A Review of Entomopathogenic Nematodes as a Biological Control Agent for Red Palm Weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae)

Simple Summary The red palm weevil, Rhynchophorus ferrugineus, is a significant pest to palm plantations globally and directly impacts economic activities. These weevils’ cryptic attack on palms is inconspicuous until the damage is irreparable. Chemical pesticides were used extensively in plantations to mitigate RPW infestation, and the results were impressive. However, their negative impact on the environment, nontarget organisms, and insecticide resistance is a primary concern. Therefore, alternative preventive and curative solutions based on the natural enemy concept are safer for the environment and more sustainable. This review highlights the use of entomopathogenic nematodes and their symbiotic bacteria as biological control agents against the red palm weevil and storage formulation. Abstract Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) is a severe pest of palm trees worldwide. The development and feeding activities of R. ferrugineus larvae inside the trunk damage palm trees. However, the absence of noticeable infestation signs at an early stage contributes to the spread of the attack. Integrated pest management (IPM) has been introduced to control R. ferrugineus infestation by implementing various approaches and techniques. The application of chemical pesticides has shown impressive results. However, biological control should be applied as an alternative solution due to adverse environmental impacts and pest resistance issues. One example is the use of entomopathogenic nematodes (EPNs) as biological control agents, which can forage and attack targeted pests without compromising the environment and other nontarget organisms. EPNs and their symbiotic bacteria have a mutualistic interaction that can kill the host within a short period of time. Therefore, this review emphasizes the effectiveness of entomopathogenic nematodes and their symbiotic bacteria against R. ferrugineus.


Introduction
The red palm weevil (RPW), Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae), is the primary pest of date palm (Phoenix dactylifera), canary palm (Phoenix canariensis), coconut (Cocos nucifera), and other palm trees [1][2][3][4]. Approximately 40 palm species are host to the RPW, with the majority being from the Arecaceae family, with one The duration of the larval development phase, which can range from 24 to 210 days, is influenced by diet and temperature, while the host plant species determines the number of larval instars [9,38]. RPWs tend to have a shorter development period if the environment and host plant are favorable to insects [39]. The plant host species influences the fertility of females RPWs [33]. RPWs are reared in laboratory facilities for their continuous supplies in various research, and their diet mainly comes from abundant and nearby sources. Norzainih et al. [40] successfully reared RPWs in the laboratory by feeding them sugarcane and reported the production of eight larval instars within 80 days. In another study, El-Zogby and Abdel-Hameid [41] demonstrated that a sugarcane diet produced 12 instar larvae in 89 days. Different ambient temperatures are likely to be the cause of these inconsistent results. Furthermore, within a year, RPWs can produce 3-4 generations or more in a single palm tree [42,43].

Red Palm Weevil Infestation
R. ferrugineus infestation on canary palms can be classified into five stages, according to Güerri-Agulló et al. [44]. The first level of early infestation begins with the absence of visual indicators of RPW assault on the palm tree and progresses to the formation of pits and notches in the leaves at the second level. The third and fourth stages are characterized by uneven leaves in the crown, with frond skirting pointing downwards. Finally, the palms die in the last stage.
In oil palm, the RPW infestation level can be classified into the early, intermediate I and II, and final stage. The intermediate stage can be detected as early as the fourth week of the attack with sawdust at the trunk's base and visible dark sap accompanied by odor from the fermentation process. Consequently, the fronds begin to collapse, and at the final stage, the collapsed fronds turn brown. Early and intermediate infestation can be treated by injecting insecticides into the trunk. The final stage requires the trees to be eradicated by cutting them down according to the guidelines provided [45].

