Sporulation Of Beauveria On Cadavers A Infestansafter Infection At Different Temperatures And Doses Of Inoculum

Journal of Invertebrate Pathology 73, 223–225 (1999) Article ID jipa.1998.4827, available online at http://www.idealibrary.com on

NOTE Sporulation of Beauveria bassiana on Cadavers of Triatoma infestans after Infection at Different Temperatures and Doses of Inoculum Beauveria bassiana is a potential candidate for biological control of triatomine bugs, vectors of Chagas disease in Latin America. Depending on the microclimatic conditions in the vector habitat, the fungus was able to develop on the cadaver after its host’s death (Luz, 1994). Sporulation of B. bassiana on cadavers of Rhodnius prolixus in the laboratory was found at a minimal humidity of 96.5% with highest rates of conidia produced between 5 and 10 days of incubation at 25°C and conidial production declined at 28–30°C and was null at 35°C (Luz and Fargues, 1998). The number of conidia produced is potentially important for transmission of the fungus to other insects. There is little information available about factors influencing fungal behavior during the saprophytic phase, which are related to the period between infection and host death. The chalkbrood fungus, Ascosphaera aggregata, has been shown to sporulate at a higher percentage on cadavers of the alfalfa leafcutting bee, Megachile rotundata, after application of intermediate doses of inoculum, than at both lower and higher doses (Vandenberg, 1992). According to Fernandez and Groden (personal communication) a significant relationship exists between initial dose and quantity of B. bassiana conidia produced on cadavers of Leptinotarsa decemlineata. We studied the influence of temperature and fungal inoculum used during the infection process on growth and sporulation of B. bassiana on cadavers of Triatoma infestans third instars. Four B. bassiana isolates were tested: CG14, CG24, CG306, and CG474, culture collection of Embrapa. Isolates originated from pentatomid hosts captured in Brazil and were known to be highly virulent to T. infestans at 25°C, 53% humidity, and a concentration of 107 conidia/ml (Luz et al., 1998). Four temperatures, 15, 20, 25, and 30°C, and a concentration of 107 conidia/ml were used for the tests. Insects were treated by immersion in conidial suspension for approximately 6 s. Seven different concentrations of conidia, 105, 3 ⫻ 105, 106, 3 ⫻ 106, 107, 3 ⫻ 107, and 108 conidia/ml, were tested at 25°C. Conditions of humidity were 53% until death. Twenty unfed, newly emerged third instar nymphs were treated in each temperature and concentration. Assays were repeated four times. Dead insects were separated daily and stored at 4°C. Tests on sporulation were done at 25°C and 97%

humidity. Insects and cadavers were kept in desiccators and humidity was maintained by saturated solutions of salt: Mg(NO3 ) 2 · 6H2O to obtain 53% and K2SO4 to obtain 97% humidity (Winston and Bates, 1965). Fungal appearance on the cadavers and external production of conidia were recorded daily. The extent of sporulation after 10 days incubation was estimated by vortexing 20 cadavers for each temperature and concentration individually in 5 ml 0.1% Tween 80 for 5 min and counting conidia using a hematocytometer. Conidia of B. bassiana isolates were observed on all killed insects from the fourth day at all temperatures tested. Quantitative sporulation was significantly different among temperatures (F ⫽ 88.2, P ⬍ 0.0001, df ⫽ 3) and among B. bassiana isolates (F ⫽ 26.2, P ⬍ 0.0001, df ⫽ 3). A temperature of 20°C during infection induced a significantly higher production of conidia with all isolates compared to the other temperatures (Table 1). Mean production of conidia at 30°C was 36% (CI at 0.95: 30.5 and 42.8%) less than at 20°C. No difference was observed between mean production of conidia at 15 and 25°C. Isolate CG474 showed a significantly higher yield of conidia over all temperatures than the other isolates.

TABLE 1 Sporulation of Beauveria bassiana Isolates on Cadavers of Triatoma infestans Third Instars after Infection at Different Temperatures a,b Temperature (°C) Isolate

15

20

25

30

CG14

3.45 b (2.91–4.10) 4.82 b (4.04–5.69) 3.99 a (3.37–4.75) 5.94 b (5.01–7.06)

6.61 a (5.54–7.88) 7.71 a (6.40–9.28) 4.47 a (3.77–5.31) 10.70 a (8.60–13.4)

5.18 a (4.36–6.15) 4.35 b (3.67–5.17) 4.40 a (3.69–5.20) 7.28 b (6.14–8.65)

2.39 c (2.02–2.85) 2.34 c (1.97–2.78) 2.22 b (1.87–2.64) 3.36 c (2.82–4.01)

CG24 CG306 CG474

a Conidia were counted 10 days after incubation of cadavers at 25°C and 97% humidity. Values are means of 20 replicates each (CI at 0.95). Means in one isolate at different temperatures followed by the same letter were not significantly different at ␣ ⫽ 0.05 (ln scaled means, Tukey test). b All values multiplied by 107.

