Effectiveness of Steinernema rarum PAM 25 (Rhabditida: Steinernematidae) Against Drosophila suzukii (Diptera: Drosophilidae)

Abstract

Drosophila suzukii (Matsumura 1931) represents one of the main pests of small fruits. The use of biological agents is very promising for insect control. In the present study, the nematode Steinernema rarum PAM 25 was evaluated for the control of D. suzukii pupae, this species has not been evaluated previously. First, we evaluated the pathogenicity of S. rarum PAM 25 at the concentration of 1,000 infective juveniles (IJs) inoculated into D. suzukii pupae. In the second bioassay, we evaluated the influence of 1,500; 2,000; 2,500; 3,000; 4,000 IJs/ml nematode concentration and temperature on D. suzukii mortality. In the third bioassay, we evaluated the influence of the isolate S. rarum PAM 25 on D. suzukii adult lifespan following pupal infection, using the concentrations with the highest mortality rate of pupae at each temperature as determined in the second experiment. The S. rarum PAM 25 isolate is pathogenic to D. suzukii. The most effective temperature for S. rarum PAM 25 activity was 14°C at a concentration of 4,000 IJs/ml. Adults infected with S. rarum PAM 25 showed a significant reduction in longevity. The results confirmed the potential of S. rarum PAM 25 for the control of D. suzukii.

Drosophila suzukii (Matsumura 1931) is a polyphagous pest with a high reproductive rate and a high dissemination capacity. The fly originates from Southeast Asia and is currently present in Europe (Calabria et al. 2012), North America (Walsh et al. 2011), South America (Santos et al. 2017), and Africa (Boughdad et al. 2021). There are still environmentally adequate areas with potential for D. suzukii occurrence in Oceania (dos Santos et al. 2017).

Female D. suzukii pierce the fruit surface for oviposition, thus enabling infestation of other opportunistic organisms, such as bacteria and fungi, leading to fruit depreciation (Vilela and Mori 2014, Renkema et al. 2018), and accelerating the deterioration process (Goodhue et al. 2011, Lee et al. 2011, Burrack et al. 2015, Diepenbrock et al. 2016).

Considering the high dispersal potential of D. suzukii and its damage to the production of small fruits as well as economic losses caused, it is necessary to seek effective control alternatives with low environmental impact for Integrated Pest Management (Garcia 2020). In this context, the use of entomopathogenic nematodes (EPNs) is promising for biological control, as they are terrestrial worms that obligatorily parasitize insects to complete their life cycle (Hominik et al. 1996, Brida et al. 2018).

Nematodes of the family Steinernematidae, genus Steinernema, have high host diversity and are described in several studies that confirm their pathogenicity to insects (Kaya and Gaugler 1993, Georgis et al. 1995). The infective juvenile phase of this worm penetrates its host and releases bacteria of the genus Xenorhabdus (Thomas and Poinar 1979), killing the insect by septicemia within 24–48 h (Mekete et al. 2005, Lewis et al. 2006, Fujimoto et al. 2007, Grewal 2012, Kucharska et al. 2015).

Although a commercial formulation has not yet been made and evaluated for the management of D. suzukii, common nematode species, Steinernema kraussei (Steiner 1923), Steinernema feltiae (Filipjev 1934), and Steinernema carpocapsae (Weiser 1955), were tested under laboratory conditions on larvae of D. suzukii, but had low mortality rates (Cuthbertson et al. 2014, Woltz et al. 2015, Cuthbertson and Audsley 2016). Heterorhabditis amazonensisAndaló et al. 2006 IBCBn 24 and Heterorhabditis indica (Poinar et al. 1992) IBCBn 05 caused 33–43% mortality in D. suzukii pupae under laboratory conditions (Brida et al. 2019). Drosophila suzukii pupae appear to be more susceptible to nematode infections than larvae (Ibouh et al. 2019). Thus, the present study evaluated the pathogenicity and virulence of S. rarum at different concentrations and temperatures on the mortality of D. suzukii pupae and the effects on the longevity of adults in laboratory. To our knowledge, S. rarum has not been tested on D. suzukii, it was not mentioned in prior D. suzukii biological control reviews (Garcia et al. 2017; Lee et al. 2019; Wang et al. 2020a, b).

