Cross-talk between immunity and behavior: insights from entomopathogenic fungi and their insect hosts

Abstract Insects are one of the most successful animals in nature, and entomopathogenic fungi play a significant role in the natural epizootic control of insect populations in many ecosystems. The interaction between insects and entomopathogenic fungi has continuously coevolved over hundreds of millions of years. Many components of the insect innate immune responses against fungal infection are conserved across phyla. Additionally, behavioral responses, which include avoidance, grooming, and/or modulation of body temperature, have been recognized as important mechanisms for opposing fungal pathogens. In an effort to investigate possible cross-talk and mediating mechanisms between these fundamental biological processes, recent studies have integrated and/or explored immune and behavioral responses. Current information indicates that during discrete stages of fungal infection, several insect behavioral and immune responses are altered simultaneously, suggesting important connections between the two systems. This review synthesizes recent advances in our understanding of the physiological and molecular aspects influencing cross-talk between behavioral and innate immune antifungal reactions, including chemical perception and olfactory pathways.


Introduction
Insects comprise ∼67% of the known global fauna, with the Insecta including at least one million species (Stork 2018 ).Insects ar e integr al to the functioning of almost all ecosystems (Crespo-Pérez et al. 2020 ).Although most insects obtain nutrients from plant lea ves , stems , roots , and/or fruits , n umerous carni vorous, omni vorous, and/or m ycophagous insects exist in high numbers in v arious envir onments, particularl y social insects.Despite being a major source of food and hence preyed upon by different animals , including amphibians , reptiles , birds , and mammals , o ver 60% of insect fatalities in natur e ar e attributed to fungal mycoses (Cole 2012 ).
Entomopathogenic fungi r epr esent a highl y div erse gr oup with ∼1600 known species in 90 genera, whose ability to infect insects has e volv ed m ultiple times in differ ent linea ges.Genera and species of entomopathogenic fungi can be found distributed within the Ascom ycota, Basidiom ycota, Blastocladiom ycota, Chytridiomycota, Entomophthor omycotina (Zoopa gomycota), Microsporidia, and Mucoromycota, as well as within the fungus-lik e Oom ycota (Hajek andSt. Leger 1994 , Boomsma et al. 2014 ).Entomopathogenic fungi infect over 18 orders of insects at all de v elopmental sta ges, fr om eggs to adults (Ar aújo and Hughes 2016 ).Most Ascomycete entomopathogenic fungi, includ-ing Metarhizium and Beauveria species, infect insects via attachment and direct hyphal penetration of the chitinous exoskeleton.Depending upon the species, this process may involve the production of cuticle-penetrating appressoria, akin to those found on certain plant pathogenic fungi, but hyphal penetration without clearly defined appressoria also occurs (Ortiz-Urquiza and Keyhani 2013 , Chethana et al. 2021 ).Subsequent to penetration, growth in the hemocoel follows, accompanied by the production of imm une-e v ading fr ee-floating yeast-like cells, termed hyphal bodies or in vivo blastospores (Wang et al. 2023 ).Se v er al other r elated entomopathogens (outside the Hypocreales) use a broadly similar str ategy, i.e. pr oduce in vivo pr otoplasts (cells without cell walls, Oomycetes) or hyphae (Chytridiomycetes and Entomophthoromycetes) during insect host colonization (Elya andDe Fine Licht 2021 , Sacco andHajek 2023 ).Postcolonization, some entomopathogenic fungal species are biotrophic or even obligate, producing spor es fr om li ving or d ying hosts that subsequently infect other hosts; ho w e v er, most ar e hemibiotr ophic, sporulating on dead hosts (Castrillo et al. 2005 ).Apart from transmission to other host insects dir ectl y, the spor es of hemibiotr ophic species can lie dormant on leaves or in the soil, grow saprophytically in the presence of suitable nutrients, or form mutualistic associations with plants, either in the rhizosphere as epiphytes or as endophytes of certain plant hosts (Araújo andHughes 2016 , Dara 2019 ).The diversity of species, broad distribution, and specialized infection r outes hav e giv en rise to a range of prolonged, complex, varied, and intriguing molecular, physiological, and ecological interactions between entomopathogenic fungi and insects.
Recent advances in the de v elopment of tools for genetic, genomic , molecular, bioinformatic , biochemical, and ecological anal yses hav e significantl y augmented our understanding of the mechanisms governing interactions between insects and pathogenic fungi (Hajek and St. Leger 1994, Roy et al. 2006, Ortiz-Urquiza and Keyhani 2013, Butt et al. 2016, Wang and Wang 2017, Wang et al. 2023, Hong et al. 2023a, Liu et al. 2023a ).Most r esearc h has pr edominantl y concentr ated on se v er al gener a of Ascomycota, e.g.Beauveria , Metarhizium , and Cordyceps , as well as members of the Entomophthor ales, se v er al of whic h hav e been globall y commercialized for pest control (de Faria and Wraight 2007 , Wang andFeng 2014 , Bamisile et al. 2021 ).The fungal-insect physiological and ecological interaction unfolds across several broad stages during the fungal infection of susceptible hosts (Fig. 1 ).These stages include: (1) precontact of the fungal spore (or mycelium) prior to attachment to the host exoskeleton.At this stage, entomopathogenic fungi produce volatile organic compounds (VOCs) that can induce behavioral or physiological responses, influencing the infection process (Baverstock et al. 2009 ).( 2) Cuticle contact: entomopathogenic fungi attach, germinate, and subsequentl y penetr ate the host exoskeleton thr ough the pr oduction of infection-related substances.Some fungal components can induce the production of antimicrobial substances in the cuticle or pr ompt gr ooming, thermor egulation, or heat-seeking behavior to r educe spor e infection on the surface (Roy et al. 2006 ).(3) Internal growth within the host hemocoel and other tissues; host cellular and humoral immune responses are activated, and/or behavior al r esponses may c hange.Entomopathogenic fungi ada pt to host antimicr obial r esponses by altering their cell wall components, producing toxins and metabolites to evade immune responses, and/or manipulating host behavior (Qu and Wang 2018 ).Infection of host tissues (beyond the hemocoel) ultimately leads to host death.(4) P enetration (outw ar ds) from inside the host, follo w ed b y gro wth and sporulation of the fungus on the surface of the insect cadaver.Within the context of the latter, some social insects exhibit sanitation beha viors , activ el y r emoving or tr eating the corpses of individuals that died from fungal infection to minimize the spread of infection (Fan et al. 2012 , Sun andZhou 2013 ).