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Testing the effect of fungal inoculum, mycelium was first observed on all cadavers within 2 days, independent of the isolate and dose, but hyphal growth was distinctly accelerated in the lower dose treatments. Early sporulation after 3 days was observed in low-dose treatments with CG24 and CG306, whereas with higher doses sporulation started only after 5 days. No dose effect was observed for isolate CG474. After 10 days exposure, the dose of conidia applied to the insect also had a significant effect on the number of conidia produced on the cadaver for isolates CG14, CG24, and CG306 (Fig. 1). Analysis by weighted least squares showed a significant quadratic response for isolate CG14 (F ⫽ 12.4, P ⫽ 0.0244, df ⫽ 6), with increasing number of conidia at lower and higher doses. A linear effect was shown for isolate CG24 (F ⫽ 10.7, P ⫽ 0.0307, df ⫽ 5), with fewer conidia produced at increasing doses, and both a linear (F ⫽ 26.6, P ⫽ 0.0067, df ⫽ 6) and a quadratic response for the isolate CG306 (F ⫽ 23.1, P ⫽ 0.0086, df ⫽ 6).

As shown by the results, the dynamics of B. bassiana during development on the host cadaver can be influenced by abiotic and biotic factors such as temperature, isolate, and inoculum dose, which are related to the infection process. Temperatures tested during infection had no effect on postmortem growth of B. bassiana isolates. However, quantitative sporulation on cadaver was influenced by the isolate and temperature, with the highest rates being found in CG474 and at 20°C in all isolates. Temperatures between 15 and 25°C during infection favored production of conidia on cadavers exposed to 25°C and 97% RH, whereas quantitative sporulation was affected when insect infection occurred at a temperature of 30°C. With the exception of CG474 high-dose treatments showed a delayed growth of all B. bassiana isolates on cadavers of T. infestans. Quantitative sporulation after different dose treatments was shown to be dependent on the isolate. Massive fungal invasion may induce precipitated host death before

FIG. 1. Sporulation of Beauveria bassiana isolates (CG14, CG24, CG306, and CG474) on cadavers of Triatoma infestans third instars (means ⫾ standard error) after 10 days exposure at 25°C and 97% humidity. Insects had been treated with different doses of conidia.

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multiplication of mycelium in the cadaver and so retard postmortem growth. Moreover, mechanisms comparable to self-inhibition at a high inoculum dose, as reported for germination of other species of fungi (Garraway and Evans, 1984), may influence initial fungal growth in a dead insect originating from a high-dose infection. T. infestans can be found in domestic and peridomestic areas, where it establishes large and dense populations. B. bassiana is able to develop on triatomine cadavers at favorable temperature and humidity. Contamination of healthy individuals exposed to the mycosed cadavers with conidia is foreseeable. Higher amounts of conidia originating from cadavers may increase transmission of the fungus to other individuals and accelerate the propagation of the disease. A highly virulent fungal isolate which produces increased amounts of conidia on cadavers should be considered for vector control. CG474 seemed to be a promising candidate to control T. infestans as the isolate produced the highest amounts of conidia on cadaver at all temperatures tested during infection. CG474 showed no influence of initial dose of conidia on postmortem growth and quantitative sporulation. Results found with B. bassiana and T. infestans suggest that more investigations should be made to understand better the influence of factors intervening during disease development on postmortem growth and sporulation of entomopathogenic fungi. Key Words: Beauveria bassiana; Triatoma infestans; sporulation; temperature; dose. The authors thank Raquel R. Silva and Alexandre M. F. Diniz for technical help, Peter Inglis for the English translation, and Bonifa´cio Magalha˜es for the critical review.

REFERENCES Garraway, M. O., and Evans, R. C. 1984. ‘‘Fungal Nutrition and Physiology.’’ Wiley, New York. Luz, C. 1994. ‘‘Biologische Bekaempfung der Uebertraeger der Chagaskrankheit (Triatominae). Einfluss von Temperatur und Luftfeuchtigkeit auf die larvale Entwicklung von Rhodnius prolixus sowie die Infektion mit Beauveria bassiana (Deuteromycetes) und Sporulation des Pilzes auf den Kadaver,’’ Ph.D. thesis. University of Tuebingen. Luz, C., Tigano, M. S., Silva, I. G., Cordeiro, C. M. T., and Aljanabi, M. A. 1998. Selection of Beauveria bassiana and Metarhizium anisopliae to control Triatoma infestans. Mem. Inst. Oswaldo Cruz, 93(6), 839–846. Luz, C., and Fargues, J. 1998. Factors affecting conidial production of Beauveria bassiana from fungus-killed cadavers of Rhodnius prolixus. J. Invertebr. Pathol., 72, 97–103. Vandenberg, J. D. 1992. Bioassay of the chalkbrood fungus Ascosphaera aggregata on larvae of the alfalfa leafcutting bee, Megachile rotundata. J. Invertebr. Pathol. 60, 159–163. Winston, P. W., and Bates, D. H. 1965. Saturated solutions for the control of humidity in biological research. Ecology 41, 232–237.

Christian Luz* Ionizete G. Silva* Celia M. T. Cordeiro† Myrian S. Tigano† *Institute of Tropical Pathology and Public Health Federal University of Goia´s CP 131 74001-970 Goiaˆnia GO, Brazil †National Research Center for Genetic Resources and Biotechnology SAIN Parque Rural, W5 Norte, CP 02372, 70849-970 Brasilia, DF, Brazil Received December 30, 1997; accepted September 9, 1998