Ecology, Adaptation, and Parasitism of Entomopathogenic Nematodes

Abstract

Entomopathogenic nematodes (EPNs) are a distinct group of insect parasitic nematodes widely used in biological pest control. Nematodes in Steinernematidae and Heterorhabditidae have a mutual association with pathogenic bacteria of Enterobacteriaceae family to kill insect hosts rapidly. In this book chapter, we would like to address the effect of ecology, behavior, symbiosis, and parasitism of EPNs for their entomopathogenic potential under field conditions in positive and negative way. Hence, this chapter will focus on four objectives—(1) The impact of biotic and abiotic factors in abundance, dispersal and persistence of EPNs, (2) the finding behavior of EPNs, (3) EPN adaptation strategies for survival during stress conditions, and (4) nature of nematode-bacterium symbiotic relationship and their role in killing insect pests. Through a comprehensive literature review and analysis, this chapter will contribute much to the existing knowledge on EPNs, emphasizing their ecological significance and the potential implications for sustainable pest control practices.

1. Introduction

In the intricate tapestry of Earth’s ecosystems, countless organisms engage in a relentless dance for survival and dominance. Among these, entomopathogenic nematodes, or nematode parasites, stand as fascinating actors in the theater of life. These diminutive yet potent creatures have evolved remarkable strategies to navigate their environments, exploit their host insects, and perpetuate their lineage. The chapter before you embarks on a journey into the captivating world of entomopathogenic nematodes, shedding light on their ecology, intricate adaptations, and their sinister yet awe-inspiring parasitic lifestyle.

From the labyrinthine soil ecosystems to the leafy canopies of towering forests, entomopathogenic nematodes have carved out niches as nature’s clandestine assassins. Among the diverse insect-parasitic nematodes, entomopathogenic nematodes (EPNs) are distinct and cooperate with insect-pathogenic symbiont bacteria to kill insect hosts. Nematodes in Steinernematidae and Heterorhabditidae have mutual association with pathogenic bacteria Xenorhabdus and Photorhabdus, respectively. EPNs kill insect hosts rapidly, usually within 48 hours of infection. Hence, they are being used worldwide for the biological management of insect pests of crops [1]. Their intricate interactions with their insect hosts, encompassing an array of nematode species and diverse host organisms, have confounded scientists for decades. In this chapter, we will unveil the enigmatic strategies these nematodes employ to locate, infect, and ultimately consume their unsuspecting prey.

The story of entomopathogenic nematodes is a story of relentless adaptation. It is a tale of microscopic creatures that have evolved an array of biological weaponry, from lethal mouthparts to symbiotic bacteria, enabling them to thrive in a world rife with challenges. Our exploration will delve into these adaptive marvels, providing insights into the evolutionary arms race that has fueled their success as formidable parasites. Yet, as we delve deeper into the world of entomopathogenic nematodes, we will also uncover the paradoxical intricacies of parasitism. While they spell doom for their insect hosts, these nematodes form intricate relationships with other organisms, particularly symbiotic bacteria, which assist them in their gruesome task. The chapter will unravel the symbiotic partnerships that sustain their parasitic lifestyle and illuminate the complex web of life in which they are entangled.

As we embark on this journey through the ecology, adaptation, and parasitism of entomopathogenic nematodes, we invite you to peer through the microscope and into the miniature worlds they inhabit. Prepare to be captivated by their astonishing strategies, awed by their adaptations, and intrigued by the profound ecological implications of their parasitic existence. These small but remarkable nematodes have much to teach us about the intricate balance of life on our planet.

1.1 Internal stress

During their free-living stage, entomopathogenic nematodes (EPNs) encounter a range of internal and environmental stressors. The capacity to effectively navigate these challenges assumes paramount importance in determining their success in infection and subsequent survival [2]. Various attributes of EPNs come into play, influencing their resilience in the face of both internal and external stressors, thereby shaping their survival as infective juveniles (IJs) within the soil.