Materials and Methods

Steinernema rarum PAM 25

The Steinernema rarum PAM 25 isolate was obtained from the ‘Oldemar Cardim Abreu’ entomopathogenic microorganism collection of the São Paulo Biological Institute, São Paulo, Brazil. Experiments were conducted at the Insect Ecology Laboratory of the Department of Ecology, Zoology and Genetics belonging to the Biology Institute of the Federal University of Pelotas, state of Rio Grande Sul, Brazil. Infective juveniles (IJs) of S. rarum PAM 25 were multiplied within fifth-instar caterpillars of Galleria mellonella (Linneaus 1758) (Lepidoptera: Pyralidae). For multiplication, five G. mellonella caterpillars were placed in a Petri dish (9 cm) with two sheets of filter paper, on which a 1.5-ml suspension was inoculated at a concentration of 500 IJs/dish. Petri dishes were sealed with PVC film and stored in an air-conditioned Bio-Oxygen Demand (BOD) chamber at 25 ± 2°C and 80 ± 10% RH. Three days after death, caterpillars were transferred to a White trap (White 1927). The IJs emerging from caterpillars were collected in distilled water (1 cm deep) in Erlemeyer flasks kept in a BOD chamber at 18 ± 1°C, 70 ± 10% RH, and used 1 d after collection (Shapiro-ilan and SEGAL 1996, Gaugler and Han 2002).

Insects

The rearing of D. suzukii was done in a temperature-controlled environment (22 ± 1°C), with a relative humidity (RH) of 70 ± 10% and a 12 h photoperiod. Adults were kept in cylindrical glass tubes with a flat bottom (85 mm × 25 mm ± 0.5 mm) and fed an artificial diet. The diet was adapted from Dalton et al. (2011) consisting of 500-ml water, agar (4 g), yeast (20 g), corn flour (40 g), sugar (50 g), 1.5-ml propionic acid, and Nipagin (10%; 3.5 ml; Schlesener et al. 2017).

Pathogenicity to D. suzukii Pupae

The experiment compared two treatments, 1,000 IJs/ml of S. rarum PAM 25 and negative control (water), with 10 replications each. For each replication, five D. suzukii pupae (48 h old) were placed in plastic containers (200 ml) with perforated lids containing 50 g of autoclaved sand substrate, with 5% humidity. A suspension of 2 ml of 1,000 IJs/ml S. rarum PAM 25 was inoculated. The control treatment consisted of 2-ml distilled water without nematodes. Afterwards, the treatments were stored in BOD at 25 ± 1°C, 70 ± 10% RH, and 12 h photoperiod. The evaluations were carried out over 10 d (mean time for total emergence of adults at 25°C), and dead pupae were counted. Pupae that did not emerge were dissected under a stereoscope in distilled water using entomological pins (40 × 0.50 mm), to observe the cause of death and IJs were counted.

EPN Concentration and Temperature on Mortality of D. suzukii

For each replication, five D. suzukii pupae (48 h old) were placed in plastic containers (200 ml) with perforated lids containing 50 g autoclaved sand substrate, with 5% humidity. A 2-ml suspension at concentrations of 1,500; 2,000; 2,500; 3,000; 4,000 IJs/ml S. rarum PAM 25 was inoculated. The control treatment consisted of 2-ml distilled water without nematodes. Subsequently, treatments were stored in BOD at 14, 22, and 25°C, 70 ± 10% RH with 12 h photoperiod. The experiment had six treatments (five concentrations + control treatment) per temperature with 10 replications per concentration–temperature combination. Evaluations were carried out over 10 d (mean time for total emergence of adults at 25°C), with dead pupae counted as described above. Based on the results, the concentrations necessary to kill 50 and 90% (lethal concentration ‘LC₅₀ and LC₉₀’) of D. suzukii pupae exposed to the S. rarum PAM 25 isolate were estimated.

Longevity of D. suzukii Adults That Survived Pupal Exposure to EPN

To assess the longevity of D. suzukii adults, several IJ concentrations and temperatures studied were used; pupae were specifically exposed to 4,000 IJs at 14°C, 1,500 IJs at 22°C, and 2,000 IJs at 25°C. The control treatment consisted of 2-ml distilled water without nematodes. In all treatments, containers with 50 g of autoclaved sand as substrate, 5% humidity, and five D. suzukii pupae (48 h old) were used. After inoculation of S. rarum PAM 25 IJs, plastic containers were covered with perforated lids (~1 mm diameter) for aeration and kept in a BOD chamber at the respective temperatures with 70 ± 10% RH and 12 h photoperiod. The evaluations were carried over 10 d, and the dead pupae were counted as described above. Adults that emerged from treatments were transferred to 500-ml plastic cups with a bottom covered with voile fabric (5 cm) and fed with artificial diet (Dalton et al. 2011), with water supplied in moistened cotton. Evaluations were carried out daily and the dead adults were transferred to Petri dishes with distilled water for dissection with the aid of entomological pins (40 × 0.50 mm) quantifying the IJs inside the insect.