Control Management of the Red Palm Weevil
Several implementations have been performed to manage RPW infestation, including trapping and monitoring, preventive and curative techniques, and plant quarantine treatments [3,38,46,47]. Preventive and curative treatment should be performed at the early stage of pest infestation to protect the palm from further damage and for recovery of the infested palm [5]. The trapping technique involves using pheromones, with both chemical synthesis and food bait showing significant decreases in RPW infestation. However, synthetic food baits are better than natural food baits since they can attract more weevils and can survive over a longer period [48]. Insecticides based on carbamate, organophosphate, phenylpyrazole, and neonicotinoid are used for preventative and curative treatments [38,47,49]. Early detection of RPW infestation can be performed using a trained dog to detect the foul odor of infested trees [9,16]. Alternatively, acoustic detection can be achieved by detecting the feeding activities of larvae within the stem [27,[50][51][52]. Several techniques have been applied to control weevil infestation in coconut plants in Malaysia. These include pheromone traps and chemical and physical controls eradicating the infested palm. The chemical controls involve spraying Cypermethrin on the crown, canopy, and stem of the palm and trunk injection with Methamidophos or Monocrotophos [53].
Management of RPW infestation within the palm trunk is also possible by using biological control agents such as EPNs, as they can penetrate, invade, and kill its prospective pests [54]. Much research has been carried out on the effectiveness of entomopathogenic nematodes (EPNs) against insect pests, especially on the RPW [55]. Besides EPNs, Metarhizhium anisopliae and Beuveria bassiana are entomopathogenic fungi commonly applied in IPM to kill RPWs in the field [56][57][58]. The pathogenicity of indigenous isolates, M. anisopliae strain MET-GRA4 against adult red palm weevils (RPWs), was investigated in vitro with different spore viabilities. The isolates were pathogenic, with 100% mortality 21 days after infection [58]. The only virus found in the RPW is the highly potent cytoplasmic polyhedrosis virus (CPV). The virus was first discovered in Kerala, India, where it infected all stages of the RPW. During the late larva stage, infection resulted in malformed adults and significantly reduced insect populations [59,60]. The efficacy of Bacillus thuringiensis (Bt), an entomopathogenic bacterium characterized by its production of insecticidal crystal proteins on larvae and adults of R. ferrugineus, was also studied. Infection with B. thuringiensis subspecies kurstaki identified from Egyptian larvae successfully controlled the RPW in laboratory conditions [60,61]. A study using commercial bacteria-based biopesticide reported the RPW larval mortality was high (>50%) at the lowest concentration (0.5 mg/mL), and it reached 85% at 2.0 mg/mL [62]. Larvae and adult mortality ranged between 46.86-58.36% and 26.79-39.04%, respectively, after 21 days of exposure to B. thuringiensis var. kurstaki [63].