Se v er al studies have shown that innate immunity and behavior al r esponses can occur sim ultaneousl y at v arious sta ges of infection and that these responses can influence each other.The central nervous system (CNS) is likely to be involved in immunity (Lampron et al. 2013 ), and conversely, k e y immune cells and tissues, such as hemocytes and the fat body, may also contribute to the behavioral responses of insects (Kamimura et al. 2020 ).Recent evidence suggests that the insect cuticle may function as a platform that potentially directs both behavioral and immune responses (Ortiz-Urquiza and Keyhani 2013 ).Generally, both behavior al imm une and innate imm une r esponses necessitate the recognition of pathogen-derived signals, presumably via specific soluble and/or membr ane-bound ligand-r eceptor pr oteins .T his recognition leads to signal transduction, output, and ultimately the induction of immune and behavior al r esponses (Fig. 2 ).In typical innate immunity pathwa ys , insects launch cellular and humor al imm une functions via r ecognition of pathogen cues that act to initiate distinct signal transduction cascades that subsequentl y activ ate physiological r esponses.Insect imm une r eactions can include the production of antimicrobial enzymes and peptides, phagoc ytosis b y immune cells, melanization, and nodule formation (Zhang et al. 2021a ).Significant pr ogr ess has been made with respect to the recognition of pathogen markers , e .g. cell wall glucan, effectors, and other pathogen-associated molecular patterns (PAMPs), via host signal transduction pathways such as the Toll, IMD (immune deficiency), and JAK/ST A T (Janus kinase/signal transducers and activators of transcription), that cooperate to initiate an immune response (Qu andWang 2018 , Zhang et al. 2021b ).Although far less understood, se v er al signals and proteins related to behavioral immunity have also been identified (Roy et al. 2006 ).In addition, se v er al compounds and pr oteins that function in beha vior ha v e been described to function in imm unity, and her e, we r e vie w the curr ent r eports examining the cr oss-talk between behavioral (including social immunity) and innate/humoral imm une r esponses during fungal infection.
Our limited understanding of host defense systems, particularly in terms of their origin and evolution with respect to fungal pathogens, has significantly impeded our ability to fully understand the complex interactions between insects and pathogenic fungi.Unr av eling the mechanisms behind the cross-talk between behavior and immunity is crucial, as it holds significant implications in immunology , behavior, ecology , and evolution and can contribute new insights to the de v elopment of the broad field of host-pathogen interactions .T his area of stud y lik el y r epr esents an emerging field that reflects the evolutionary selection pressur es r esulting fr om the ongoing "arms r ace" between insects and fungal pathogens.Additionally, it is widely acknowledged in terms of practical applications that fungal-based "bio-pesticides" often exhibit slow mortality rates and inconsistent efficacy in field application for control of agricultural, in vasive , human healthr ele v ant, and other insect pests, potentially due to the insect behavior al imm une r esponses, innate imm une r esponses, or cr osstalk between these defenses .T he de v elopment and use of mor e potent fungal biological control agents for pest management, in particular, may gr eatl y benefit fr om an understanding of the interactions between immune and behavioral responses.

Cross-talk between olfaction, behavior, and immunity: before contact of host cuticle with entomopathogenic fungi
Before entomopathogenic fungi engage , i.e .attach to the host cuticle , insects ma y engage in a voidance beha viors to minimize contact (and hence infection).For example, the se v en-spotted ladybird, Coccinella septempunctata , can detect and avoid leaves and soil containing Beauveria bassiana (Ormond et al. 2011 ).Mole crickets also avoid contact with soil harboring B. bassiana (Thompson and Br andenbur g 2013 ).Suc h envir onmental avoidance behaviors can impact fungal pesticide efficac y.The J apanese beetle, Popillia japonica (Villani et al. 1994 ), and termites (Staples and Milner 2000 ) exhibit avoidance behaviors to w ar d soil containing entomopathogenic fungi, likely providing one mechanism that can diminish the effectiveness of the application of fungal pesticides to w ar d these insects.Beyond triggering behavioral effects, se v er al VOCs emitted by fungi can induce physiological effects on insects without direct contact.The termite Macrotermes michaelseni can discriminate between virulent and avirulent strains of Metarhizium anisopliae and B. bassiana based on emitted VOC profiles (Mburu et al. 2011 ).Ho w ever, arthropods vary in their ability to detect and avoid entomopathogenic fungi based on species, de v elopmental sta ge , sex, and ecological location (e .g. soil, rhizosphere, and plant phylloplane) of the fungus (Bav erstoc k Figure 1.Simplified infection processes of entomopathogenic fungi to insect hosts .T he processes of fungal infection of insect hosts can be divided into se v er al sta ges: (1) conidial contact and subsequent adhesion to the host cuticle, (2) conidial germination, germ tube/a ppr essorium formation, and penetration into the insect host, (3) hyphal body formation, pr olifer ation in within the insect hemocoel and surrounding tissues, hyphal extension outw ar ds, and death of the insect, and (4) fungal sporulation and shedding from the cadaver.et al. 2009 ).For instance , Agriotes obscurus larvae , but not male adults , a void M. anisopliae (Janmaat et al. 2022 ).Flower beetles a void B .bassiana when present on lea ves but not in soil (Meyling and Pell 2006 ), whereas Japanese beetle larvae avoid soil-borne M. anisopliae (Villani et al. 1994 ).Some social insect species, such as the termites Reticulotermes flavipes , M. michaelseni , Zootermopsis augusticollis, and the ants Acromyrmex striatus , and F ormica rufa , a void fungal-infected areas or nestmates (Myles 2002, Mburu et al. 2009, Liu et al. 2019a ).Ho w e v er, other species, such as Myrmica ruba, do not avoid fungal contamination, including M .brunneum found in surr ounding ar eas (Per eir a et al. 2021 ).In some instances, social insects can detect but r eact differ entiall y to diseased workers outside versus inside the nest, with health-detectable cues tunable within the social context (e.g.M. ruba infected by M. anisopliae ) (Leclerc and Detrain 2016 ).Reticulitermes flavipes termite workers can e v en be r eintegr ated into a colon y after infection with Metarhizium fungi (Moran et al. 2022 ).Founding queens of the ant Formica sel ysi a ppear attr acted to soil "contaminated" with Beauveria or Metarhizium (Bruetsch et al. 2014 ).Whether this repr esents par asite manipulation or benefits the ant via the selection of suitable nesting sites remains unclear.Nematophagous fungi, suc h as Poc honia c hlam ydosporia (intriguingl y with a similar genome composition as Metarhizium ), produce VOCs that are activ e a gainst both nematodes and insects, indicating potential coe volutionary r esemblances toward differ ent hosts by fungal pathogens (Lozano-Soria et al. 2020 ).
