Introduction

The confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae), is one of the most harmful pests of stored grain beetle, native to Africa, but usually found all over the world (Hagstrum and Subramanyam 2009). As a secondary pest, the confused flour beetle primarily prefers infested or mechanically damaged grains. Both adults and larvae are responsible for severe post-harvest losses, particularly in stored products, such as wheat, maize, barley, sorghum, oilseeds, spices, bran, and dried herbs. (Boyer et al. 2012; Hagstrum et al. 2013). Post-harvest losses in developed countries are estimated that 5–10%, whereas this rate rises to 75% in developing countries (Boxall 2001; Hodges et al. 2011; Mason and McDonough 2012).

Chemical insecticides are considered the primary control method for insect pests. However, the high frequency and widespread use of chemical insecticides stimulates the development of resistance in insects and causes environmental pollution as they cannot be broken down easily. The demand for pesticide-free products in society, and environmental problems caused by the use of synthetic chemical pesticides against such pests, encourage scientists to search for different control options (Lu and Wu 2010; Alkan 2020; Ertürk et al. 2020).

The EPNs are widely used in many countries and have attracted interest as potential biocontrol agents. They can be preferred in the control of harmful insects due to their wide host range, active host seeking, easy and low cost mass production, long-term efficacy, easy application, compatibility with most chemicals and environmentally safe for humans and other non-target organisms (Kaya 1990; Shields et al. 1999; Rumbos and Athanassiou 2017; Vitta et al. 2017; Belien 2018; Yuksel et al. 2019; Yağci et al. 2021a, b). EPNs are obligate parasites and they have the ability to actively search for their host. A number of stimuli factors such as the body temperature of the host insect, the CO2 released by the host, the host’s feces, and kairomones are affecting the effectiveness of infective juveniles (IJs) (Athanassiou et al. 2008; Rasmann and Turlings 2008; Rumbos and Athanassiou 2012; Tülek et al. 2015; Yağcı et al. 2021c). The IJs, the only free life stage of the EPNs, enter the host through natural openings or penetrate into the insect cuticle. The toxins produced by the symbiotic bacteria, Xenorhabdus spp. (Enterobacterales: Morganellaceae) and Photorhabdus spp. (Enterobacterales: Morganellaceae) associated with EPNs sacrifice the host within between 24 and 48 h (Woodring and Kaya 1988; Shapiro and Lewis 1999; Forst and Clarke 2002; Tülek et al. 2015; Canhilal 2016; Alotaibi et al. 2022).

EPNs are promising alternatives to chemical pesticide that are widely used in agricultural biological control programs (Ffrench-Constant et al. 2007). Steinernematidae and Heterorhabditidae are well known and utilized endoparasites for numerous agricultural insect pests. However, the pathogenicity of EPNs on insect pests of stored commodities are limited despite that EPNs have the potential for control of coleopteran pests (Duncan and McCoy 1996; Laznik et al. 2010; Javed et al. 2020). EPNs have received little attention in post-harvest protection except for Steinernema carpocapsae and S. feltiae that so far are the most commonly tested steinernematid nematode species against stored product pests (Rumbos and Athanassiou 2017).

It is clear that in vitro experiments are primary and essential for the success of field experiments. Therefore, the present study aimed to determine the pathogenicity, virulence, and biocontrol potential of different native EPNs isolates from Türkiye of S. carpocapsae (Tokat-Bakışlı05) (Weiser) (Rhabditita: Steinernematidae) and S. feltiae (Tokat-Emir) (Filipjev) (Rhabditita: Steinernematidae), Heterorhabditis bacteriophora (TOK-20) Poinar (Rhabditita: Heterorhabditidae), H. bacteriophora (11KG) Poinar (Rhabditita: Heterorhabditidae) at different concentrations and temperature on the mortality of T. confusum adults under laboratory conditions.

Materials and methods

Insect rearing

Tribolium confusum

Adults of T. confusum were grown on cracked wheat-containing 5% brewing yeast (w/w), aged between 7 and 28 days (Athanassiou et al. 2016) in a 1000 ml capacity glass jar. The insect culture was raised in continuous darkness in a climate chamber at 25 ± 1 °C and 60 ± 5% relative humidity (R.H.) (Nüve ID 501, Ankara, Türkiye). The culture has been rearing since 2010 in the Entomology Laboratory of the Plant Protection Central Research Institute, Ankara, Türkiye.

Galleria mellonella

Wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) larvae, which are known susceptible to infection of entomopathogenic nematode species, were used in the experiments (Barrón-Bravo et al. 2021). For this purpose, the original stock culture of G. mellonella was received from the Department of Plant Protection, Faculty of Agriculture, Tokat Gaziosmanpaşa University, Türkiye. All EPNs cultures were raised in the Nematology Laboratory of the Plant Protection Central Research Institute, Ankara, Türkiye since 2018. The larvae of G. mellonella were fed on a diet containing glycerin (500 g), honey (500 g), wheat flour (890 g), dry baker’s yeast (222 g), milk powder (445 g), and beeswax (125 g). Beeswax and dry baker's yeast were melted and then mixed with other materials. The prepared artificial diet has been stored in the refrigerator for the intended use.

