Selection for Entomopathogenic Fungi and LD 50 of Metarhizium anisopliae ( Metsch . ) Sorok . for the Lesser Mealworm Alphitobius diaperinus ( Panzer ) ( Coleoptera : Tenebrionidae )

The pathogenicity of insect-pathogenic hyphomycetous isolates to the lesser mealworm Alphitobius diaperinus (Panzer) was tested in this study. Thirty isolates of Beauveria bassiana (Balsamo) Vuillemin, Paecilomyces amoenoroseus (Hennings), P. fumosoroseus (Wize) Brown & Smith, P. lilacinus (Thom) Samson, P. tenuipes (Petch) Samson, Metarhizium anisopliae (Metschnikoff) Sorokin, and M. anisopliae var. acridum were initially screened by sprinkling dry conidia onto adult A. diaperinus or by allowing adults to walk on Petri dishes with sporulating fungal isolates. The two most virulent isolates, CNPSo-Ma352 (M. anisopliae) and CNPSo-Ma356 (M. anisopliae), killed 30% and 26.7% of the dry-conidia treated adults, respectively. These two isolates were selected for LD 50 bioassays. LD 50 of CNPSo-Ma352 was 4.5 x 10 conidia per larva, and 2.1 x 10 conidia per adult, and for strain CNPSo-Ma356, LD 50 was 2.2 x 10 conidia per larva and 1.3 x 10 conidia per adult. Larvae were 5-6 times more susceptible than adults. A larger number of conidia required to cause 50% mortality in insect evaluates, suggesting the reduced susceptibility of A. diaperinus to entomopathogenic fungi. Nevertheless, these and other strains of fungus offer an alternative method for controlling of lesser mealworm in poultry houses when associated to integrated management.


INTRODUCTION
The lesser mealworm or little beetle, Alphitobius diaperinus (Panzer) is the most important poultry-house pest in Brazil, where it successfully breeds, feeds, and develops in the mixture of warm litter and poultry droppings.Environmental conditions in the poultry house (25-30 ºC, humidity between 50% and 70%) allows beetles to complete one life cycle during the chicken growing cycle (approximately 50 days) (Salin et al., 2000;Chernaki & Almeida, 2001).Beetles (adults and larvae) feed on feed residues, feces, and dead or dying birds (Pfeiffer & Axtell, 1980).They are considered to be important vectors of pathogens, such as Salmonella typhimurium (Loeffler), Escherichia coli Lignieris, avian leukosis virus, and turkey enterovirus and rotavirus (McAllister et al., 1994;1995).Additionally, due to high beetle population levels, adult insects may become local nuisances during downtime, when adults often leave the poultry house and enter nearby residences.The control of A. diaperinus is important for poultry health and production, as well as for reducing the nuisance they cause.It is difficult, however, to obtain good control because chemical insecticides are not effective, and repeated applications may cause the development of resistance.In addition, this may cause chemical insecticide residue problems.Therefore, other alternatives must be developed to control this important pest.
LD 50 is often used to determine the concentration of the pathogen or of the compound will kill 50% of the population in question.Thus, LD 50 provides a useful value to compare results from different laboratories, hosts, and entomopathogenic strains or species.The aim of this study was to assess the susceptibility of A. diaperinus to the entomopathogenic fungi: Beauveria bassiana, Paecilomyces amonoeroseus, P. tenuipes, P. lilacinus, P. fumosoroseus, Metarhizium anisopliae and M. anisopliae var.acridum, and to determine the lethal doses (LD 50 ) of the most pathogenic isolates.

Alphitobius diaperinus
Larvae and adult lesser mealworms were collected from poultry litter in a broiler farm in the municipalities of Cascavel and Curitiba, state of Paraná, southern Brazil.Colonies were kept in plastic trays (37 x 26 x 13 cm) with poultry litter and rabbit feed, at 24 ± 2 °C, in continuous darkness, and 50 -70% relative humidity.
Tests were conducted using randomly selected adults of approximately the same size (sex unknown), and 1.0 to 1.3-cm long.

Selection and virulence of fungi isolates
Dry conidia from 1-2 week-old colonies were sprinkled onto the surface of the adult beetles.For the hydrophilic isolates CNPSo-Bb11, CNPSo-Bb13, LPB, CNPSo-Ma4, CNPSo-Ma5, CNPSo-Ma358, and NRRL-895, with low spore formation, insects were placed directly on the fungal colonies for 60 sec.Fifteen insects were used for each isolate, and replicated twice (total of 30 adults per isolate), including the control group.After inoculation (or no treatment, in the case of controls), insects were maintained in sealed (with fine textured cloth) 500-mL plastic containers with rabbit feed and slightly moistened sawdust, and kept in a climate chamber at 27 °C in constant darkness for 20 days.Dead insects were daily removed, and placed on Petri dishes with moistened cotton and filter paper, at room temperature, until signs of fungal infection appeared, or for 7 days, whichever came first.For counting, the 30 isolates were prepared by aerosol conidia suspension on slides with a PDA culture film, and incubated in the dark for 20 hours, at 26º-28 ºC.The two most virulent isolates were chosen for further bioassays (LD 50 ).