Various VOCs have been identified from entomopathogenic fungi, including fatty and other acids , esters , terpenes , aldehydes , ketones , alcohols , and other organic compounds (Bojke et al. 2018 ).These compounds r epr esent both primary and secondary metabolites and are synthesized through diverse biochemical pathwa ys , with esters , acids , and terpenoids being the most predominant (Jele ń and Wasowicz 1998 ).Some VOCs have been shown to affect insect behavior.For example, the terpenoid geosmin, pr e v alent in many fungi, along with benzaldehyde, induces avoidance behavior in the fruit fly Drosophila melanogaster (Störtkuhl et al 2005 ).Fruit flies can detect dimethyl trisulfide and 2-phenylethanol, avoiding ovipositing in contaminated areas (Holighaus and Rohlfs 2016 ).Octanol and hexanol in M. anisopliae and other fungi attract sand fly females (Machado et al. 2015 ).Some m ushr ooms hav e been r eported to r e pel fungi vorous insects by producing 1-octen-3-ol (Holighaus et al. 2014 ).1-Octen-3-ol can also modulate insect behavior by acting as a repellent or an attractant to different insects (Fäldt et al. 1999, El Jaddaoui et al. 2023 ).Although 1-octen-3-ol is also produced by entomopathogenic fungi, its potential role in mediating insect behavior and immunity remains unknown.Intriguingly, 1-octen-3-ol can serve as an inhibitor of fungal spore germination, indicating its potential role in microbial ecology through the reduction of intraspecific or interspecific competition when spore concentr ations ar e high (Chitarr a et al.As opposed to repelling, certain entomopathogenic fungi are able to attract insect hosts, indicating a distinctl y differ ent e volutionary pathwa y.T he gr een peac h a phid, Myzus persicae , is attracted by B. bassiana conidia (Geedi et al. 2023 ), and M. brunneum rhizospher e inter actions with cabba ge ( Br assica oler acea ) appear to manipulate plant VOC production to attract herbivorous insects (Cotes et al. 2020 ).In addition to short-and mediumchain volatiles, fungi produce a diverse mix of terpenes, such as γ -gurjunen by B. bassiana , β-elemene by Isaria fumosorosea , and α-farnesene by Hirsutella danubiensis (Bojke et al. 2018 ).Essential oils containing terpenes exhibit antimicrobial and antioxidant properties (Bozin et al. 2007, Diao et al. 2013 ).The dominant fatty acid, palmitic acid (C16:0), in B. bassiana , Batkoa spp., I. fumosorosea , and Metarhizium flavoviride plays crucial roles in intermediary metabolism (Bojke et al. 2018 ) and affects the growth of the cotton bollworm, Helicoverpa armigera (Satyan et al. 2009 ).Additionally, certain VOCs, such as 3-methyl-1-butanol, and compounds with limited volatility, including oc hr ato xin, aflato xins, penicillic acid, and various Fusarium toxins and deriv ativ es, exhibit cytotoxic properties toward insect cells (Holighaus and Rohlfs 2016 ).These findings suggest that fungal VOCs or nonvolatiles likely participate in behavioral, toxicity, and immune responses (Fig. 2 ).Ho w e v er, the underl ying mec hanisms driving these actions are poorly characterized.Biotic and abiotic environmental factors are known to trigger the differential production of metabolites, whic h likel y r esults in alter ed VOC pr ofiles .T hese altered profiles could induce varying behavioral responses in different target insects (Bennett et al. 2012 ).Despite the potential significance of these interactions, they remain relatively underexplored in terms of underlying genetic and biochemical mechanisms, not only due to their often being overlooked but potentially to difficulties in experimental models and a ppr oac hes to examine these interactions.
Ho w e v er, se v er al important insights have been gained.Gas phase 1-octen-3-ol acts on D .melanogaster dopaminergic neurons in labor atory tests, e v en at low concentrations, causing neurotoxic and cytotoxic effects (Morath et al. 2012 ), indicating that dopamine and dopamine receptors ma y pla y a role in mediating immunity and behavioral responses.Phenethyl ethanol, produced by M. anisopliae , has been shown to affect both avoidance behavior and immune inhibition in locusts.Mor eov er, a specific locust odorant binding protein (OBP; LmigOBP11) has been identified as a mediator of 2-phenylethanol detection, influencing insect innate immune responses in the hemolymph (Zhang et al. 2023a ).Intriguingly, LmigOBP11 inhibits insect imm une r esponses and is upregulated during M. anisopliae infection, suggesting that the fungus manipulates OBP expression to enhance successful mycosis .T hese findings support the emergence of a field exploring molecular cross-talk between insect detection of fungal VOCs via odorant/ligand binding pathwa ys , subsequent a voidance beha viors , and imm une activ ation.Identifying additional insect and fungal molecular determinants involved in VOC production (fungal) and detection (insect) associated with specific behaviors will likely reveal crucial layers to these interactions .T he notion that the fungal pathogen exploits host detection to suppress immunity adds a novel dimension to the coevolutionary arms race between these organisms.