The last instar larvae of G. mellonella were used, according to Mohamed and Coppel (1983). Wax moth eggs were kept in the diet in one liter glass jar under 16:8 h light and dark under 23–24 ± 1 °C degree (Nüve ID 501, Ankara, Türkiye). After about 30 days, the wax moth’s last larvae were obtained for mass rearing.

Entomopathogenic nematodes culture

Steinernema carpocapsae (Tokat-Bakışlı05), S. feltiae (Tokat-Emir), Heterorhabditis bacteriophora (TOK-20), and H. bacteriophora (11KG) were obtained from the Plant Protection Department of Tokat Gaziosmanpaşa University, Türkiye. The nematode species were identified by Dr. Selçuk HAZIR and Dr. Harun ÇİMEN (Department of Biology, Faculty of Arts and Sciences, Aydin Adnan Menderes University, Aydin, Turkey) based on morphometric and molecular analyzes (Kepenekci et al. 2017, 2018; Çağlayan et al. 2020). EPNs were gathered on the last stage larvae of the greater wax moth at 25 ± 1 °C and 60 ± 5% R.H. under laboratory conditions according to Kaya and Stock (1997). For mass rearing of EPNs isolates; ten instar larvae of G. mellonella were placed in six cm diameter Petri-dishes lined with Whatman paper (No.1, Whatman International, Maidstone, UK), and wetted with distilled water. Suspension of juveniles of each nematode isolate were prepared, and then applied on the greater wax moth larvae. Petri-dishes were sealed with a parafilm, and then placed in the incubator at 25 ± 1 °C and 60 ± 5% R.H. IJs were collected from infected larvae by using the “White trap” method (White 1927).

Biossays

Experiments were conducted in 6 cm Petri-dishes under controlled conditions (15, 25 °C and at constant 65% R.H.). The dishes lined with Whatman paper (No.1 Whatman International, Maidstone, UK) and 0, 25, 50, and 100 IJs/larva concentrations were prepared. The determined nematode concentrations were added to the filter paper with a pipette in 1 ml of distilled water to each Petri-dish. Ten T. confusum adults were introduced into Petri-dishes containing 5 g sterilized cracked wheat. For control treatments, only distilled water was used. Petri-dishes were sealed and then wrapped with Parafilm to prevent insects from escaping. The Petri-dishes were put in a rearing chamber. Mortality of beetles was checked at 48, 72, and 96 h after the inoculation. Dead beetles were counted at each time interval (Yuksel et al. 2019; Yağci et al. 2021a, b, c). The whole experiment was repeated three times with different dates. Each treatment was replicated five times and 10 individuals were used in each replicate.

Data analysis

The data obtained from the concentrations screening tests were first converted to percentage mortality, and then arcsine transformation was carried out (Zar 1999; Warton and Hui 2011). To separate the mean differences, Tukey’s multiple range test was used at 5% significance levels. All statistical analyses were performed using MINITAB Release 18 statistical software with the general linear model in order to determine the statistical interactions between treatments (Mckenzie and Goldman 2005).

Results

The insecticidal activity of EPNs isolates against T. confusum was determined, in order to be used as biological control agents. All tested EPNs isolates caused varying levels of death and were found significant (P < 0.05). Also, the mortality rate increased with increasing concentrations, temperature, and exposure times (Tables 1, 2). The adults of T. confusum were found susceptible to all EPNs isolates. The results show that all EPNs isolates increased the mortality of T. confusum (P < 0.05) when compared with the control group, which recorded a maximum mortality of 0.7% at all temperatures, exposure times, and concentrations.

Table 1 Efficacy of entomopathogenic nematode isolates on mortality rate of confused flour beetle at 15 °C
Full size table
Table 2 Efficacy of entomopathogenic nematode isolates on mortality rate of confused flour beetle 25 °C
Full size table

Mortalities of confused flour beetle varied strongly at 15 °C (Table 1). H. bacteriophora (TOK-20), recorded the highest mortality rate with 57.7% at a concentration of 100 IJs/adult after 96 h of exposure. H. bacteriophora (TOK-20) isolate was the most effective, which caused 17.4% mortality at 50 IJs/adult concentration after 96 h of exposure. S. carpocapsae (Tokat-Bakışlı05) showed the highest efficacy of 36.5% at the concentration of 100 IJs/adult after 96 h of exposure.The highest mortality rate for S. feltiae (Tokat-Emir) was 36.6% at the concentration of 100 IJs/adult after 96 h of exposure. However, the lowest effect at the concentration 25 IJs/adult was recorded with 1.6% mortality after 48 h. The highest mortality rate of 55.6% was resulted at a concentration of 100 IJs/ml from H. bacteriophora (11KG) isolate at 15 °C after 96 h of exposure.

