Resumen

Pineda Gutiérrez F, Saldarriega Osorio Y, Calle Osorno J, Uribe Soto S. 2003. Susceptibilidad de Rhodnius pallescens (Hemiptera: Reduviidae) de V estadío de desarrollo a la acción de Beauveria spp. Entomotropica 18(3):163-168.

Se realizaron ensayos de laboratorio para determinar la susceptibilidad del V estadio de desarrollo del insecto Rhodnius pallescens Barber al hongo entomopatógeno Beauveria spp. Los insectos se criaron y mantuvieron en el laboratorio de Micología del Instituto de Biología de la Universidad de Antioquia. Se aislaron dos cepas del hongo entomopatógeno B. bassiana del insecto triatomino R. pallescens y dos de B. brongniartii del curculionido Homalinotous validus (Olivier), habitantes de las palmeras de Attalea butyracea en la región de San Onofre, Departamento de Sucre, Colombia. La patogenicidad de Beauveria bassiana a R. pallescens fue estudiada exponiendo los insectos a concentraciones del hongo de 1 x 10⁷ esporas/ml impregnadas en pedazos de lona. Los diferentes aislamientos produjeron varios grados de mortalidad en los insectos, desde 77,5% B. bassiana (Bb UdeA₁₄) a 100% B. brongniartii (Bt UdeA₁₆) a los 22 days. Los valores de TL₅₀ de 10,11 y 12 días fue estimado para los aislamientos B. bassiana (Bb UdeA₁₄), B. brongniartii (Bt UdeA₁₅) and B. bassiana (Bb UdeA₁₃) respectivamente_. El valor TL₅₀ más bajo fue de 7 días para B. brongniartii (Bb UdeA₁₆), siendo este aislamiento más efectivo que los otros y el control (X² = 200.84, p< 0.0001). Ninfas de quinto estadio de R. pallescens fueron más susceptibles a B. brongniartii (Bt UdeA₁₆) que a las otras cepas probadas, y 50% de los insectos mostraron micosis a los 10 días después de haber sido expuestos a esta cepa (X² = 47.08, p < 0.0001).

Palabras clave adicionales: Beauveria bassiana, Beauveria brongniartii, hongos entomopatógenos, Homalinotus validus.

Introducción

Chagas' disease caused by Trypanosoma cruzi and transmitted by triatomine bugs of the subfamily Reduviidae is a serious public health problem in Latin America. It affects 3.4% of the population of Colombia, where 3.6 million people inhabit high-risk areas (Moncayo & Guhl 1997) in the center, south and Caribbean coast of the country (D' Alessandro et al. 1981; Corredor et al. 1990). 

Table 1. Isolates and species of Beauveria, evaluated during the present study, toghether with
insects hosts collected in San Onofre (Sucre).

Host species	Beauveria sp.	Isolate
Rhodnius pallescens (Hemiptera: Reduviidae)	B. bassiana	Bb UdeA13
Rhodnius pallescens (Hemiptera: Reduviidae)	B. bassiana	Bb UdeA14
Homalinotus validus (Coleoptera: Curculionidae)	B. brongniartii	Bt UdeA15
Homalinotus validus (Coleoptera: Curculionidae)	B. brongniartii	Bt UdeA16

The entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. Criv. is a natural pathogen of many insect species (Couteaudier & Viaud 1997) which has been used for many years as a biological pest control agent (Ferron et al. 1991).

The mycelium of the fungus penetrates the insect cuticle, invading the tissues and causing death (Hajek & St Leger 1994). Pathogenecity is also due to mycotoxins that block the insect inmune system (Kachatourians 1991; Uribe et al. 1997).

Romaña & Romaña (1981); Romaña et al. (1987); Romaña & Fargues (1987); Romaña (1992), reported the susceptibility of triatomines to entomopathogenic fungi in nature. A total of 23 species of This insects are known from Colombia, including Rhodnius prolixus Stål.; R. robustus Larrouse; R. pallescens Barber; R. brethesi Matta; Triatoma dimidiata Latreille; T. maculata Erichson; and T. venosa Stål.; all of which have been found in houses (Molina et al. 2000). The principal vector of Chagas' disease in Colombia is R. prolixus which is anthropophilic, occurs a high population densities, has a short life cycle, and is highly susceptible to infection with T. cruzi. This species has a high dispersal capacity, which together with is ability to adapt to a variety of ecological conditions explains its wide geographical distribution in Colombia (D'Alessandro et al. 1984; Schofield & Dujardin 1997).

Other triatomine species occur in forested areas, such as R. pallescens which lives in Attalea butyracea (Muttis ex l. f.) Wess. Boer (Arecaceae) palms in the San Onofre region of the Colombian department of Sucre (Romaña et al. 1999) and is the main vector of T. cruzi in Panama. This species also occurs in Belize where it lives in chicken houses, as well as being associated with opossums and sloths (Lent & Wygodzinsky 1979, Christensen & De Vasquez 1981). Homalinotus validus (Coleoptera: Curculionidae) is an inhabitant of the Attalea butyracea palms in the San Onofre region of the Colombian department of Sucre.