1.1.1 Oxidative stress

Oxidative stress occurs when there is a disparity in the generation of reactive species (RS), including reactive oxygen species (ROS) and reactive carbonyl species (RCS), resulting from mitochondrial respiration and the antioxidant mechanisms of the nematode and its host. Due to their high reactivity, RS can interfere with cellular metabolism, and an overabundance or imbalance in their production can potentially lead to cellular demise [3]. To mitigate the effects of RS, diverse mechanisms are employed for detoxification, encompassing processes such as oxidation (e.g., facilitated by aldehyde dehydrogenases), conjugation (e.g., through interaction with glutathione), or reduction (e.g., facilitated by aldoketoreductases) [4]. In Heterorhabditis bacteriophora, IJ tolerance to H₂O₂ exposure correlates with their lifespan in sand [5]. Nematodes produce numerous enzymes dedicated to neutralizing reactive oxygen species (ROS) throughout their life cycle. While the effects of ROS and their detoxification mechanisms on Caenorhabditis elegans are well-documented, the influence of oxidative stress on the endurance of EPN infective juveniles (IJs) in soil is still not fully understood and warrants further exploration [6].

1.1.2 Nutritional stress

Infective juveniles (IJs) are the non-feeding stage and instead rely on their internal stored reserves of lipids, mainly triacylglycerols, and glycogen for their survival. These lipids make up roughly 20 to 31% of the nematode’s dry weight. In Heterorhabditis bacteriophora, the amount of unsaturated fatty acids accounts for 57% of the total fatty acids. But Steinernematid species tend to have a higher amount of saturated fatty acids, at times up to 70% of their total lipid content.

Among the 18 fatty acids detected in Steinernema species, oleic acid, stearic acid, and palmitic acid decrease over time, showing that these fatty acids are used as primary energy sources by the nematodes. On the contrary, glycogen reserves range from 10 to 18% and they appear to deplete more slowly than lipids in early stage of IJs but more quickly following lipid depletion. As a result, it is proposed that glycogen might serving as an alternate energy source if lipid reserves in older IJs are exhausted [7].

2. Abiotic stress

2.1 Temperature

In temperate regions, Arctic, sub-Arctic, and high-altitude environments, nematodes that live in soil may experience temperatures below zero [8]. These organisms can persist in these circumstances by either tolerating the ice that forms on the exterior of their bodies (freezing tolerance) or by supercooling to prevent freezing at subzero temperatures (freezing avoidance). Nematodes exercising freeze avoidance technique perish when their bodies freeze. Supercooling is the capacity of an organism to maintain its body fluids in a liquid condition even at the temperatures below the freezing point. This ability can be enhanced by the production of the cryoprotectants such as trehalose and glycerol [8].

Entomopathogenic nematodes have been observed in polar regions, signifying their capacity to endure subzero temperatures. Steinernematids have been identified in multiple locales across northern Europe and Canada, characterized by their exposure to freezing climatic conditions. While Heterorhabditids have been isolated in temperate regions, the mechanisms underlying their cold tolerance remain unelucidated. Notably, Steinernema feltiae, S. anomali, and Heterorhabditis bacteriophora exhibit resistance to freezing, with recorded lower lethal temperatures of −22, −14, and −19°C, respectively [9].

It was also found that entomopathogenic nematodes can be cryopreserved using liquid nitrogen. The strains used nowadays for commercial applications are active at temperatures of 18 to 30 degrees [10]. However, some of the studies have shown that the new isolates from temperate regions can also effectively kill insects at temperatures ranging from 6 to 12°C [11].

2.2 Heat tolerance

Extreme temperatures, exceeding 32°C, have detrimental effects on various living organisms, including nematodes [8]. Heat shock proteins are stress-related proteins that play an important role in the survival of organisms at elevated temperatures. Genes related to the production of these heat shock proteins were highly conserved across various species of EPNs and other free-living nematodes like C. elegans. The most commonly studied HSP that has been detected is HP88 in H. bacteriophora [12].