Statistical Analysis

In the first bioassay, data were observed to confirm pathogenicity and virulence of S. rarum PAM 25 on D. suzukii pupae. From the second bioassay, for analysis of the studied variables (concentration and temperature), the generalized linear models (GLMs) of the exponential distribution model (Nelder and Wedderbum 1972) were used. To compare treatments, Tukey multiple comparisons (P < 0.05) were performed using the glht function of the Multicomp package, with adjustment of P values. In the third bioassay, separate t-tests were used to compare the control versus nematode treatment at each temperature/concentration. All analyses were performed using statistical software (R Development Core Team 2019). A binomial model with a log–log complementary function (gompit model) was used to estimate lethal concentrations (LC₅₀ and LC₉₀), using the Probit Procedure in SAS 9.2 (SAS Institute 2011).

Results

Pathogenicity to D. suzukii Pupae

The isolate S. rarum PAM 25 was pathogenic to D. suzukii pupae, with a pupal mortality rate of 48% (24 of 50 died). The number of IJs recorded for S. rarum was 18.1 ± 1.2 IJs/pupae. In the control treatment (water), there was 100% pupae viability.

EPN Concentration and Temperature on Mortality of D. suzukii

At a temperature of 14°C, the mortality of D. suzukii pupae was 70%, at a concentration of 4,000 IJs/ml with 33.9 IJs/pupae (Table 1). There was no difference in the mortality rate in the other concentrations, which ranged from 28 to 44%. However, the number of IJs in pupae varied from 5.90 IJs/pupae at the concentration of 1,500 IJs/ml to 23.6 IJs/pupae at the concentration of 3,000 IJs/ml. Lethal concentrations (LC₅₀ and LC₉₀) were obtained in smaller numbers of IJs for the S. rarum PAM 25 isolate at a temperature of 22°C with 1,436.4 IJs for LC₅₀ and 3,711 IJs for LC₉₀ (Table 2).

Longevity of D. suzukii Adults That Survived Pupal Exposure to EPN

The longevity of D. suzukii adults was influenced by the presence of S. rarum PAM 25 IJs (Fig. 1). At 14°C with a concentration of 4,000 IJs/ml, the mean longevity of infected adults was 5.18 d, differing from the control group with 8.97 d and a mean value of 1.67 IJs/adult. At 22°C with a concentration of 1,500 IJs/ml, the mean longevity of infected adults was 6.26 d, differing from the control with 10.10 d and a mean of 5.59 IJs/adult. At 25 with a concentration of 2,000 IJs/ml, the mean longevity of infected adults was 6.79 d, differing from the control group with 9.53 d and a mean value of 8.08 IJs/adult.

Discussion

Among the nematode species already reported, S. rarum has shown promise for pest management of other pests (Cagnolo and Almiran 2010), and may be a potential biological agent for the management of D. suzukii. In the present study, D. suzukii pupae were susceptible to S. rarum PAM 25 infective juveniles in the concentration of 1,000 IJs/ml. In Brazil, S. rarum PAM 25 was isolated by Barbosa-Negrisoli et al. (2010) in native areas of the state of Rio Grande do Sul and corresponded to the most common nematode found in soil samples (36.8%), with high prevalence in sandy soils at average temperatures of 25°C. The exploration of native EPN species for pest management is one of the main factors to achieve successful biological control (Brida et al. 2017). This species of nematode had pathogenicity and virulence evaluated in several insect pests such as Anthonomus grandis (Boh, 1843) (Coleoptera: Curculionidae), Spodoptera frugiperda Smith, 1979 (Lepidoptera: Noctuidae), Cullex pipens L. (Diptera: Culicidae) (Doucet (1999), Culex apicinus (Diptera: Culicidae) (Cagnolo and Almirón 2010), and Ceratitis capitata (Wiedemann 1824) pupae (Jean-Baptiste et al. 2021). Other EPN species have been tested on D. suzukii pupae with mortality below 60% This includes S. kraussei at 22°C with 33% mortality and at 2 °C with 57%, S. feltiae at 22°C and 25°C both with 34% mortality, and S. carpocapsae at 22°C with 43% and at 25°C with 52% mortality. All cited species were tested at high concentrations of 10,000 IJs/ml (Cuthbertson et al. 2014, Cuthbertson and Audsley 2016).