Biology of Entomopathogenic Nematodes
Nematodes are microscopic, multicellular, and nonsegmented bodies of worms under the phylum Nematoda. Nematodes have adapted to live in various environments and have symbiotic relationships with other organisms [73]. The life cycle consists of the egg stage, four larval stages, and adult stage [74]. The life cycle of EPN is 5-10 days, depending on temperature, bacterial symbiont, and ability to suppress the immunity of the insect host [75,76]. EPNs are widely distributed, but the species varies according to geographic regions and habitats [77,78]. There are 30 families of nematodes associated with insects, plants, and vertebrates [79]. Steinermatidae and Heterorhabditidae are significant families widely used as control agents of insects. Currently, there are two genera in Steinernematidae, Steinernema Travassos, 1927 (comprises over 30 species), and Neosteinernema Nguyen and Smart, 1994 (one species, Neosteinernema longicurvicauda). On the other hand, Heterorhabditidae is solely represented by the genus Heterorhabditis Poinar 1976 and one species, Heterorhabditis bacteriophora [80,81]. However, Hunt and Nguyen reported that 95 valid species of Steinernema and 16 species of Heterohabditis were described by the end of 2015 [82].
A symbiotic Gram-negative bacterium, genus Xenorhabdus in Steinernema and Photorhabdus in Heterorhabditis, lives in the modified intestines of infective juvenile (IJ) nematodes [83,84]. When the IJ invade their specific host after entering via the anus, spiracle, and mouth, the symbiotic bacteria released by the IJs in the hemocoel of the target insect multiply and cause the death of the insects within 24-48 h due to septicemia [85,86]. In addition, the symbiotic bacteria produce secondary metabolites that cause cytotoxic activity. It will cause the hemolymph immunodepression of the insect, thus leading to septicemia and death of the host [87]. The secondary metabolites produced by the symbiotic bacteria also inhibit other bacteria, fungus, and protists from developing in the nutrient-rich hemolymph of the dead cadaver, thus providing a suitable condition for the nematodes to reproduce [88,89]. The IJs feed on the multiplied bacteria and digested host tissue until all the sources are depleted and begin to search for a new host [90]. As a result, only infective juvenile (IJ) or dauer larva can survive outside the host [86] (Figure 1). The brown color of the insect cadaver indicates the insect was killed by Steinernematid nematodes, while the red color indicates Heterorhabditid species. The color difference is due to the pigment released by the symbiotic bacteria in the cadaver [80,85]. nematodes, while the red color indicates Heterorhabditid species. The color difference is due to the pigment released by the symbiotic bacteria in the cadaver [80,85].
The symbiotic bacteria, Xenorhabdus and Photorhabdus, are essential to the EPN. They kill the insect host in a short period, providing a suitable environment for the EPN to reproduce and produce antibiotics and secondary metabolites that prevent any development of other microorganisms and convert the host tissue into food [96]. In exchange, the EPN provides protection and access to the host's hemolymph [97]. However, in this bacterium-nematode complex, nematodes are responsible for overcoming the host immunological defense by making lipid or protein [98][99][100]. Thus, the bacterium-nematode complex plays a vital role against insect hosts.
The bacteria Xenorhabdus spp. and Photorhabdus spp. exist in two phenotypes, primary and secondary [101]. The first-phase bacteria are smaller, 3-4 µm in length, and induce more secretory enzymes, toxins, antibiotics, and protein with the oval or circular shape. The secondary bacteria are larger, 6-7 µm in length, and do not produce enzymes or antibiotics or flat colonies. The primary bacteria only reside in the insect's body for a few hours after being released by the nematodes before being converted to secondary bacteria. The transformation is believed to adapt to the external environment [102]. Research conducted on X. stockiae and P. luminescens subsp. Akhurstii [88] against Aedes aegypti and Aedes albopictus has shown that the symbiotic bacteria are malicious against mosquitoes and demonstrated that they could be utilized as biological control agents.

Application of Entomopathogenic Nematodes as Biological Control Agent
The nematodes use two basic strategies in finding a host: active searching, i.e., cruising or foraging, and the passive method by waiting for the host to contact with the nematodes and ambush [94]. As a result, the EPN can reach its specific host even in a
The symbiotic bacteria, Xenorhabdus and Photorhabdus, are essential to the EPN. They kill the insect host in a short period, providing a suitable environment for the EPN to reproduce and produce antibiotics and secondary metabolites that prevent any development of other microorganisms and convert the host tissue into food [96]. In exchange, the EPN provides protection and access to the host's hemolymph [97]. However, in this bacteriumnematode complex, nematodes are responsible for overcoming the host immunological defense by making lipid or protein [98][99][100]. Thus, the bacterium-nematode complex plays a vital role against insect hosts.
The bacteria Xenorhabdus spp. and Photorhabdus spp. exist in two phenotypes, primary and secondary [101]. The first-phase bacteria are smaller, 3-4 µm in length, and induce more secretory enzymes, toxins, antibiotics, and protein with the oval or circular shape. The secondary bacteria are larger, 6-7 µm in length, and do not produce enzymes or antibiotics or flat colonies. The primary bacteria only reside in the insect's body for a few hours after being released by the nematodes before being converted to secondary bacteria. The transformation is believed to adapt to the external environment [102]. Research conducted on X. stockiae and P. luminescens subsp. Akhurstii [88] against Aedes aegypti and Aedes albopictus has shown that the symbiotic bacteria are malicious against mosquitoes and demonstrated that they could be utilized as biological control agents.

