On the efficiency of entomopathogenic nematodes (Rhabditida: Heterorhabditidae and Steinernematidae) on rust red flour beetle, Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae)

The rust red flour beetle, Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae) is a serious pest of stored grains and grain products across the world. This beetle is hold a significant place in Turkey by causing damages on stored products. T. castaneum primarily attacks milled grain and its derivates. Entomopathogenic nematodes (EPNs) are regarded as extremely an important biological control agent. EPNs kills their hosts within 48 h by the bacteria they carry. Efficacies of 4 isolates of EPNs Steinernema carpocapsae (Tokat Bakisli 05), S. feltiae (Tokat-Emir), Heterorhabditis bacteriophora (TOK-20) and H. bacteriophora (11-KG) against T. castaneum was investigated under laboratory conditions. The experiments were carried out thrice with 10 replicates at 2 different temperatures (15 and 25 ºC). EPNs isolates were tested at 3 different concentrations (250, 500 and 1000 IJs/ml) with a pure water as control. The overall mortality caused by H. bacteriophora (Kg11) was significantly higher than the other EPN species. At 25 ºC, H. bacteriophora (Kg11) at the highest concentration (1000 IJs/ml) caused 87.6% mortality after 120 h., followed by S. carpocapsae and S. feltiae with 79.22 and 75.3% mortality rates, respectively. The mortality percentages exhibited by all nematodes’ species at both temperatures were lowest at the concentration of (250 IJs/ml). At 15 °C, H. bacteriophora (Kg11) caused (55.2%) mortality rate at the highest concentration (1000 IJs / ml) after 120 h. The study suggested that these nematodes were efficient and could be recommended to control T. castaneum in its biological control programs.


Background
The red flour beetle, Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae) is a cosmopolitan pest of stored products (Nenaah 2014), with a polyphagous feeding habit (Bachrouch et al. 2010), attacking widespread variety of stored products and their by-products causing a loss in both quantity and quality (Obeng-Ofori and Reichmuth 1999). Product attacked by T. castaneum usually contains carcasses, and exuviae. Both larvae and adults cause serious losses by feeding on product and processed foods. The damage of larvae are mostly confined to the germ of the grains in wheat (White and Lambkin 1988). Fumigation by chemical pesticides still the main method for control stored grain pests. These Erdoğuş Egypt J Biol Pest Control (2021) 31:116 chemical pesticides have hazard effects on both the environment and the consumers, on the other hand caused insect resistance (Lu and Wu 2010), therefore, scientists work to use other methods to control store insects rather than chemical pesticides.
Entomopathogenic nematodes (EPNs) are an important biological control agent found in soil all over the world. Today, in many countries, EPNs are used in biological control programs of many economic insect pests. They are a group of soil dwelling nematodes that parasitize insects pests. Use of these nematodes is economical and eco-friendly, since they are harmless to non-target organisms, human health and the environment (Gulcu et al. 2017). These organisms infect insects in soil through natural openings or thin parts of the cuticle and release their symbiont bacteria in insect hemolymph. Death occurs within 16-24 h due to the bacteria reproducing in the insect's body (Kepenekci 2012).
This study aimed to evaluate the virulence of 4 EPNs' isolates against T. castaneum under laboratory conditions.

Methods
EPNs, T. castaneum adults, the great wax moth, Galleria mellonella L. cultures constituted the main materials of the study. Experiments carried out under laboratory conditions in the Nematology Laboratory of the Central Research Institute of Plant Protection (Ministry of Agriculture and Forestry of the Republic of Turkey) in 2020.

Rearing Galleria mellonella larvae
A special nutrient diet, containing 890 g of flour, 222 g of dry baker's yeast, 500 g of glycerin, 500 g of honey, 445 g of milk powder and 445 g Flour, bran, milk powder and yeast, mixed and then poured on a mixture of honey and glycerin, was prepared (Mohammed and Coppel, 1983). Galleria mellonella eggs were placed on a food medium in 1 lt glass jars and then placed in the incubator (16/8 h lighting set at 23-24 °C) for hatching and development of the larvae.

Rearing of entomopathogenic nematode species
Mature instar larvae of G. mellonella were used for rearing the EPNs. The larvae were placed on Whatman paper (White, 1927), soaked in sterile water, in small Petri dishes (diameter of 6 cm). The 2nd and 3rd stages of infective nematode were collected from the water by a dropper and applied on the G. mellonella larvae. Then, the lids of the Petri dishes were wrapped by a parafilm and placed in the incubator (20-23 °C). The Petri dishes were inspected every 10 days. The obtained juveniles were kept in a refrigerator (10 ºC).

Rearing of T. castaneum
Tribolium population grown in the Stored Crop Pests Unit of the Entomology Department, Ankara Plant Protection Central Research Institute, Turkey was used in the study. A mixture of dry yeast and wheat flour (1: 3) was used in the cultivation of Tribolium. 250 g of the nutrient mixture, sterilized at −18 °C for 120 h, was placed in a 1 L glass jar with a perforated lid for ventilation. Glass jars were kept in climate chambers at 25 ± 1 °C and 60 ± 5% RH. 500-750 adults were placed in each jar for oviposition and after a week, adults were removed from by passing through a 30 mesh sieve. The screened adult individuals were placed to a new jar containing food, and the continuity of the cultures was ensured.

Laboratory experiments of EPNs
For each nematode species, the experiments were carried out in plastic Petri dishes (9 cm) under the same conditions 3 times on different dates with 10 replicates per repeat and at 2 different temperatures (25 and 15 °C) in a climate chamber. T. castaneum adults (10 individuals) were placed by the help of soft forceps into Petri dishes with 5 g sterilized wheat crumbs bedding. Then, the EPN isolates prepared, using distilled water at 250, 500 and 1000 IJs/ml, were applied directly into the Petri dish. After the application, the Petri dishes were covered by a parafilm. Only pure water was used in the control group. The vitality of the adult individuals in the Petri dishes was counted regularly at the end of 48, 72 and 120 h and the mortality % were calculated. Cadavers were taken place "White trap" and were examined under a stereomicroscope. After one week, EPNs juveniles were obtained from infected T. castaneum adults.

Statistical analysis
Duncan multiple comparison test was performed with SPSS statistics 17. Square root transformation was applied to non-normally distributed data, followed by ANOVA (Duncan test).

Results
Statistical evaluations of the activitiy of the EPNs (48, 72 and 120 h post-inoculation), all the 3 parameters (nematode species, levels and temperatures) and their interactions were significant. The of all EPNs species was directly proportional to the mortality percentage of pest adults as presented in tables (1 and 2). The lowest mortality % was recorded at the lowest applied concentration (250 IJs/ml) and at 15 °C for the 4 EPNs species.

Discussion
Although EPNs are most effective in the larval stage, they can be effective on all insect stages. They enter the host's hemocell through the host's natural openings such as the mouth, anus, stigmas, hair follicles, or through thin parts of the cuticle (only in Heterorhabdites) (Bedding and Molyneux 1982;Wang and Gaugler 1998). EPNs are in a symbiotic relationship with bacteria of the genus Xenorhabdus (in Steinernematids) and Photorhabdus (in Heterorhabdites). IJs entering