Susceptibility of nymphs of A. albofasciata towards entomopathogenic nematodes nymphs of A. albofasciata

The third and fourth instar nymphs of A. albofasciata were collected from field with cane cultivated in Atoyaquillo of sugarmill El Potrero, coordinates 18° 59’ 38.74 North latitude and 96° 46’ 50 West longitude. The nymphs were collected with the support of stainless-steel spatulas, were separated by gender and placed individually in plastic plates with cavities, which contained moist moistened paper toweling and stolons of star grass (Cynodon sp.). A preliminary test with grass stolons showed that rootlets of different lengths are formed in the area of the nodes, which remain active for several days (10-12). It was considered that if the nymphs could adhere to the rootlets and stay at least 4-5 days producing the excretion of saliva, it would constitute a favorable period for the trials proposed in this investigation.

The nymphs collected were transferred to the laboratory and kept under observation for 24 h, those that survived the management were selected. Subsequently, each nymph was confined individually in boxes Costar^® (Corning Inc. NY USA) with 6 cavities, covered with moistened filter paper and a grass stolon (as food). During the experiment, the nymphs were kept at room temperature. The nymphs that survived and continued to produce saliva were used in the experiment, due to their adaptation to space and food exchange.

Entomopathogenic nematodes

The nematode isolates used for this research were obtained from larvae of G. mellonella that were used as bait insects in soil samples from three sugar mills in the state of Veracruz, Mexico (Central Progreso, Constancia and La Gloria). The experiments were carried out with two isolates of Steinernema sp. (CPVC12, CPVC13) and one of Heterorhabditis sp. (CPVG13) (Table 1). The morphological and molecular taxonomic determination of the isolates is currently in process and will be reported in a later article.

The nematodes were propagated on last instar larvae of G. mellonella (L.) and harvested as infective juvenile (JI) by the White trap method (^{Kaya and Stock, 1997}), sterilized with 0.01% benzalkonium chloride and two changes of sterile distilled water. The suspension of infective juvenile (JI) was centrifuged at 500 rpm for 5 min, in order to eliminate dead nematodes by decanting the supernatant. The nematodes were kept at room temperature in distilled water, oxygenating them with a fish tank pump continuously to reduce mortality. The concentration of JI in suspension was estimated using aliquots of 20 μL, using the average of 7 counts, adjusting the concentration required in the experiment.

Experimental procedure

The process of inoculation of the different isolates was the same (Table 1). Costar^® plates with 6 cavities were used. Each cavity was covered with two layers of filter paper previously sterilized and moistened. In each cavity, a stolon (3.5 cm) of star grass (Cynodon sp.) where the formation of rootlets was induced, on these a nymph of A. albofasciata was placed. The inoculation was carried out applying a single concentration of infective juvenile, 120 JI cm^-2 according to ^{Alves et al. (2005)}. The application was made on the filter paper, allowing the displacement of the JI and search for the host insect.

The treatments were incubated at 25 ±2 °C and relative humidity (74% min and 84% max) and photoperiod of 9 and 15 h dark-light. In the control, the nymphs were treated only with sterile water. The nymphs were reviewed every 48 h and mortality was determined by plaque taking into account the symptoms (red or beige color, flaccidity) of the nymphs. Each dead nymph was removed from the plate and placed in a humid chamber, which consisted of a Petri dish with moistened filter paper, the emergence of JI was determined by infected nymph. The JI were collected during a period of 4 to 5 days and the total JI was determined in 10 ml of the suspension collected by nymph. This value was taken as indicative of the reproductive capacity of the nematodes. Each treatment had four repetitions (four plates/24 nymphs) and the whole experiment was repeated on three different occasions. The experiment lasted 7 days after the application of infective juvenile. The entire laboratory phase was carried out under aseptic conditions.

Effect of soil with the persistence of entomopathogenic nematodes

Soil samples from 8 sites were selected, two belonging to the Constancia sugarmill, two to Central Motzorongo and four to El Potrero. The soil samples were taken in an area 30 x 30 cm long by wide and approximately 10 cm deep using a shovel. From each site thirteen samples (a total of 5 kg) were obtained and were stored at 4 °C for no more than two weeks. Composite samples were prepared, combining the 13 sub samples of each site by ingenuity.