LD 50 estimation
The two isolates chosen for further bioassays were plated on Petri dishes with spell-out medium (PDA) or in adult beetles, and were incubated at 26 ºC, 60-65% relative humidity, and complete darkness.When isolates were sporulating, conidia were collected by scraping dry conidia from the surface of the culture plate.Dry conidia were then placed in test tubes with sterile distilled water containing 0.01% Tween 80. Conidial suspensions were then filtered, homogenized, and adjusted to the correct concentrations with the use of a hemacytometer.
The concentrations used were from 2.8 x 10 3 conidia/ mL to 1.8 x 10 5 conidia/mL.Each adult or larva was inoculated by pipetting 2 µl of the relevant concentration of the isolates CNPSo-Ma352 or CNPSo-Ma356 (by monocanal micropipette) onto the ventral side of the insect.Control insects received the same volume of sterile water (0.01 % Tween 80).Inoculated insects were then placed in 500 mL plastic containers as described above.
Each treatment comprised four replicates, with 640 adults and 520 larvae tested for each isolate.Dead insects were daily removed, and individually placed on Petri dishes with moistened cotton and filter paper, at room temperature, until signs of fungal infection appeared, or for 7 days, whichever came first.Samples of conidia from the dead animals were treated and counted as described above.

Statistical analysis
Probit analysis was used to determine LD 50 , based on cumulative mortality per treatment (LeOra Software, 1987).Slopes and y-intercepts of the mortality regressions of the two treatments were compared (α = 0.05).

Entomopathogenic fungus selection
All isolates were viable.CNPSo-Ma5 had the lowest germination rate (72%), while all other isolates presented germination rates of 85% or higher.The two most virulent fungi were isolate CNPSo-Ma356, which killed 30% of the adults and all insects killed by this isolate developed mycosis, and isolate CNPSo-Ma352, which killed 26.7% of the adults, and 75% of the insects killed by this isolate developed mycosis.These two isolates were therefore used in further bioassays.All other fungal isolates caused much lower or no mortality (Table 1).

DISCUSSION
Many authors have stressed that fungal susceptibility is reasonably well-known for beetles, and in general, when beetles are exposed to large concentrations of conidia (10 7 conidia/mL), mortality can be very high (up to 98%, Steinkraus et al., 1991).A. diaperinus larvae and pupae are more susceptible than adults to a variety of isolates of B. bassiana, B. brongniartii, P. farinosus, P. fumosoroseus, M. anisopliae, Verticillium lecanii, Acremonium sp., and Fusarium sp.(Steenberg & Jespersen, 1997).It was shown that B. bassiana isolates under poultry-house conditions can cause high mortality rates in A. diaperinus (Crawford et al., 1998).Recently, Alexandre et al. (2006) evaluated the effect ofpoultry litter (new and used) by submerging insects in a fungal suspension (10 8 conidia/mL), and observed that B. bassiana isolates were more sensitive to high temperature than M. anisopliae because conidia viability, vegetative growth, and virulence were negatively affected.
Average LD 50 doses were 2.2 x 10 4 and 4.5 x 10 4 conidia per larva, and 13.0 x 10 4 and 21.2 x 10 4 conidia per adult (CNPSo-Ma356 and CNPSo-Ma352, respectively).These doses are quite high as compared to other studies.For example, LD 50 of Metarhizium spp. in Locusta migratoria migratorioides is from 60 to 300 conidia per grasshopper (Nowierski et al., 1996).Therefore, the use of fungi to control the lesser mealworm in poultry houses in Brazil will require further field studies to determine the cost-benefit ratio of each strategy and the virulence of the fungus (or conversely, susceptibility of the host).
Although a larger number of conidia is required to cause 50% mortality of the evaluated insect, this research study provides data that indicates that these and other fungus strains offer an alternative method for controlling the lesser mealworm in poultry houses.We recommend the controlled and studied application of fungi to poultry houses with significant infestations, perhaps simultaneously with studies on the impact of chemical as well as botanical or desiccants pesticides (diatomaceous earth) and growth regulators on fungal efficiency.Also, we believe that research studies on the best way to apply fungi to pests is an area that may yield important results in terms of cost reduction (for instance, Arends traps impregnated with conidia, according to Geden & Steinkraus (2003).DR, Almeida LM Selection for Entomopathogenic Fungi and LD 50 of Metarhizium anisopliae (Metsch.)Sorok.for the Lesser Mealworm Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

Figure 1 -
Figure 1 -A comparison of LD 50 between MA352 and MA356 fungal strains, and adult and larval lesser mealworm beetles.Adults are less susceptible than larvae (p < 0.05), and strains present similar virulence.Lethal Dose 50% axis is shown on a logarithmic scale, as LD 50

Table 1 -
Assay for virulence of pathogenic fungus.Number of deaths and percentage of deaths with evidence of fungus in 30 trials of Alphitobius diaperinus adults inoculated with different isolates of Beauveria bassiana, Paecilomyces spp and Metarhizium spp dry conidia.