Cross-talk: during fungal cuticle contact and penetr a tion
Insects possess mechanisms for detecting and initiating hygienic behaviors when entomopathogenic fungal spores attach to their cuticle.Self-gr ooming incr eases in F. sel ysi ant workers exposed to M. brunneum (Tr a gust et al. 2013 ).Other ant species, such as the r ed imported fir e ant, Solenopsis invicta (Qiu et al. 2014 ), the formic acid spraying Asian ant, Lasius japonicus (Okuno et al. 2012 ), and the carnivorous ant, Platythyrea punctata (Westhus et al. 2014 ), also display nestmate grooming behaviors in response to detection of fungal pathogens .In termites , alarm pheromone can act as a trigger for hygienic grooming, e.g. in R. flavipes infected with Metarhizium (Bulmer et al. 2019 ).T his beha vior in volv es not onl y physicall y r emoving spor es but also pr oducing antimicr obial pr oteins , lipids , and metabolites in the epicuticle.In the cotton aphid (Kim and Roberts 2012 ) and diamondback moth (Vandenberg et al. 1998 ), infection by B .bassiana and/or Lecanicillium attenuatum speeds up instar de v elopment, potentiall y as a way to elude infection into the next life stage by shedding infected structures.Some insects, including grasshoppers ( Schistocerca gregaria ), exhibit "behavioral fe v er" by r aising their body temper atur e thr ough sun basking to combat Metarhizium infection (Ouedraogo et al. 2004 ).These behaviors are critical for some insects, resulting in significant reductions in infecting conidial numbers and thus improving infected insect survival rates.Physiological immune responses are also initiated at this sta ge, typicall y inv olving (i) hemoc ytes and fat bodies migrating to the wound site to promote repair and prev ent/r educe infection; (ii) melanization to sequester and kill the inv ading micr obe; (iii) induction of pr ophenoloxidase and other imm une r elated enzymes; and/or (iv) antimicrobial peptide (AMP) production within the hemocoel and potentially on the cuticle (Qu and Wang 2018 ).Although only limited information is available , beha vior al and imm unological r esponses may compete with each other.For example, in termites, intr a gr ooming behaviors in response to Metarhizium exposure lead to reduced AMP production, indicating optimization or trade-off effects between behavioral and immune responses (Liu et al. 2019b ).
Depending on the insect species, the cuticle is considered to contain an outermost "waxy layer" with various long-to-midc hain hydr ocarbons , including alkanes , alkenes , and their methylbr anc hed deriv ativ es, fatty acids and esters, alcohols, ketones, and aldehydes, as well as minor components, including triacylglycerols, e po xides, and ethers (Chung andCarroll 2015 , Golian et al. 2022 ) (Fig. 2 ).Some insect epicuticular lipids are useful substrates for entomopathogenic fungi and contribute to crucial prepenetr ation e v ents during infection (Jarr old et al. 2007, Zhang et al. 2012, Pedrini et al. 2013 ).Under the waxy layer, the highl y scler otized portion of the cuticle consists of c hitin-cr oss-linked tanned pr oteins, with fungal c hitinases long consider ed important virulence factors (Charnley 2003 ).Insect cuticular constituents play crucial roles in environmental adaptation and act as informa-tion and communication signaling molecules, mediating insect behavior al r esponses (Ho w ar d and Blomquist 2005 ).Fungal spores can attach to the hydrophobic cuticle using adhesins and/or hydr ophobin pr oteins (Wang and Wang 2017 ).Degradation of the w axy lay er and subsequent cuticular compounds involves the activities of numer ous "cuticle-degr ading" enzymes, including secr eted lipases, pr oteases, c hitinases, and gl ycosidases (Wang and Wang 2017 ) (Fig. 2 ).Degr adativ e pr oducts and some components, like hydr ocarbons, ar e metabolized b y the fungus via pathw ays that involve the action of peroxisomes and cytochrome P450 enzymes (Ortiz-Urquiza and Keyhani 2013 ).Additionall y, penetr ation may involve direct mechanical pressure from growing appressoria (Wang and Leger 2007 ).Grooming-induced spore removal from the cuticle is likely stimulated by the production of fungal compounds.Extracts of fungal volatiles markedly increase both termite grooming and a ggr essiv e behaviors (Yana gawa et al. 2011a ).Aggression in some ant and termite species intensifies tow ar d fungus-infected w orkers, excluding them from other group members (Yanagawa et al. 2011b, Cremer et al. 2018 ).
Regarding fungal signals potentiall y r ecognized and affecting host beha viors , extr acellular-associated pr oteins on the fungus surface induce grooming behavior in Drosophila (Shang et al. 2023 ).Additionally, fungal cell wall components, whether released or secr eted, suc h as β-1,3 glucan, c hitin, and secr eted pr oteins, including effectors , hydrophobins , and virulence-r elated pr oteases, can potentiall y activ ate host insect innate imm une pathways befor e penetrating the host cuticle (Li and Xia 2022 ).Ho w e v er, whether these compounds affect behaviors such as grooming, burrowing, heat seeking, or molting remains to be determined.The fungal membr ane ster ol, er goster ol, has emer ged as a potentiall y k e y fungal molecule recognized by the host, inducing behavioral and imm une r esponses (Rodrigues 2018 ).Coptotermes formosanus termite workers respond to ergosterol levels with increased grooming behavior to w ar d Metarhizium -infected nestmates (Chen et al. 2023 ).In the ant Linepithema humile , Metarhizium fungal spores producing lo w er le v els of er goster ol a ppear to av oid detection b y the insect, with the induction of grooming and other sanitary behaviors suppr essed (Stoc k et al. 2023 ).Alter ed cuticle components, particularly fatty acids on the host surface used by entomopathogenic fungi during germination, ar e belie v ed to affect insect behavior al r esponses (Zhang et al. 2012 ).It is unclear whether insect compounds, derived from the degradative activities of the infecting fungus and/or released during infection, may stimulate either immunological or behavioral responses.