2.3 Desiccation tolerance

Besides extreme temperatures, dry conditions can also have a negative impact on the survival of the nematodes [13]. For movement and efficient survival of nematodes, they require at least a thin film of water surrounding the body. But some of the stages in their life cycle can withstand the absence of water for a prolonged period. This can be possible by the process called anhydrobiosis, a type of cryptobiosis that is induced by prolonged exposure to dry conditions. Anhydrobiosis is achieved by the gradual loss of body water [14]. Besides, these nematodes also show some structural and behavioral changes such as clumping and coiling to reduce the exposed cuticular surface area, thus reduced the loss of water through the cuticle. While the specific biochemical pathways of anhydrobiosis are unknown, it has been found that during a progressive water loss process, the levels of trehalose and glycerol grow dramatically, while glycogen and lipid levels are dropped. Glycerol and trehalose act as the protectants [13].

In cases of severe dehydration, entomopathogenic nematodes (EPNs) undergo a process known as anhydrobiosis [15]. Anhydrobiosis is a reversible physiological phenomenon where an organism can lose as much as 98% of its body water without metabolic arrest, entering a state referred to as cryptobiosis. This concept was initially described by Cooper and van Gundy in 1971 and further elucidated by Womersley in 1981. While EPNs are capable of achieving partial anhydrobiosis, characterized by an approximate 80% reduction in oxygen consumption [16], they do not progress to full cryptobiosis. Therefore, they are categorized as quiescent anhydrobiotes [16, 17, 18]. Desiccation, the process of drying out, initially prompts a temporary increase in EPN metabolism before gradually slowing it down to levels below the normal metabolic rate [16]. During this phase, there is a decrease in glycogen production, indicated by the down-regulation of glycogen synthase (gsy-1), while the synthesis of trehalose and glycerol from existing glycogen and neutral lipid reserves increases. These findings have been supported by research from refs. [13, 15, 19, 20, 21]. Trehalose gradually replaces water in cell membranes and plays a crucial role in preserving cell structures and stabilizing proteins [14, 19, 20, 22]. In the case of S. feltiae, desiccation results in a twofold increase in trehalose content [23]. Furthermore, desiccation induces the expression of casein kinase (CK2), leading to the transcriptional activation of a nucleosome-assembly protein (NAP-1) through physical interaction [15, 24, 25]. In response to desiccation, EPNs also synthesize osmoregulant molecules (e.g., produced by ALDH) and antioxidants (e.g., Gg., DESC47, HSP40), which may serve to provide additional protection against damage caused by the drying process [15, 24, 25].

3. Biotic stress

3.1 Fungi

In fungi, nematophagous fungi are the common adversaries of nematodes, and they are classified under different categories such as endoparasites, nematode trapping, and parasites of cysts and eggs. If these fungi are present in the soil, this will be detrimental to the survival and ability of infection of EPNs [26]. In order to protect themselves from these fungi EPNs employ several mechanisms including, high cruising moment of nematodes to evade the traps formed by the fungi secondly some EPNs such as Heterorhabditids retain their second-stage cuticle until they enter the insect body, thus protecting their body from fungal traps [27]. Third, EPNs can detect the chemical signals from fungal traps and avoid those [28]. In addition, when the nematodes are encountered by the fungal traps, their defense system is activated leading to the production of antimicrobial peptides through PRRs, thereby ensuring the efficacy of EPN-based pest control strategies in the future.

3.2 Bacteria

Pathogenic soil bacteria can attach to the surface of the EPN, FLN, and plant parasitic nematodes (PPN) [29]. Main bacteria attacking these nematodes are Pasteuria sp., Kaistia sp., Lysinibacillus fusiformis, Enterobacter sp., Bacillus cereus, Klebsiella quasipneumoniae, and Pseudomonas aeruginosa [30]. Endospore-forming bacteria have been related to the reduction of virulence in EPNs. The immune response of EPNs to bacterial agents remains unclear. In the case of C. elegans, which is a model organism often used for studying nematode biology, pathogen attacks are detected through pattern recognition receptors (PRRs) that identify pathogen-associated molecular patterns and/or disruptions in cellular homeostasis. This detection triggers both cell-autonomous and non-autonomous responses. Understanding how EPNs identify and respond to various pathogens may give important insights about target markers that might improve EPN survival in soil [30].

