overall, the longevity of adult parasitoids emerging from control treatments was significantly longer than for parasitoids emerging from the fungal treatments (F1,
943
=
23.35, P <0.001; Fig. 3A). Parasitoid longevity was not significantly different amongst treatments with different parasitoid developmental times, either for the control treatments (F3, 943 = 2.27, P = 0.079) or the fungus treatments (F3, 943 = 2.52, P = 0.057) (Fig. 3B, C). Within the fungus-inoculated treatments, the longevity of the adult parasitoids was not significantly different for the two isolates used (F1, concentrations of conidia used (F2,
943
943
= 0.28, P = 0.600) and the three
= 1.34, P = 0.262) (Fig. 3B, C). There was a
significant interaction between developmental time and isolate that affected longevity (F3, 943
= 2.78, P = 0.040), where the greatest longevity was observed after 8 days of
development for isolate BB40 (Fig 3B) and 12 days of development for isolate GHA (Fig 3C). When all the dead adult parasitoids from the fungal treatments were incubated, we generally observed sporulation on only a few. The percentage of sporulation increased according to the LCs with 4, 7 and 26% for the LC20, LC50 and LC90 respectively. For the different developmental stage of the parasitoid, we observed a decrease in the proportion sporulating as the developmental stage at the time of inoculation increased: 21, 14, 9 and 8% sporulated in the 3, 5, 8 and 12 days of development treatments respectively.
4. Discussion
In a previous study BB40, was shown to be more virulent against 4th instar B. cockerelli nymphs than the GHA isolate, but less virulent than GHA against adult T. triozae (TamayoMejía et al., 2015). In the current study we found that the two B. bassiana isolates were 13
similar to each other in terms of infection rates against parasitoids; Tamayo-Mejía et al. (2015) reported differences in virulence against the adult parasitoid using the same isolates. However, we consider that a direct comparison between the results from these two studies is not possible because there were two fundamental differences; each study used different developmental stages of the parasitoid (adult vs larvae), and secondly, the first study made dose-response assays while in the current study we tested the effect of only three conidial concentrations. However, it is important to note that while the overall infection rates observed in parasitoids were statistically similar, we did find a significant effect of conidial concentration, with the LC90 concentration, estimated against B. cockerelli by TamayoMejía et al. (2015), causing the greatest parasitoid mortality.
Due to their speed of kill, entomopathogenic fungi generally have a competitive advantage over parasitoids when they attack the host at the same time (Furlong and Pell, 2005). Our study showed that, even when parasitoids had the advantage of prior residency on the host, the fungus still infected them resulting in reduced emergence of adult parasitoids, especially when treated with high concentrations of conidia. However, when the parasitoid was very close to emerging as an adult (after 12 days of development) when the inoculum was applied, there was no evident effect of B. bassiana and the emergence rate was similar to the control treatment (Fig. 2). Overall we observed that the length of time that the parasitoid had been developing on B. cockerelli nymphs strongly influenced its susceptibility to infection; the longer the developmental time of the parasitoid, the less likely for it to become infected (Fig. 1). Although this result is consistent with other host/ fungus/ parasitoid studies (Fransen and van Lenteren 1994; Fuentes-Contreras et al., 1998; 14
Hamdi et al., 2011; Kim et al., 2005), it is difficult for us to provide a specific reason for this effect. It has been reported that the production of phenoloxidase may increase during host development (González-Santoyo and Córdoba-Aguilar, 2012), which may contribute to why later stages of T. triozae were less susceptible. Furthermore, the parasitoid may be secreting immunosuppressive substances in response to the defense mechanisms of its host (Hartzer et al., 2005), since with increasing age the cuticle thickness also increases, which is the first barrier that the pathogen must breach to achieve infection (Hajek and St Leger, 1994).
The overall emergence of adult parasitoids was not affected when fungus was inoculated after 12 days of development (Fig 2B, C), as emergence seemed similar to the control treatment (Fig 2A). This result suggests that time is a key factor in the outcomes of the interactions between and parasitoids, and entomopathogenic fungi.
Parasitoid emergence from inoculated hosts is commonly used as the indicator of parasitoid survival in studies evaluating the susceptibility of natural enemies to entomopathogenic fungal infection (e.g. Avery et al., 2008; Ren et al., 2010), since infected parasitoids cannot emerge from their hosts. However, in our study, despite many parasitoids emerging as adults, their longevity was reduced compared to parasitoids that emerged from non-fungal-inoculated B. cockerelli nymphs (Fig 3A), so measuring emergence only could underestimate the negative effects of the fungus. We observed that only a relatively low percentage of parasitoids emerging from fungal-treated nymphs became infected, where the highest percentage was obtained in the highest conidial 15
concentrations (Fig 2b) and early stages of parasitoid development (Fig 2c). The reduction in the longevity of adults that emerged in fungus-inoculated treatments but did not sporulate after death (i.e. were not infected), could be related to the potentially lower quality of the infected prey they ingested compared with uninfected prey (Lee et al., 2004). When an entomopathogen temporally coincides with a parasitoid on the same host, interspecific competition for food can inevitably arises (Furlong and Pell, 2005). Our results are similar to those reported by Brodeur and Rosenheim (2000) and FuentesContreras et al., (1998), but differ from the results of Aiuchi et al., (2012), who reported that longevity of Aphidius colemani (Dalman) females emerging from Aphis gossypii Glover nymphs treated with Lecanicillium spp. were no different to the longevity of control parasitoids. This may be due to the specificity of the fungi used or the concentrations applied as, in our study, the longevity of emerging parasitoids was greater when the concentration of B. bassiana applied was low, and when the developmental stage of the parasitoid was more advanced at the time of fungal exposure (Fig 3B, C). This observation was also reported by Ren et al., (2010), who evaluated the use of L. muscarium and Eretmocerus sp. nr furuhashii Rose & Zolnerowich against Bemisia tabaci (Gennadius).