After adhesion consolidation, the next fungal infection phase involv es germination, a ppr essoria formation, and the initiation of penetration.Detecting and eliminating the pathogen before it r eac hes the hemocoel, i.e. at the epidermal stage, would represent another line of defense (Pedrini 2018 ).The epidermis (below the cuticle) and associated cell types, such as oenocytes (also present in the fat body), constitute the outermost aspect of insect tissues (Makki et al. 2014 ).These cells support various specialized sensory structur es extending fr om the cuticle, suc h as bristles , hairs , and antennae, and are implicated in immunity (Davis andEngström 2012 , Martins andRamalho-Ortigao 2012 ).Ho w e v er, little is known about epidermal-le v el defenses a gainst fungal pathogens aside fr om their r ole in supporting cuticle scler otization and molting, e.g.thr ough r egulation and expr ession of laccases and c hitin synthases/chitinases (Merzendorfer andZimoch 2003 , Dittmer andKanost 2010 ).Penetrating hyphae from entomopathogenic fungi fr equentl y form close to bristles (Sahayaraj et al. 2014 ).This may be because the cuticle's thinner components make these areas easier to penetrate.Ho w ever, Drosophila mechanosensory bristles exhibit nonselective detection of dust, including fungal spores, landing on the fly, which can then trigger grooming/cleaning behaviors (Zhang et al. 2020a ).In the earl y sta ges of fungal infection, specific olfactory-related proteins in the insect host can show significant differ ential expr ession (Le vy et al. 2004, Zheng et al. 2021, Zhang et al. 2021a ).A mutant of the Drosophila odorant rece ptor corece ptor , ORCO , results in the inability of the fly to recognize chemicals associated with eliciting grooming behavior (Yanagawa et al. 2018 ).Additionally, the Drosophila chemosensory protein CheA75a has been shown to recognize the Metarhizium robertsii spore surface, specifically the fungal extracellular membrane protein Mcdc9, inducing self-hygienic behavior that involves the r emov al of fungal spores from the insect body (Shang et al. 2023 ).In tsetse flies, two odorant-binding proteins, OBP6 and OBP28a, are upregulated during symbiont ( Wigglesworthia ) colonization, influencing melanization and cellular immunity through various insect transcription factors, including lozenge (Benoit et al. 2017 ).Apart from olfactory proteins , locusts , which can transition between solitary and gregarious social lifestyles, activate the upstr eam imm une modulator Gr am-negativ e binding pr otein 3 in the gregarious lifestyle, likely establishing prophylactic immunity and reducing fungal spread, suggesting a role for pattern receptor proteins in both behavior and immunity (Wang et al. 2013 ).Similarly, two compounds secreted by termite salivary glands, termicin and Gr am-negativ e binding pr otein 2, exhibit antifungal activity (Hamilton and Bulmer 2012 ).These data support the idea that insects can detect and respond to fungal components such as VOCs via specific chemoperception (olfaction) or immunity-recognizing proteins to affect behavioral and immune communication during fungal contact with the host (Fig. 3 ).

Cross-talk: during the internal growth stage of entomopathogenic fungi in insects
After penetrating the integument and entering the hemocoel, in most cases examined thus far, the infecting fungus undergoes a dimor phic tr ansition, fr om hyphal gr owth to the pr oduction of free-floating single-celled hyphal bodies .T hese cells tra vel within the insect (open) circulatory system, using hemolymph nutrients and accessing/infecting internal tissues that can include the trac heae, m uscles, digestiv e tr act, and r epr oductiv e or gans (Toledo et al. 2010 ).During this in vivo growth stage, various innate imm une r esponses in the insect ar e activ ated.The fat body and hemocytes mediate humoral and cellular processes that are part of insect innate immunity.These processes have well-described responses to a variety of microbes, including fungi (Lemaitre and Hoffmann 2007 ).Humoral immune-related pathway components, such as those of the pattern recognition receptors, Toll, IMD, and JAK/ST A T pathwa ys , and imm une-r elated factors , including AMPs , l ysozymes, a polipophorin III, hemoc y anin, and tr ansferrin, ar e produced in insects in response to fungal infection (Eleftherianos et al. 2021 ).Host cellular immunity includes coagulation, hemocyte activation and phagocytosis, encapsulation, melanization, prophenolo xidase cascade, and o xidati ve burst production, which have been well-characterized as antimicrobial responses against fungi at this stage of infection processes (Li and Xia 2022 ).Howe v er, entomopathogenic fungal hyphal bodies have evolved various strategies to evade the insect innate immune system, including escaping encapsulation and phagocytosis, masking the fungal cell surface to avoid detection, and producing toxins and other compounds that suppress the host immune reaction (Huang et al. 2023 ).Indeed, Metarhizium and Beauveria can grow within insect cells as well as inside soil predatory amoeba, suggesting links between insect pathology and survival in soils (Kurtti andKeyhani 2008 , Bidochka et al. 2010 ).As fungal cells pr olifer ate within the hemocoel, the host immune system is ov er po w ered b y the activity of the fungus and fungal pr olifer ation.In addition to innate imm unity, behavior al alter ations ar e typicall y observ ed during this infection sta ge, ther eby affecting feeding, a ggr ession, r esponse to semioc hemicals, and r epr oductiv e behavior.Notabl y, certain fungal entomopathogens, particularly those with narrow host ranges, induce c har acteristic behavior al alter ations aimed at incr easing transmission.Host "zombification" can lead to diverse beha viors , from height seeking follo w ed b y mandibular fixation (as observed in ants infected by Ophiocordyceps and flies infected by Entomophthora ) to increased or frenzied mating (shown in cicadas infected by Massospora ) (Macias et al. 2020, de Bekker et al. 2021, Elya and De Fine Licht 2021 ).