4. Chemoreception behavior in entomopathogenic nematodes

Chemoreception in entomopathogenic nematodes plays a critical role in their survival, host-finding, and infection process. Chemoreception is the ability of organisms to detect and respond to chemical stimuli in their environment. EPNs use chemoreception to navigate through the soil in search of suitable insect hosts and to locate their hosts for infection. Here is how chemoreception works in entomopathogenic nematodes:

4.1 Host location

EPNs are free-living in the soil, and they actively search for suitable insect hosts to infect. To find hosts, they rely on chemical cues emitted by insects or decaying organic matter associated with the hosts. These chemical cues may include volatile compounds released by insect larvae, fecal matter, or other organic substances.

4.2 Movement toward host

Once the nematode detects the chemical cues associated with a potential host, it exhibits positive chemotaxis, which means it moves toward the source of the attractive chemical stimuli. This helps the nematode to approach the host effectively.

4.3 Recognition and penetration

When an EPN reaches an insect host, it needs to recognize and penetrate the insect’s body to initiate infection. The nematode uses chemoreceptors located on its mouthparts to sense specific chemicals on the insect’s surface, helping it to identify suitable entry points, such as natural body openings or softer cuticle areas.

4.4 Infection

After penetrating the host, the EPN releases symbiotic bacteria from its gut into the insect’s body cavity. These bacteria are responsible for producing toxins that kill the insect and also help in breaking down the host tissues, creating a suitable environment for the nematodes to feed and reproduce.

Overall, chemoreception is a fundamental aspect of the biology of entomopathogenic nematodes as it allows them to locate, infect, and successfully parasitize their insect hosts. Understanding the chemoreception mechanisms in EPNs can aid in the development of more effective biocontrol strategies and improve their application in agricultural and pest management practices.

4.5 Foraging strategies

Infective juvenile (J3) is the only free-living stage of EPNs which are non-feeding, having thick cuticle, non-developing and non-reproductive stage called the dauer stage. There are two major types of foraging strategies in IJs, ambush (surprise attack by jumping on the host) and cruise (movement in the soil in search of host) [6]. The actively moving hosts are attacked by ambush foragers, while sedentary hosts are attacked by cruise foragers [31].

The EPN juveniles are attracted toward their host through volatile cues produced by host insect such as CO₂ [32, 33], excretory products and fecal matters [34], Heat [35], and pH gradient [36]. The EPN Juveniles are attracted to plant root diffusates also, because such plant is considered [10] as habitat for the host insect [37, 38]. For example, Heterorhabditis megadis which is attracted to plant diffusates when damaged by its host insect beetle Diabrotica virgifera [39] and by weevil larvae Otiorhynchus sulcatus [37].

5. GPCRs

G protein-coupled receptors (GPCRs) also called as seven transmembrane receptors which are located in the cell membrane of the nematode sensory structures. Signal transduction in G protein-coupled receptors (GPCRs) is a complex process by which extracellular signals are converted into intracellular responses. GPCRs are cell membrane receptors that transmit signals from various ligands, such as hormones, neurotransmitters, and sensory stimuli, to the inside of the cell [40]. Here is a general overview of the signal transduction process in GPCRs [41]:

5.1 Ligand binding

The process begins when a specific ligand binds to the extracellular domain of a GPCR. This binding induces a conformational change in the receptor, leading to activation.

5.2 G protein activation

Upon activation, the GPCR interacts with and activates a heterotrimeric G protein complex located on the intracellular side of the cell membrane. The G protein consists of three subunits: α, β, and γ.