It has been reported that T. triozae could not recognize infected B. cockrelli nymphs, as parasitoids became infected when they manipulated fungal-inoculated nymphs (Tamayo et al., 2015). This suggests a potential negative interaction between these two biological control agents. However, if the nymphs were parasitized first, as we demonstrated in this study, the parasitoids did have a greater opportunity to survive, especially if the parasitoid had been developing for 12 days before any fungus was inoculated. It would be important 16
to follow up our results by studying other factors such as the parasitism rates achieved by adult parasitoids that had emerged from B. bassiana-infected B. cockerelli nymphs, and also fungal transmission from parasitized hosts. This would provide further information on the potential for using these two biological control agents together.
Overall, our results showed that the two B. bassiana isolates studied here, had negative effects on the survival of T. triozae by causing direct infection of larvae parasitizing B. cockerelli nymphs. However, if a time separation between applications of the two agents could be allowed, we consider that a greater reduction in the pest population could be achieved compared with when each agent is used separately. Our results also show that it is important to release the parasitoid first; doing the contrary results in greater negative impacts on the parasitoid because it cannot effectively distinguish between healthy and infected nymphs (Tamayo-Mejía et al., 2015).
5. Conclusion
It is possible to use the pathogen B. bassiana in combination with the parasitoid T. triozae to control B. cockerelli populations, but the parasitoid must be released first. In this scenario, it is only feasible to apply the selected B. bassiana between 8 to 11 days after release of T. triozae to ensure parasitoid survival is not compromised. The use of this time synchronization may limit competition between these two control agents; however, field evaluations are required to confirm this.
17
Acknowledgements
This study was supported by Laboratorio de Inmunología y Virología, DEMI, FCB-UANL, by Fundación Guanajuato Produce A.C. (grant 542/09 to PT); and by CONACYT (grant 155771 to PTG and scholarship 79390 to FTM). We thank Suzanne Clark for her valuable help on the statistical analyses and Robert W. Behle and Julissa Ek-Ramos for manuscript draft review.
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24
Tables
Table 1. LC20, LC50 and LC90 values estimated previously for isolates BB40 and GHA against B. cockerelli (Tamayo-Mejía et al., 2015), which were used in this study to inoculate B. cockerelli nymphs parasitized with T. triozae. LC=Lethal concentration.
BB40
GHA
LC20 (conidia mL-1)
3.61 x 104
7.46x105
LC50 (conidia mL-1)
9.54 x 105
1.97x107
LC90 (conidia mL-1)
1.39 x 108
2.88x109
25
Figure legends
Figure 1. Proportion of Tamarixia triozae infected by the two Beauveria bassiana isolates BB40 (A) and GHA (B). Four experimental units (Petri dishes) were used to produce each bar. The LC concentrations applied were those determined previously against B. cockerelli.Error bars represent 95% confidence intervals back-transformed from the logistic scale.
Figure 2. Average number of Tamarixia triozae adults emerging from 4th instar Bactericera cockerelli nymphs. (A) Control treatment vs B. bassiana-treated nymphs (combining results from the two isolates). (B) Nymphs treated with isolate BB40 at three different concentrations and carrying parasitoids at different developmental stages. (C) Nymphs treated with isolate GHA at three different concentrations and carrying parasitoids at different developmental stages. For Figure A, the number of experimental units (Petri dishes) used is showed at the bottom of each bar, for Figures B and C, four experimental units (Petri dishes) were used to produce each bar. The LC concentrations applied were those determined previously against B. cockerelli. Error bars represent SEM.
Figure 3. Longevity (days) of Tamarixia triozae adults emerging from Bactericera cockerelli nymphs. (A) Untreated vs B. bassiana-treated B. cockerelli nymphs. (B) Nymphs treated with isolate BB40 at three different concentrations and carrying parasitoids at different developmental stages. (C) Nymphs treated with isolate GHA at three different concentrations and carrying parasitoids at different developmental stages. The number of 26
adults parasitoids used is showed at the bottom of each bar. The LC concentrations applied were those determined previously against B. cockerelli. Error bars represent SEM.
27
28
29
31
Highlights • • • • •
B. cockerelli nymphs parasitized with T. triozae were treated with B. bassiana Fungus was applied after parasitoids had developed for 3, 5, 8 or 12 days Infection rate decreased with parasitoid age at the highest fungal dose More adult parasitoids emerged at the lower fungal doses Adult parasitoids from untreated hosts lived longer than those from treated hosts
33