Fungal PAMPs, cell wall components, proteins, effectors, metabolites, and e v en miRNAs hav e been shown to be secreted by fungal pathogens, along with other unc har acterized factors that can either activate and/or facilitate evasion of host chemoperception, innate immunity, and antifungal behaviors (Qu and Wang 2018 ) (Fig. 2 ).Strategies emplo y ed b y the fungus to shield itself from host defenses include the production of a collagenous coat within the hemocoel (Wang and Leger 2006 ), e v asion of host innate immunity via antioxidant enzyme production, and cell wall protein and glucan rearrangements (Lu and Leger 2016 ).A fungal laccase secreted during this stage also functions in innate immunity (Lu et al. 2021 ).Mor eov er, v arious entomopathogenic fungi produce an array of secondary metabolites during growth in the insect hemocoel.These include products of nonribosomal polyketide synthetases and secondary metabolite cluster pathways (Gibson et al. 2014 ).In Metarhizium , destruxins, a group of cyclohexade psipe ptides, exhibit di verse insect to xic effects, including the inhibition of humor al imm une pathways (such as Toll/Imd), reduction of AMP production, inhibition of host prophenoloxidase and o xidati v e burst r esponses, and c ytotoxicity to hemoc ytes and other cells (Hu et al. 2016 ).Destruxins have immunosuppressive effects, including cellular immune system dysregulation in insects, pr e v enting nodule formation, and disrupting phagocytosis (Vey et al. 2002 ).They also affect Ca 2 + influx, decrease intracellular fr ee H + le v els, affect m uscle function, and induce par al ysis and sluggishness (Ruiz-Sanchez et al. 2010 ).Furthermore, these toxins can induce cell apoptosis through increased o xidati ve stress and interfere with mitochondrial and hormonal signaling pathwa ys , as well as acetylcholine receptors (Butt et al. 2016 ).Certain destruxins serve to inhibit behavioral fever (Hunt and Charnley 2011 ), and mutants that produce enhanced levels of destruxins have been shown to be more virulent (Huang et al. 2022, Li et al. 2022 ).Ad ditionally, other to xic metabolites isolated fr om v arious insect pathogenic Beauveria , Metarhizium , and Isaria species, such as beauverolides, beauvericin, tennelin, isarolides, brassinolide , cytochalasins , and oosporein, exhibit diverse behavioral and immune effects (Sinha et al. 2016 ).These findings highlight the important role of secondary metabolite toxins in potentially mediating cross-talk between the antifungal behaviors and the antifungal immunity.
As a consequence of infection, host-derived metabolites also undergo marked changes in content and concentration as the fungus grows inside the host.These changes are driven in part by fungal degradation and consumption of host tissues, including alter ations in carbohydr ate, pr otein/peptide/amino acids, fatty acid, lipid, ar ac hidonic acid, and amine profiles, as well as by host responses to the invading fungus (Xu et al. 2015, Zhang et al. 2023b ) (Fig. 3 ).In certain instances, different metabolites and degradativ e pr oducts can feed into the regulatory pathways that control innate immune responses.For example, entomopathogenic fungi can suppress host innate immunity by inhibiting the production of host ar ac hidonic acid during infection (Dean et al. 2002 ).Inter estingl y, imm une activ ation components, suc h as the microbial cell wall components laminarin and lipopolysaccharide , can induce beha vior al fe v er in locusts (Bundey et al. 2003 ).Eicosanoid biosynthesis is implicated in this r esponse, likel y r egulating the transformation of arachidonic acid to prostaglandins, with the latter controlling body temper atur e in both mammals and arthropods (Bundey et al. 2003 ).These data establish aspects of the biochemical link between behavioral and immunological responses , i.e .mediated by either the presence or detection of microbial components and/or circulating insect and fungal metabolites (e.g.eicosanoids).Tryptamine production by M. robertsii has been shown to induce the expression of immune signaling-related genes and r eactiv e oxygen species production in grasshoppers via the IMD and Toll pathways by activating the host aromatic hydrocarbon receptor LmAhR (Tong et al. 2020 ); ho w ever, this inhibits the expression of the AMP defensin, thus tryptamine production by the fungus acts to suppress host antifungal responses.In plants, tryptamine functions in r epr essing herbivor e feeding and r epr oduction (Gill et al. 2003 ).Additional fungal components, including effector proteins that are secreted and target specific host proteins (e .g. signal pathwa y-interfering toxins , serine proteases , ec kinase, and tyr osine phosphatase) and secondary metabolites (e.g.nonribosomal peptide and polyketide mycotoxins, destruxins , and alkaloids), ha ve been shown to affect insect beha vior, with such effects primarily explored in terms of antifeedant and lethargic activities (Gibson et al. 2014, Will et al. 2020, Toopaang et al. 2023 ).