5.3 G protein activation and dissociation

The binding of the activated GPCR to the G protein causes the exchange of GDP (guanosine diphosphate) on the α-subunit for GTP (guanosine triphosphate), leading to the dissociation of the α-subunit from the βγ-subunits.

5.4 Effector activation

Both the α-subunit and the βγ-subunits can independently regulate downstream signaling pathways. The α-subunit can directly interact with various effector proteins, such as adenylyl cyclase or phospholipase C, depending on the specific GPCR and the type of G protein involved.

5.5 Second messenger production

Activation of effector proteins leads to the production of second messengers, such as cAMP (cyclic adenosine monophosphate) or IP3 (inositol trisphosphate), depending on the pathway. These second messengers act as intracellular signaling molecules that mediate the transmission of the signal to various downstream effectors.

5.6 Activation of downstream pathways

The second messengers activate downstream signaling pathways that often involve protein kinases, ion channels, and other intracellular effectors. These pathways ultimately lead to changes in cellular responses, such as alterations in gene expression, enzyme activity, ion channel conductance, and more.

5.7 Termination of signal

The duration of the signaling response is tightly regulated to prevent continuous activation. The GTP-bound α-subunit of the G protein has intrinsic GTPase activity, which hydrolyzes GTP to GDP, resulting in the inactivation of the α-subunit. Once inactive, the α-subunit reassociates with the βγ-subunits to reform the inactive G protein complex.

5.8 Receptor desensitization and internalization

Prolonged or repeated stimulation of GPCRs can lead to desensitization, where the responsiveness of the receptor to ligand binding is reduced. This can involve processes such as phosphorylation of the receptor by kinases, leading to recruitment of β-arrestins that inhibit further G protein signaling. Additionally, the desensitized receptor can be internalized into the cell through endocytosis.

5.9 Receptor recycling or degradation

Following internalization, the receptor can be either recycled back to the cell membrane after ligand dissociation or targeted for degradation in lysosomes. The recycling and degradation processes help regulate the availability of active receptors on the cell surface.

Overall, GPCR signal transduction is a dynamic and highly regulated process that enables cells to respond to a diverse array of extracellular signals and adapt to changing environmental conditions. This process plays a pivotal role in various physiological and pathophysiological processes throughout the nematode body. These GPCRs will help in drug discovery and novel method of developing nematode resistance in plants for nematode management in case of PPNs and effective in host finding for beneficial nematodes like EPNs.

6. Insect parasitism and symbiosis

6.1 Parasitic potential of EPNs

After the application of EPNs in the field, their numbers decrease rapidly within the first few days, followed by a more gradual decline over the course of a month, influenced by several factors. Approximately 80 percent of the reduction in EPN populations post-application can be attributed to factors such as dehydration and exposure to UV radiation. Additionally, energy depletion caused by a lack of food resources and the presence of antagonists has a minor but significant negative impact on EPN survival once they are introduced into the field. For example, out of 1000 IJs that are initially applied, only around ten of them are capable of effectively overcoming these abiotic challenges and are available for insect biocontrol purposes. What makes EPNs unique is their ability to achieve successful insect pest control with a very small quantity of EPNs within 24–72 hours. Furthermore, this population can persist for years through recycling mechanisms [10].

6.2 Host penetration by EPNs

The one percent population of EPNs is also subjected to the insect’s immune system as part of the process for achieving successful insect biocontrol. The primary role of the nematode is to effectively introduce symbiotic bacteria into the insect’s hemocoel, which results in the successful elimination of insects through septicemia. The insect employs various defenses to counter the entry of the nematode, including behavioral, mechanical, and cellular responses. Typically, infective juveniles (IJs) initially enter the insect’s body through natural openings such as the anus, mouth, or spiracles. However, there is an exception in the case of Heterorhabditis, which, while also using natural openings, can additionally pierce the insect’s cuticle with their specialized tooth. This cuticle-piercing method allows them to gain entry rapidly, typically within 30 minutes to 1 hour, as opposed to the conventional route, which takes more than 3 hours to access the insect’s hemocoel [2].