Although S. rarum PAM 25 has convenient thermal requirements at 25°C (where the species was isolated from the ground, Barbosa-Negrisoli et al. 2010), in the present study, the higher mortality rates were found at temperatures of 14 and 22°C, the latter considered optimal for D. suzukii development. At 25°C, 60–76% of adults emerged, this temperature did not show great results for the potential pathogenicity of the nematode. An important factor to consider is how low temperatures influence the metabolism of insects. As D. suzukii enters diapause, ceasing its metabolic activities and extending its time at the pupae stage, and consequently prolongs its period of exposure to parasite. This likely explains the high mortality rate (72%) at the lowest temperature tested, 14°C. IJs at temperatures between 8 and 20°C conserve lipid reserves longer, so the ability of IJs to infect is greater, as they have energy reserves to better search for their hosts (Andaló et al. 2011). At 22°C, S. rarum PAM 25 had a rapid emergence in Galleria mellonella (4.75 d), significantly responding at temperatures around 22°C, the EPNs of this species show greater activity (Norton and Garcia-del-Pin 2009, Andaló et al. 2011, Brida et al. 2017). Steinernema rarum maintains its infectivity at 23 ± 2°C (Cagnolo 2008). However, in the 25°C bioassay, the mortality rate ranged from 18 to 30%, indicating that this temperature can be favorable to both nematode and insect, but does not lead to high mortality rates, and consequently leading to a higher emergence rate of D. suzukii.

The longevity of D. suzukii adults was influenced by the presence of IJs of S. rarum PAM 25. When penetrating the insect’s integument, IJs usually cause mortality between 24 and 48 h; however, the emergence of infected adults indicates an insect resistance to infection during the pupal period (Woltz and Lee 2017). The resistance of D. suzukii is due to its rapid immune response with high production of hemocytes, this species is capable of producing five times more than D. melanogaster, which makes it significantly more resistant to parasitism (Kacsoh and Schlenke 2012). Nevertheless, at its larval stage, D. suzukii demonstrates no immunological efficiency to symbiotic bacteria of S. carpocapsae and X. nematophila, affecting the host humoral response (Garriga et al. 2020). The synthesis of these genes in fruit flies was observed in adults of C. capitata after infection by S. carpocapsae, which produced specific genes, aiming at the inhibition of the microorganism (Garriga et al. 2020). Inactivation of the pathogen during the pupal phase of the insect and reestablishment of its activity during the adult phase indicate the ability of these organisms to overcome the insect’s immune system.

The inactivation of the pathogen present in the nematode during the pupal stage and its presence in the adult stage indicates the ability of these organisms to persist in the insect (Nomano et al. 2015). In D. suzukii, the average time from pupae to adults at temperatures of 14°C is 8.97 d, at 22°C is 10.10 d, and at 25°C is 9.53 d (Tochen et al. 2014). After exposure to EPNs in the present study, there was a reduction in the longevity of D. suzukii adults to 5.18 d at 14°C, 6.26 d at 22°C; and 6.79 d at 25°C. EPNs reduced the longevity of adults, shortening the life span of flies and, consequently, lowering the damage they can cause to fruit. Although the inoculated concentration of IJs is of great importance for the success of EPNs infection (Brida et al. 2017), we verified here that regardless of concentration or temperature, IJs were able to resist the immune system of D. suzukii, persisting in adulthood and causing mortality in a short period.

In Brazil, the search for nematode-based products with potential for pest control is growing annually (Brida et al., 2017). and studies that characterize the natural occurrence of nematodes in fruit orchards in Brazil (Barbosa-Negrisoli et al. 2009) provide potential candidate species. It is important to continue efficacy trials in the field, where various environmental parameters such as temperature, moisture, vegetation types, and soil properties can affect the survival and virulence of nematodes in the field. However, based on our results, S. rarum shows promise as a tool for an integrated management program of D. suzukii.

Acknowledgments

This study was financed in part by the Federal University of Pelotas (UFPel)—PIB-M/D Program. The author thank the National Council of Technological and Scientific Development (CNPq) for the productivity scholarship provided to FRMG and Federal University of Pelotas for providing the space for this study.

References Cited