Application of Entomopathogenic Nematodes as Biological Control Agent
The nematodes use two basic strategies in finding a host: active searching, i.e., cruising or foraging, and the passive method by waiting for the host to contact with the nematodes and ambush [94]. As a result, the EPN can reach its specific host even in a sealed area such as a tree trunk. Major target insects of EPNs in IPM are Coleoptera and Lepidoptera, but it also applies to other orders, such as Thysanoptera, Diptera, Orthoptera, Blattodea, Hymenoptera, and Siphonaptera [81]. Besides that, the application of EPNs against insect pests in the orchard system also contributes to promising results [103]. Shahina et al. [104] conducted a laboratory bioassay of seven EPNs species against all life stages of the RPW, including the eggs. All seven EPNs species killed all stages of the RPW, but the emergence

Biological Assay on Pathogenicity of Entomopathogenic Nematodes against R. ferrugineus
Various studies on the efficacy of EPNs against the RPW have been conducted, particularly in the Mediterranean, Middle East, and Southern Asia (Table 1). Steinernema carpocapsae caused the highest mortality of RPW larvae at 98.9% after eight days of treatment under laboratory conditions, followed by H. bacteriophora at 86.9% and Steinernema feltiae at 38.9% [55]. The efficacy assessment of EPNs was conducted in the date palm field in the UAE [106], where a local isolate of Heterorhabditis indicus appeared to kill larvae, adults, and cocoons, of the RPW successfully within a short period when compared to Steinernema riobrave and S. abbasi. The RPW population in the date palm field also showed a successful decline after the second treatment within two months. Due to their high pathogenicity against insect hosts [34], the two common EPNs are utilized as biological control agents in IPM are S. carpocapsae Weiser and H. bacteriophora Poinar [107], as shown in Table 1.

Formulation
EPNs can be stored and large-scale produced as biopesticides in two ways, in vivo and in vitro [117]. The production of EPNs has enabled at least 13 species of Steinernematids and Heterorhabditids to be commercialized for biological insect control purposes [118]. For small-scale experiments such as laboratory bioassay with low dosage and mortality, the EPN can be inoculated on a dish and absorbent paper before being transferred to White Trap for in vivo harvesting. This method is dependent on environmental conditions that affect yields, such as optimal temperature, proper aeration, and moisture [117]. On the other hand, in vitro methods can offer mass production of the nematodes to fulfill the demanding needs for EPN application in the field. The rearing of nematodes via in vitro methods does not require insects as hosts but through solid or liquid culture [119]. In vitro reliable culture methods are performed by rearing the nematode with respective symbiotic bacteria in a growth medium. In contrast, nematodes are cultured in vitro liquid culture methods after the symbiotic bacteria were introduced in the liquid culture [120]. Next, the mass-produced nematode will be transformed into a new entity or product that practical methods can apply. This process is known as EPN formulation [121] and involves inclusions of carriers, additives, and active ingredients. Various forms and mediums have been used to store and transport EPNs, including aqueous suspension, synthetic sponges, gels, clay, powder, and infected cadaver [122]. These formulations are widely used and commercialized in other countries; for instance, S. carpocapsae was commercialized byproduct Sanoplant from Switzerland, Helix from Canada, ORTHO Biosafe USA from the USA, and BASF from Germany [97,122].

Conclusions
Rhynchophorus ferrugineus is indeed a ferocious pest in palm trees worldwide. R. ferrugineus infestation creates a substantial economic impact on the plantation and food supply. Many infested trees need to be cut down to prevent dispersion towards other trees. Moreover, farmers and planters have to spend more on preventive and curative treatments to prevent the infestation from spreading. Although chemical treatments are effective, they have numerous drawbacks, including pest resistance, worker's health, and environmental concerns. Therefore, alternative methods can provide a safer approach yet more effective in overcoming this infestation case of R. ferrugineus. Microbial entomopathogens are effective biological control agents since each organism has its approach and strategies for invading its potential host. Entomopathogenic nematodes (EPNs) are proven to be effective biological control agents in managing RPW infestation in various studies. EPNs and their symbiotic bacteria are the significant mutualistic duo that can give promising results in overcoming this infestation issue of R. ferrugineus. For a more specific outcome, many approaches can be explored in leveraging the pathogenicity properties of the EPNs and their symbiotic bacteria.