To avoid the effect of native nematodes in the persistence experiment, soil sampling was carried out using the bait insect method with G. mellonella (^{Zimmerman, 1986}). Each soil sample was moistened with sterile distilled water at field capacity (60%). Samples of 500 g of each locality were taken by placing them in plastic containers of 500 mL of capacity (experimental unit), seven larvae of G. mellonella of the last instar were individually confined in metal mesh cages and introduced in the containers with soil and distributed randomly. Each container was covered with a lid with perforations to allow constant aeration, the vessels were inverted (^{Miduturi and Moens, 1997}) and incubated in a breeding chamber at 25 ±2 °C for 7 days, to confirm the presence of native nematodes. This procedure was repeated twice.

Cultivable microbial populations

Within the first week of collection, the natural populations of bacteria, fungi and actinomycetes of each soil sample were estimated. Samples of 10 g of soil from each location, sieved in No. 10 mesh, were placed in a 250 mL Erlenmeyer flask containing 90 mL of sterile distilled water. The samples were shaken for 18 minutes in an orbital shaker (Thermolyne Cimarec^® 3), to subsequently prepare serial dilutions (^{Dhingra and Sinclair, 1985}). A final dilution factor of 10^-3 and 10^-5 was considered adequate for fungal isolation, 10^-4 and 10^-6 for bacteria and actinomycetes 10^-3 and 10^-5.

Planting was done by plaque extension in Petri dishes with nutritive agar (NA) for bacteria, potato dextrose agar (PDA) for fungi and Czapeck Dox agar with pH 8.0 for actinomycetes with three replications by dilution. Plates were incubated at 27 °C/5d, 7d and 10d for bacteria, fungi and actinomycetes, respectively. After the incubation period, the number of colony forming units (UFC) was estimated by selecting the dilution in each medium that favored the proper enumeration (10^-3 for bacteria and fungi and 10^-6 for actinomycetes). The UFC/gram of dry soil (air) was calculated by multiplying the arithmetic average of the number of UFC per plate by the dilution factor. The result was divided by the estimated dry soil weight. The physical and chemical characteristics of the soil were carried out by the Soil Fertility Laboratory of the Edaphology Program, Postgraduate School.

Experimental procedure

For this experiment, soil from El Potrero, Central Motzorongo and Constancia sugarmills were used, these were selected because the presence of nematodes was not recorded. Two isolates, CPVC12 (Steinernema spp.) and CPVG13 (Heterorhabditis sp.) were used. The procedure was the same for each type of soil and nematode evaluated. For each soil and nematode combination, 22 containers with 500 g of soil each were prepared, in the center of 11 of the containers, 5 000 infective juveniles were suspended, suspended in 2.5 mL of sterile distilled water (ADE), in the 11 remaining containers only 2.5 mL of ADE was deposited, these being the control treatment.

The survival of the nematodes was quantified by the proportion of larvae of G. mellonella infected when in contact with soil inoculated by nematodes. For this, in two containers, with and without nematodes, they were introduced in seven cages made of metal mesh in each container. Cages 1 cm in diameter by 4 cm in length contained a larva of G. mellonella. Seven days later, this procedure was repeated using two different containers (with and without nematodes) and so on at 14, 21, 28, 35, 42, 49, 56, 63 and 70d after inoculation of the nematodes. The experiment was incubated at 25 °C in total darkness. The larvae of G. mellonella were left in contact with the soil for 6 d and then they were removed from the soil and the number of dead larvae was quantified.

Each larva was placed in a cavity of a plate of six cavities containing moist filter paper, and incubated to the same experimental conditions. The presence of infective juvenile (JI) was determined by observations between 10 a 15d. The experiment was carried out under a completely randomized design, where each soil/nematode combination had two replicates, and the experiment was repeated twice (four repetitions in total).

The data were analyzed using logistic regression (assuming that the data had a binomial distribution), where each number of dead larvae was a proportion of the total larvae evaluated. When it was necessary, the presence of greater dispersion of the data under the assumption of the binomial distribution was allowed, comparing the ratio of the mean deviation of the treatments and the residual mean deviation against a F distribution, instead of comparing the deviation of the treatments with a distribution of χ². Before comparing between treatments, the results obtained between repetitions in time were compared to be combined. With the combined repetition data, survival between nematode isolates, between the 4 soil types, between the evaluation times and finally the interaction between these factors was first compared. The analysis was carried out in the GenStat v. 8 (^{Payne et al., 2005}).