In addition, during the internal growth phase of the fungal pathogen in host insects, the expression of various neuronalrelated genes in the CNS, particularly those associated with morphogenesis, de v elopment, behavior, cognition, learning, and memory, is significantl y alter ed (Zhang et al. 2017 ) (Fig. 3 ).The consequence of these c hanges r emains unknown but may either involv e (i) behavior al attempts to hinder infection (suc h as heatseeking or grooming), (ii) neurological impairment and/or degradation due to extensive fungal growth, and/or (iii) pathogen efforts to manipulate host behavior in order to benefit sporulation and/or dissemination of the pathogen upon host death.Some of the differ entiall y expr essed genes ma y be in volved in both beha vioral and immune functioning, with modulation of neuronal signaling occurring during infection.For example, the octopamine and dopamine receptors ( Dop1R1 ) in Drosophila , known to function in the formation of a ppetitiv e memories for sugar and other foods , pla y a role in learning (Burke et al. 2012 ).Octopamine, with contr asting imm unoenhancing and imm unosuppr essiv e effects, is a k e y hormone in acute stress responses related to flight or fight behavior, and injecting locusts with octopamine has been shown to increase their susceptibility to M. anisopliae (Goldsworthy et al. 2005 ).Giv en that both neur ons and imm une cells hav e octopamine receptors, octopamine and dopamine ligand/receptors may contribute to cross-talk between behavioral and immune responses.Another example is the upregulation of the activated metabotropic glutamate receptor 2/3 in M. acridum -infected locusts at different fungal infection time points, suggesting the existence of a mechanism for CNS sensing of infection and induction of neur opr otectiv e pathways (Zhang et al. 2017 ).Additional affected neur ological-r elated pr ocesses include the expr ession of glutaminase, implicated in immunological challenge protection, and calmodulin, involved in inflammation, promoting immune signaling by controlling nitric oxide production (Zhang et al. 2017 ).
The CNS itself includes resident immune surveillance mechanisms, such as microglia and Toll-like receptors (Fig. 3 ).Ho w ever, the roles and responses of these mechanisms in fungal-mediated infection remain to be explored.γ -Aminobutyric acid type A receptors expressed on T cells inhibit the cell responses to antigens (Tian et al. 1999 ).The r eported decr ease in γ -aminobutyric acid type A expression during infection in locusts may inhibit such imm une r esponses, indicating a potential link to imm unity in both the CNS and immune tissues.Regarding links to c hemor eception, little evidence exists; ho w ever, environmental odors can influence immune components , e .g. during hematopoiesis .Sensing food odors via the Or42a odor ant r eceptor in Drosophila stimulates pr ojection neur ons (Asahina et al. 2009 ), leading to downstr eam activ ation of neur osecr etory cells, whic h in turn mediates the release of GABA into the hemol ymph, potentiall y functioning in immunity.

Cross-talk: pre-and postmortem
In se v er al systems, suc h as "zombie" ants, infected insects exhibit ele v ation-seeking behaviors consider ed to aid in the dispersal of emerging fungal spores (de Bekker et al. 2021 ).Social insects , notably ants , termites , and honeybees , displa y various forms of sanitation behavior to w ar d sick and deceased nest mates, in volving self-remo val, conspecific remo val, burial, or dismemberment of dead or dying nestmates (Sun and Zhou 2013 ).Additionall y, fungal anta gonistic micr obes that pr oduce antifungal compounds may be recruited, constituting a form of "biological warfare" (Mattoso et al. 2012, Hong et al. 2023b ).Dying bees, for instance, activ el y self-exclude fr om the hiv e, pr esumabl y to r educe the risk of infecting nestmates (Rueppell et al. 2010 ).In the garden ant, Lasius neglectus , workers enhance brood care and hygienic behaviors in the presence of M. anisopliae -contaminated workers (Ugelvig and Cremer 2007 ).These behaviors seek to limit fungal growth in cadavers and reduce the likelihood of infection of healthy nestmates .T his can be particularly important as the fungal spor es pr oduced on cadav ers show gr eater virulence compared with those isolated from artificial media, suggesting optimization of virulence-r elated pr ocesses in the presence of specific hosts (Hussain et al. 2010 ).Additionally, the fungus produces specific compounds, such as oosporein by B. bassiana , that help maximize cadaver utilization by suppressing competing microbes (Fan et al. 2017 ).
Various compounds induce cleaning and hygienic behaviors in insects .For example , phenethyl acetate , 2-phenylethanol, and benzyl alcohol released by fungal-infected bees induce nest cleaning behavior (Swanson et al. 2009 ).Fungal infection of ants, including pupae, leads to alterations in surface fatty acids, which then act as chemical signals to induce cadaver removal behaviors (Qiu et al. 2015 ).Similarly, octanol, octanone, and other com-pounds released from dead termite cadavers have been shown to induce corpse removal (Sun et al. 2017 ).Some chemicals not only modulate behavior but also function in insect innate imm unity, e.g.2-phen ylethanol pr oduction by Metarhizium inhibits AMP production in locusts (Zhang et al. 2023a ).The VOC profiles of entomopathogenic fungi cultivated in artificial culture and on insect cadavers differ (Hussain et al. 2010 ), indicating potential variations in effects on behavioral and immune responses based on growth substrates.Further exploration is needed to understand these sanitation and hygienic beha viors , including identifying critical chemicals and genetic pathways that modulate these behaviors and determining their relationship with innate immunity.

Future challenges
The interaction between entomopathogenic fungi and insects occurs on spatial and temporal scales , in volving strategic maneuv ers fr om both participants.Spanning initial contact, germination, penetration, dimorphic transition, internal proliferation, extension outw ar d, and sporulation on the cadaver, this dynamic inter action occurs ov er hours , da ys , or longer.T his permits substantial le v els of selection and coe volutionary r elationships to de v elop, intertwining insect behavior and innate imm une r esponses .T hese ar e likel y dual str ategies that determine whether hosts succumb to pathogen infection or overcome such infections (Fig. 3 ).During the early phases of the inter action, befor e the hemocoel produces hyphal bodies, insects primarily use beha vioral defenses , like avoidance or grooming, to stop the infection from spreading.These earl y-sta ge behavior al defenses ar e vital for social insects in minimizing exposur e, potentiall y r esulting fr om the success of their social adaptations in nature.Evidence suggests the initiation of innate imm unity activ ation as earl y as possible , i.e .prior to cuticle penetration, can function to "prime" the organism for combating infection (Zhang et al. 2020b ).Detection and response to fungal compounds (e.g.cell wall components, secreted products, and VOCs) likely undergo strong selection.Indeed, investing in behavioral defenses becomes a strategic approach to preventing infection and reducing reliance on innate immunity.The insect lifestyle markedly influences the balance between behavioral and innate immunity mechanisms, with soil-dwelling and social insects favoring behavior al ada ptations (suc h as sanitation), whereas solitary or more mobile insects may opt for avoidance strategies (Meunier 2015 ).Further examination of these issues is likely to yield novel information on the evolutionary strategies that hav e emer ged in response to fungal infection.As fungal penetration occurs, the options available to the insect become constrained.Some insects may attempt additional behavioral responses, such as grooming or heat seeking, whereas others invest in resistant physical barriers (such as the epicuticle) to limit water and nutrient availability and potentially as a reservoir for antiseptic compounds (Ortiz-Urquiza and Keyhani 2013 ).Further research is needed to determine whether de v elopmental r esponses such as shedding of the cuticle or early pupation are positive or passiv e str ategies that contribute to r educing pathogen infection and/or increasing survival.