The objective of the present study were to isolate, cultivate, identify Beauveria spp. and to determine the susceptibility of R. pallescens to these fungi.

Materials and Methods

Origin of insects and fungal isolates: The insects isolates were obtained from the palm trees A. butyracea and the fungus from the triatomine bug Rhodnius pallescens and the weevil homalinotus validus in the municipality of San Onofre, which has an area of 30 km² and includes the rural sectors of Las Brisas and Vista Hermosa (lat 09°60'0'' and 09°80'11''N, long 75°28'05'' and 75°42'00'' W respectively), between Higuerón, Plan Parejo and La Libertad (Calle 2000).

Specimens of R. pallescens and H. validus were captured in the field by dismantling the crowns of 30 A. butyracea. The triatomines were sorted by instars and nutritional status. The live insects were placed in plastic tubes sealed with muslin and stored unfed for forty days. Specimens that died during this period were placed in plastic Petri dishes with a diameter of 5.5 cm, together with a small piece of dampened cotton to induce fungal sporulation.

Isolates of Beauveria spp.: The isolates obtained and their origins are listed in Table 1.

Isolating fungi: Dead insects invaded by the fungus were observed under the stereomicroscope and mycelium removed for inoculation on Petri dishes of Sabouraud dextrose and malt extract agar. The Petri dishes were incubated for one week at 25 °C. Slides preparations of the cultures were stained with lactophenol blue and mounted for detailed examination. Fungi were identified according to De Hoog (1972), Hugues (1973) and Mangenot & Reisenger (1976). All fungi isolated from weevils were found on dead specimens within the crowns of palm trees, so that no laboratory rearing of H. validus was carried out. The isolates of B. bassiana and B. brongniartii used in this study were preserved in a cold chamber at 4 °C at the Mycology Laboratory at the Biology Institute of the Unversity of Antioquia.

Culture media: The fungi were kept in malt extract agar (MEA) and Sabouraud dextrose agar (SDA) (Oxoid LTD., Basingstoke, Hampshire, UK). The technique of Romaña & Fargues 1987 was used to obtained the spores. The spores were inoculated in an inclined semi-synthetic medium (Romaña & Fargues 1987) for two weeks at 25 °C. Spores concentrations were determined with a Neubauer haemocytometer, counting the central square of 16 cells in two drops of suspension for each replicate. A spore concentration of 1 x 10⁷ spores/ml was used in the bioassays.

Table 2. Proportions of Rodnius pallescens of diferent developmental stages invaded by
fungi during quarantine

Developmental stage	Showing mycosis (%)	Not showing mycosis (%)	Total (%)
Nymphal instar 1	10 (7.94)	5 (5.38)	15 (6.85)
Nymphal instar 2	22 (17.46)	14 (15.05)	36 (16.43)
Nymphal instar 3	26 (20.63)	16 (17.20)	42 (19.18)
Nymphal instar 4	11 (8.73)	13 (13.98)	24 (10.96)
Nymphal instar 5	33 (26.19)	26 (27.96)	59 (26.94)
Adult	24 (19.05)	19 (20.43)	43 (19.63)
Totals	126 (100.00)	93 (100.00)	219 (100.00)

Table 3. Proportions of adult male and female Rodnius pallescens invaded
by fungi during quarantine

Sex	With mycosis (%)	Without mycosis (%)	Total
Females	10 (41.67)	8 (42.11)	18 (41.86)
Males	14 (58.33)	11 (57.89)	25 (58.14)
Total	24 (100.00)	19 (100.00)	43 (100.00)

Insect rearing: Live specimens of R. pallescens were maintained in darkness in an air conditioned chamber (WTBbinder 78532 (Tuttlingen/Germany), at 26.5 °C and 80% relative humidity (Romaña & Fargues 1987). The insects were kept in plastic containers of dimensions 16x12 cm containing a central opening covered with muslin. Cross-shaped cardboard ladders were placed inside the containers as refuges for the bugs, which were allowed to take blood from mice (Mus musculus Linneaus) for 2-3 h twice per week.

Direct contamination of insects: Four replicates of 20 fith instar nymphs of R. pallescens and a control proup were exposed to 1 x 10⁷ spores/ml. They were kept in complete darkness in an air conditioned chamber (WTBbinder 78532 (Tuttlingen/Germany), at 26.5°C and 80% relative humidity. Live insects were counted and dead ones placed in sterile containers with a small piece of moist cotton, then stored in an incubator at "25 ºC" for 7 days. The insects were observed daily. All dead insects were assumed to have succumbed to the fungi that had developed on their cadavers (Romaña & Romaña 1981). Samples of the fungal mycelium were stained in lactophenol blue and mounted on slides for examination under the microscope to confirm that the fungi responsible were Beauveria spp.

Statistical analysis: Data on survival rate of R. pallescens and mycosis were analysed using Freeman Halton's Test on the Statxact-4 stastistical programme.