6.3 EPNs breaking the insect mechanical barriers

When insects detect the penetration of infective juveniles (IJs) through their cuticle, they exhibit aggressive behavioral defenses to avoid the nematodes. An example of this can be seen in white grubs, which employ grooming behavior using their rasters and legs, a behavior that can even result in the death of IJs [42]. Additionally, the epi-cuticular wall of mealy bugs has been observed to act as a barrier against Heterorhabditis nematodes. Furthermore, if the entry of EPNs through spiracles is detected by insects, they utilize sieve plates to protect their spiracles and cuticular hairs as mechanical resistance tools against IJ entry. For instance, white grubs, which possess long hairs, are capable of trapping IJs with these hairs [11]. Nevertheless, the mechanical pressure exerted by the IJs assists them in breaking through the relatively weak tracheolar wall of the insect [43]. Thirdly, although infective juveniles (IJs) prefer to enter the white grub through its mouth, caterpillars employ their sharp mandibles to kill these nematodes. Additionally, the passage of IJs into the mouth of wireworms is restricted due to the narrow width of their mouths. Therefore, in the case of most wireworms, houseflies, and leaf miners, IJs utilize the path provided through the anus for entry. However, this preference for entry through the anus can pose challenges for IJs, particularly when the insect frequently defecates. Once the nematodes successfully overcome these barriers, they enter the insect’s gut. Nevertheless, within the foregut of the insect, gastric juices are produced, and these juices can result in the mortality of approximately 40 percent of the nematode population [44].

6.4 EPN shattering peritrophic membrane to gain entry into host midgut

After escaping from the foregut, the nematodes find themselves in the insect’s midgut, which contains structures like gastric caecae, malphigian tubules, and midgut epithelium. This is where the discharge of waste materials with feces is terminated. At this stage, the nematodes will attempt to move deeper into the insect’s hemocoel. However, their progress is often impeded by the insect’s peritrophic membrane [43]. This peritrophic membrane serves to delay the penetration of nematodes into the insect’s hemocoel. Within the midgut of the insect, epithelial cells secrete this chitinous envelope, which functions as an ultra-filter. Consequently, this layer restricts the passage of particles larger than 20 nanometers, effectively preventing nematodes from reaching the hemocoel. Despite the obstacle presented by the peritrophic membrane, Heterorhabditis and Steinernema can breach it by puncturing it using a mural tooth and creating holes through the application of mechanical pressure, given that the membrane is quite delicate. Additionally, hystolytic enzymes aid the nematode in breaking down and tearing the peritrophic membrane [45].

6.5 A look on nematode-bacteria synchronized life cycle

Once the infective juveniles (IJs) reach the insect’s hemocoel, they undergo a complete recovery from the diapause stage. At this point, the nematodes feel secure and regurgitate their monospecific gut bacteria into the insect’s hemocoel. The life cycle of these symbiotic bacteria becomes synchronized with that of their host. This is exemplified by Heterorhabditis and its monospecific symbiotic association with the Photorhabdus bacterium in their gut. Although host-restricted Photorhabdus bacteria are typically transmitted maternally in Heterorhabditis through endotokia matricida, the infective juveniles of these nematodes (J3), after recovering from diapause, regurgitate their entire gut bacteria into the insect’s hemocoel as part of a selective mechanism (to distinguish between P form and M form bacterial cells). They then retrieve a portion of these bacteria, specifically the M form population, to form a persistent biofilm in their posterior intestinal cells, INT9L and INT9R [46]. As a result of both growth and the fresh adherence of bacteria, they form a mass consisting of more than 50 cells, firmly attaching themselves to the intestinal lumen on the posterior side of the nematode. Subsequently, these bacterial cells breach the gland epithelium and invade the cytoplasm of rectal gland cells. Each rectal gland cell of the maternal nematode contains at least one symbiont. These bacterial cells multiply within the rectal cells, resulting in around 10–30 vacuoles per cell, causing the rectal gland cells to enlarge. In parallel, the next generation of pre-infective juveniles (pre-IJs) develops inside the maternal nematode through a process known as endotokia matricida. These pre-IJs typically consume the protoplasm of the mother and develop within her body cavity. Meanwhile, the rectal gland cell undergoes lysis, and the vacuoles containing symbionts from the rectal cells enter the body cavity of the mother. Once the bacterial cells reach the maternal pseudocoelom, they attach themselves to the cardia of the freshly formed infective juveniles (IJs) and multiply in this location. Eventually, they reach the anterior intestine of the fresh IJs, which serves as their habitat for further reproduction and multiplication through the intestinal lumen [47, 48].