Susceptibility of nymphs of A. albofasciata against Heterorhabditis sp. and Steinernema spp.

In general, differences were observed in mortality caused by Steinernema spp. (CPVC12; CPVC13) and Heterorhabditis sp. (CPVG13). The percentage of mortality (62.5%) was higher for Heterorhabditis sp. at 25 °C, compared to Steinernema (CPVC12) (56.25%) and Steinernema sp. (CPVC13) (33.33%). Although mortality was observed in the treatments, it was not possible to perform a statistical analysis, since the control treatment showed high mortality from other causes. However, this experiment showed that the isolates of both Steinernema and Heterorhabditis had the ability to cross the saliva produced by the nymphs, penetrate the insect cavity and cause infection (Figure 1b, 1c).

Figure 1 Spittlebug nymphs: a) healthy nymph; b) nymph infected by Heterorhabditis. (CPVG13); c) infected by Steinernema (CPV12). Dissections made to parasitized nymphs; d) adult nematodes of (CPVG13) Heterorhbaditis; e) adult nematodes of (CPV12) Steinernema; f) meristemed nematodes; and g) nymphs with presence of necrosis, possible presence of bacteria.  

The dissections of the corpses of infected nymphs allowed to verify the reproductive capacity of the entomopathogenic nematodes (Figure 1d, 1e). The nymphs exposed to Heterorhabditis (CPVG13) and Steinernema sp. (CPVC12) showed differences in abundance of infective juvenile (Table 2). In the case of Heterorhabditis, the highest proportion corresponded to 3 500 JI per nymph (Table 2).

Cultivable microbial populations

The largest population of bacteria and fungi was present in Constancia soil (3X10⁹ UFC g^-1 of soil and 1.5 x 10⁴ UFC g^-1 of soil for bacteria and fungi, respectively) (Figure 2a) while in Central Motzorongo the proportion of bacteria (3 x 10⁸ UFC) and fungi (4 x 10⁴ UFC g^-1 of soil) was considered low (Figure 2b). The largest actinomycete population was found in site 2 of El Potrero plantation (7 x 10⁵ UFC g^-1 of soil) and the lowest population was obtained in Constancia (8.6 x 10⁴ UFC g^-1 of soil) and Motzorongo (1.7 x 19⁵ UFC g^-1 of soil) (Figure 2c). The physical and chemical characteristics of the soil (Table 3).

Figure 2 Colony forming units. a) bacteria; b) fungi; and c) actinomycetes, obtained from cane soil cultivated in the Constancia, Motzorongo plant and El Potrero sugarmills.  

Effect of soil with the persistence of entomopathogenic nematodes

There were no significant differences between the data of the four repetitions ( χ32 = 1.3215 , p= 0.427), which allowed the data to be combined to make a joint analysis and see the effect of treatments. Significant differences were found between the persistence profiles between Heterorhabditis (CPVG13) and Steinernema (CPVC12) ( χ12 = 64.3496 p< 0.001). The isolation that persisted the most was the (CPVC12) of Steinernema (Figure 3B) compared to the isolation (CPVG 13) of Heterorhabditis. A significant soil effect was found in the survival of both nematodes ( χ32 = 11.2522 , p= 0.01); however, this effect was different for each nematode isolation ( χ32 = 17.3337 , p< 0.001), where Steinernema at 8 weeks showed high proportion of dead insects indicating high persistence of the JI, while in the inoculations with Heterorhabditis the persistence of JI began to decline from the 5th week after the inoculation occurred. The time that the nematodes spent in the soil had a significant effect on their survival ( χ92 = 462.3188 , p< 0.001) and this effect was similar for the two species of nematodes ( χ92 = 13.9253 , p= 0.125).

Figure 3 Proportions of mortality a) Heterorhabditis sp.; and b) Steinernema sp. in larvae of G. mellonella. The error bars represent confidence limits of 95%, transformed from the logistic scale. The persistence of JI constitutes the average value of dead larvae of G. mellonella by date of evaluation at 25 °C.