The results showed that the highest mortality rate was 91% for H. bacteriophora (11KG) at 100 IJs/adults concentration after 96 h post treatment at 25 °C (Table 2). This mortality rate of H. bacteriophora (TOK-20), S. feltiae (Tokat-Emir), S. carpocapsae (Tokat-Bakışlı05) depicted 81.2, 74.1, 56.1%, respectively. The lowest mortality rate in all isolates at 25 IJs/adult were less at 25 °C temperature and all exposure period. It was found that the mortality rate was increased proportionally as exposure time passed from the inoculation.

Discussion

This research studies showed that the virulence and biocontrol potantial of different EPNs isolates against T. confusum is dependent on ambient temperature and nematode concentration. Rumbos and Athanassiou (2012) found that S. carpocapsae caused low mortality rate (15.2 and 22.4%) on T. confusum after 4 and 8 days of exposure at 27 °C and 70% R.H. Alikhan et al. (1985) noted that the effect of S. carpocapsae against Tribolium confusum at two different concentration and calculated 48–56 h after death, and they caused low mortality levels. In the previous studies, Majić et al. (2021), studied the pathogenicity of different strains of S. feltiae at different concentrations of 0, 300, and 700 IJs insect−1 at 15 and 25 °C temperatures against T. castaneum. They found that the activity did not exceed than 82.0% depending on the insect biological stage, temperature, and EPNs concentration. Furthermore, Kepenekci et al. (2015) tested the virulence of H. bacteriophora, S. carpocapsae, and S. feltiae against the T. castaneum and T. confusum adults under three different temperatures (15, 20, and 25 °C) and found that S. carpocapsae was the most effective to the T. confusum (85.35%) and T. castaneum (86.47%) adults and other isolates were less effective. Javed et al. (2020) reported that different concentrations of S. pakistanenses (LM07), S. bifurcatum (LM-30) depicted higher mortality rates against T. confusum, at 30 ºC than at 20 ºC. They added that the same EPNs species were found to increase mortality rates with an increase in temperatures. Also, Laznik et al. (2010) reported that three different isolates of S. feltia showed varying efficacy against S. oryzae adults, and this difference is due to the origin of the isolates. It was concluded that different isolates of the same EPNs species showed different activity for the target insect.

In the present study, especially in the Heterorhabditis species were observed of weak mortality rates even at the highest concentrations, time, and temperature. On the contrary, Eldefrawy and Raheem (2017) found 100% mortality of T. confusum at variable doses of 1000, 2000, and 3000 IJs of H. bacteriophora.

Another comment to be made on this subject is that the smaller the host's biomass, the lower the infection density by EPNs (Wójcik 1986). T. confusum adults are small-sized insects. Due to the low biomass of this insect, it is thought that nematodes cannot produce new generations in high numbers within the insect body and this causes low mortality rates. As it is known, the bacteria that carry by the nematodes transform the insect body mass into a nutrient medium to complete the nematode life cycle. According to the obtained results, the mortality occurring in the exposed to EPNs insects varies depending on virulency, nematode species and strains, insect species, insect biological stages, EPNs concentrations, exposure time, and temperature. Unlike synthetic pesticides, EPNs like other insect pathogens, cause slow mortality in insects (Moore et al. 2000). Although the insect biological stage is considered an essential parameter in insect individual’s rapid or slow death, the larval stage is out of the scope of this study. Athanassiou et al. (2008) revealed that T. confusum larvae are more sensitive than adults to EPNs. They added that S. feltiae caused larval stage mortality rates ranging from 79 to 100% after 7 to 14 days of nematode exposure at the highest dose, respectively. One could say that an adult insect body was assessed to be less likely to be invaded by IJs due to the dense sclerotized integument, which presents a significant barrier to penetration of EPNs, when compared to a larval body structure.

Conclusions

The present study indicated that the S. feltiae (Tokat-Emir), S. carpocapsae (Tokat-Bakışlı05), H. bacteriophora (11KG), and H. bacteriophora (TOK-20) isolates can be used for control of T. confusum adults under controlled conditions. H. bacteriophora (11KG) was the most effective species against the confused flour beetle at 25 °C. EPNs are considered to have a high potential for biological control agents in storage facilities. Besides that, a few species have also been reported as commercially available (Grewal 2002; Georgis et al. 2006). However, there may be some difficulties in the effective use of nematodes as biocontrol agents under storage conditions. In order for nematodes to survive in these conditions, adequate humidity levels must be provided (Doberski 1981). These difficulties require efforts to develop new formulations that will enable nematodes to survive until they find their hosts and need to be tested under field conditions.

Author contribution

MY: Supervision, Conceptualization, Methodology, Investigation, Writing—original draft, SE: Conceptualization, Investigation, Writing—original draft, Supervision, Data curation, FDE: Methodology, Writing—review and editing, TAF: Methodology, Writing—review and editing.