6.6 How nematode escapes encapsulation?

Conversely, you might be curious about the role of the P form population of bacterial cells, which will be discussed in the following paragraphs. Currently, both the nematode and bacteria are located in the midgut of the insect. However, it has been documented that it takes the nematode a minimum of 30 minutes to release the bacteria after entering the insect’s hemocoel. In other words, the nematode has to contend with the insect’s immune system for a brief period on its own. Once the infective juveniles (IJs) are detected within the insect’s hemolymph, the insect’s immune system initiates nematode encapsulation. This process involves the IJs becoming trapped within melanin-hardened cellular capsules, which have been observed in orthoptera, diptera, lepidoptera, and coleopteran insects [44]. Nematodes employ various strategies, including evasion, tolerance, and suppression, to escape from encapsulation by the insect’s immune system. Firstly, infective juveniles (IJs) of S. carpocapsae secrete lipids that assist the nematode in evading recognition by the insect [49]. Secondly, at least a significant minority population of Steinernema nematodes can tolerate encapsulation and continue with their parasitic activities [50]. Lastly, sometimes, nematodes use a tactic of confusing the insect by regurgitating bacteria just before being encapsulated by the insect. This causes the insect’s immune system to prepare for encapsulating the nematode, but the presence of bacteria goes unnoticed by the insect, allowing the bacteria to start killing hemocytes without interruption [51].

6.7 How bacteria respond to insect immune system?

After being recognized by the host’s immune system, the bacteria undergo hemocyte aggregation and subsequent nodulation, processes partially triggered by eicosanoids. Photorhabdus and Xenorhabdus disrupt hemocyte aggregation and the formation of nodules by inhibiting the activity of phospholipase A2, an enzyme responsible for initiating the insect eicosanoid pathway. Additionally, Photorhabdus, similar to many other gram-negative pathogenic bacteria, possesses a type III secretion system (TTSS) that transfers effector proteins into the host’s eukaryotic cells. One of these effectors, known as LopT, shields the cells by suppressing phagocytosis and diminishing nodulation. While Xenorhabdus lacks a dedicated TTSS, it relies on a flagellar TTSS for the secretion of lipase [52].

Lipopolysaccharide (LPS) is a significant component of the outer cell membrane found in both Photorhabdus and Xenorhabdus. In Xenorhabdus, LPS exhibits cytotoxic properties through its lipid A component, which binds to and damages insect hemocytes. Additionally, LPS has the ability to inhibit phenoloxidase activity, thereby suppressing melanization. In the case of Photorhabdus, the precise role of LPS is not entirely clear, but it may play a crucial role in countering antimicrobial peptides produced by the insect’s immune system [53]. Conversely, Xenorhabdus takes a different approach by suppressing the transcription of insect genes responsible for encoding antimicrobial peptides. In the early stages of infection, Photorhabdus produces a trans stilbene antibiotic known as (E)-1,3-dihydroxy-2-(isopropyl)-5-(2-phenylethenyl) benzene. This compound serves a dual purpose in combating the host’s immune response and providing protection against microbial competitors by inhibiting phenoloxidase activity [54].