Results

Of the four autochthonous isolates of Beauveria from San Onofre (Sucre), two were B. bassiana and two (2) B. brongniartii (Sacc.) Petch, obtained respectively from R. pallescens and H. validus (Table 1). A total of 376 specimens of R. pallescens were collected in palm trees, of which 271 were unfed and 105 fed. In all 210 unfed and nine fed triatomines died during quarantine, of which 126 (124 unfed) presented some type of mycosis while 93 showed no signs of fungus (Table 2).

Among the adult bugs, 14 males and 10 females showed some sort of mycosis while 11 males and eight females were fungus-free (Table 3). There was no significant association between mycosis and the stage of nymphal development (X² = 2.44, p = 0.79) or sex (X² = 0.32, p = 0.064) but an association was observed between mycosis and unfed insects (X² = 4.79, p = 0.028).

With respect to insect mortality due to Beauveria (strains Bb UdeA₁₃, Bb UdeA₁₄, Bt UdeA₁₅, Bt UdeA₁₆), significant differences were found (X² = 200.84, p < 0.0001) between treated and control groups for fifth instar nymphs of R. pallescens (Figure. 1). The LT₅₀ value for this group was seven days. Mortality of 100% was obtained with the Bt UdeA₁₆ isolate 21 days after exposure (Figure 1).

Fith instar nymphs of R. pallescens were more susceptible to B. brongniartii (UdeA₁₆) than the other strains tested and 50% of the insects showed mycosis 10 days after being exposed to this strain (X² = 47.08, p < 0.0001) (Figure 2).

Discussion

The isolates obtained during the present study belonged to two species of B. bassiana originating from triatomine bugs and two of B. brongniartii from weevils. Even if the isolates of B. bassiana did indeed originate exclusively from triatomines and those of B. brongniartii only from weevils in the present study, it cannot be concluded that these fungi are pathogenic only to these insects. Workers in previous studies carried out in the San Onofre region have isolated B. bassiana both from weevils and triatomines (Arroyave 1995; Calle 2.000).

These insects are often naturally infected by the fungi, irrespective of sex or developmental stage, as confirmed by the laboratory quarantine data (Table 2 and Table 3).

A higher infection rate by some type of fungus was seen in the triatomines classified as unfed, suggesting that these were more susceptible to mycosis.

All the Beauveria isolates used in the laboratory assays produced a high mortality and fungal esporulation in fifth instar nymphs of R. pallescens.

Infection was produced by invasión across the integument, a feature that distinguishes entomomycosis from viral, bacterial or protozoal infections (Romaña & Romaña 1981).

Mortality in adults of R. pallescens was attributed to mycosis caused by B. bassiana or B. brongniartii, since these were recuperated from the dead insects.

The susceptibility of adult triatomines to mycosis has been little studied (Ferron 1985). In susceptibility tests carried out with first instar nymphs of R. prolixus, R. pallescens, R. robustus, R. negletus and Eratyrus cuspidatus, concentrations of 3x 10⁸ spores/ml of the B. bassiana strains INRA 297 and UdeA₁, induced 100% mortality in all the insects. The LT₅₀ values varied according to species, including 7.5 days for R. pallescens infected with INRA 297 and only 4.6 days for the same species esposed to the isolate UdeA₁ (Arroyave 1995). The LT₅₀ value for fifth instar nymphs of R. pallescens obtained during the present study (7 days) was some what lower than that obtained by these authors with the strain INRA 297. When exposed to the UdeA₁ strain, fifth instar nymphs showed LT₅₀ values a little greater than those obtained for first instar nymphs exposed to a higher concentration of the fungus. These results show that the susceptibility may be affected by the developmental stage, the concentration of the spores and the species of fungus, as well as factors such as the geographical origin of the fungus, and the physiological state of the insects (Romaña & Fargues 1992; Arroyave 1995; Romaña 1995).

The pathogenecity of fungi of the genus Beauveria to triatomines was demonstrated in previous studies (Romaña & Romaña 1981; Romaña & Fargues 1987; Romaña 1992). The results of the present study are very similar to those obtained by these researchers and provide further information on the potential of entomopathogenic fungi for the microbiological control of triatomines in their own habitat.

At present three major programmes are directed towards the control of Chagas' diseases, i.e., the Southern Cone Initiative and similar multinational efforts involving countries of the Andean region and Central America. These have produced excellent results in the control of triatomines at the domiciliary level.

However wild foci of these insects remain, presenting the threat of reinfestation. This might best be countered by means of microbiological control using entomopathogenic fungi such as Beauveria spp.

Acknowledgements

The authors thank the Corporación de Patologías Tropicales, Facultad de Ciencias Exactas y Naturales, Instituto de Biología and the committee for Research Development of the University of Antioquia for financial and logistic support in carrying out this research and the following people for their assistance through assessment, technical support or field work: Abel Díaz Cadavid, Cristina Romaña, Gustavo García G., Paula Takegamy, Juan Guillermo Penagos, Jean Paul Delgado Charry, Diego Salazar, Carmen Vázquez, Ana Isabel Gutiérrez, Inés Elena Giraldo and Beatríz Cardona.

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