Once they have successfully evaded the immune system, bacteria deploy various toxins to eliminate their insect hosts. Among the most potent weapons in their arsenal are the Tc (toxin complex) protein toxins. Tc proteins are high-molecular-weight insecticidal toxins that can cause lethal effects in insects, even when ingested orally. In the case of Photorhabdus, four distinct toxin complexes (Tca, Tcb, Tcc, and Tcd) have been identified. Notably, Tca shares similarities with Bacillus thuringiensis’ d-endotoxin in its ability to disrupt the insect midgut epithelium [55]. In Xenorhabdus, the counterparts of Tc proteins are referred to as Xpt (Xenorhabdus protein toxin) and include XptA1, XptA2, XptB1, and XptC1. Among these, XptA1 plays a central role in exerting insecticidal activity, while XptB1 and XptC1 are crucial for full virulence. XptA1 possesses a unique “hollow box” structure that allows it to act as a receptacle for XptB1 and XptC1 proteins, enhancing its ability to bind effectively to the host gut. This cooperative action makes XptA1 a staggering 300 times more toxic to lepidopterous larvae when compared to its standalone potency. It is worth noting that the mode of action of Xpt toxins in Xenorhabdus differs from the toxin system employed by Bacillus thuringiensis. In other words, once bacteria bypass the host’s immune defenses, they deploy Tc and Xpt protein toxins to target and kill their insect hosts. These toxins have evolved to be exceptionally potent, often disrupting the insect’s gut and causing lethal effects, with XptA1 playing a central role in the process. Unlike similar toxins found in Bacillus thuringiensis, Xenorhabdus’s Xpt toxins employ a different mechanism of action [56].

Both of these bacterial genera release extracellular, cytotoxic proteins known as hemolysins. In Xenorhabdus, the xenorhabdolysin (C1) hemolysin stands out for its extremely high virulence, capable of triggering apoptosis in both insect and mammalian cells. This cytotoxin is a crucial component for Xenorhabdus to achieve full virulence. The role of hemolysins in Photorhabdus is somewhat less clear, as these bacteria can retain their virulence even without the secretion of hemolysins. Nonetheless, Photorhabdus produces other toxins that serve as significant virulence factors [54].

In Photorhabdus, the toxins Mcf1 and Mcf2 (makes caterpillars floppy) play a pivotal role in causing rapid loss of insect body turgor and ultimately death. Mcf1 accomplishes this by destroying hemocytes and the insect midgut through the induction of massive apoptosis. Meanwhile, the exact site and mode of action of Mcf2 remain unknown. These toxins share strong homology with each other but diverge at their N-termini, encoding different effector domains. These distinct effector domains may enable them to target different sites within the insect. Additionally, other Photorhabdus toxins are involved in damaging the insect gut. Photorhabdus takes up residence between the basal membrane and midgut epithelium and expresses the gut-active toxin Tca, along with PrtA, an RTX-like metalloprotease. Together, they induce extensive programmed cell death of the midgut epithelium [57].

Beyond causing the demise of the insect host, these toxins may aid in bioconverting the insect tissue to provide nourishment for the developing nematodes. Numerous virulence factors are engaged in the nematode-bacterial infection process. However, it remains unclear which mechanisms are universal and which are specific to nematodes, bacteria, or insect hosts. As new nematode species are being discovered, each with its unique association with specific bacteria, more opportunities arise to uncover the specificities involved in bacterial virulence.

7. Conclusion

Entomopathogenic nematodes exemplify the complexity of parasitic relationships in nature. Their remarkable adaptations and multifaceted parasitism strategies have far-reaching ecological implications, from influencing insect populations in natural ecosystems to providing sustainable solutions for pest management in agriculture. Understanding the ecology, adaptations, and parasitism mechanisms of EPNs is not only a subject of scientific interest but also holds promise for the development of eco-friendly pest control strategies.

Acknowledgments

We heartily thank the academic editor Soumalya Mukherjee, for providing us with this opportunity.

Conflict of interest

The authors declare no conflict of interest.