Ad ditional aven ues of r esearc h include addr essing the ecological roles and evolutionary mechanisms of cross-talk between imm unity and behavior, particularl y befor e physical contact and during initial spore attachment to the cuticle.Unresolved questions include why the fungus would produce VOCs that lead to insect a voidance , potentiall y r educing their c hances of finding a host, i.e. what role do these VOCs play natur all y, and/or whether some entomopathogenic fungi hav e e volv ed mec hanisms to mask VOCs that repel hosts.One aspect that r emains poorl y examined is that this phenomenon is likely impacted within the context of plant associations.In this case, plants may seek to emit VOCs to repel them, but the consequence of the fungal-plant association on such activities remains unknown (Branine et al. 2019, Thompson 2022 ).Entomopathogenic fungi are proposed to hav e e volv ed fr om gr ass endophytes ∼100 million years ago, and the differentiation or coselection from endophytic colonization is suggested to have helped promote the acquisition of insect pathogenic genes (Quesada Mor a ga 2020 ).One hypothesis is that entomopathogenic fungi with impr ov ed pathogenicity result in plant pr otection fr om herbivor ous insects and access to pr e viously non utilizable n utrient r esources, and in exc hange, the fungus obtains carbon within a semiprotected ecological niche (i.e.rhizosphere , epiphyte , and/or endophyte) (Iwanicki et al. 2022 ).T hus , the selection for maintaining and/or enhancing the ancestral fungal plant remains strong.Genetic mechanisms, including horizontal gene transfer (from plant to fungus and/or from insect to fungus), may facilitate the emergence of pathogens with br oad host r anges fr om ancestors with narrow and restricted host ranges (Zhang et al. 2019 ).Given the large diversity and distribution of fungal entomopathogens, it is important to consider both conv er gent and div er gent e volutionary mec hanisms (Arnesen et al. 2018 ).
Once the fungus has r eac hed the hemocoel, morbidity becomes almost inevitable .Beha vioral responses , such as ingesting antimicrobial components or dietary modification, can support immune defenses (Leal et al. 2022 ), but at this advanced stage, the infection process likely affects insect behavior, with innate immune defenses becoming less effective.Chitin (including variously deacetylated forms and, to a lesser extent, chitosan) acts as an important component due to its central role in the structure of insect cuticles and presence in the fungal cell walls, with c hitin oligosacc harides also found in plants (Sánc hez-Vallet et al. 2015, Pusztahelyi 2018, Moussian 2019 ).Chitin functions as a microbe-associated molecular pattern in plant pathogenic fungi and is recognized as a nonself-component by plant lysin motif (LysM)-containing receptor kinases .T his recognition triggers host imm une r esponses during micr obe-plant inter actions (Hu et al. 2021 ).LysM pr oteins ar e also found thr oughout the fungal kingdom, including plant and insect pathogens, and are thought to be produced by pathogenic fungi to mask their cell wall chitin from detection by host (whether plant or insect) defense systems (Akcapinar et al. 2015 ).Functional aspects of LysM effectors in entomopathogenic fungi have been characterized; e.g. a family of 12 LysM proteins is found in B .bassiana .These pr oteins hav e been shown to suppress chitin-induced insect immunity, help protect the fungal cells from phagocytosis and nodulation, and hence act to promote fungal virulence.Another strategy involves deacetylation of chitin oligomers, as seen in a number of fungal plant pathogens, which helps the fungus evade recognition by host c hitin r ece ptors, allowing for survi v al in host plants (Sánc hez-Vallet et al. 2015 ).Similarly, the deacetylation of chitin in the entomopathogenic fungus B .bassiana has been r ecentl y r eported to contribute to fungal virulence (Liu et al. 2023b ).Ho w e v er, the r ole of chitin in mediating the cross-talk between behavioral and imm une r esponses in fungal-insect inter actions r emains unknown.
Following the death of a host, healthy insects either avoid or r emov e the cor pses, especiall y in the case of social insects .T his cor pse mana gement and the v arious str ategies emplo y ed can significantly impact pathogen dispersal and insect prophylaxis.In response, certain entomopathogenic fungi are known to hijack host behavior to increase transmission (de Bekker et al. 2021 ).A prime example of this is the host-specific parasitic "zombie"-ant fungus, Ophiocor dyceps unilater alis ( sensu lato ), which manipulates ant behavior to facilitate fungal de v elopment and spor e dispersal, a phenomenon known as the fungal extended phenotype (Andersen et al. 2009, Hughes et al. 2011a ).Zombie ant fungi may target host phototaxis in a circadian manner to increase locomotor activity (Andriolli et al. 2019 ), causing the ant to deviate from foraging trails and exhibit convulsions and twitches as it attempts to climb foliage (elevation seeking) (Pontoppidan et al. 2009, Hughes et al. 2011a, de Bekker et al. 2015 ).The invasion of mandibular muscle tissues causes the ant to cling to the vegetation (Pontoppidan et al. 2009, Hughes et al. 2011a ), resulting in a final "death grip" or manipulated bite that causes mandibular muscle atrophy, locking the jaw and pr e v enting the cadav er fr om falling (Hughes et al. 2011a ).Fungal growth during the ant's death forms a sexual structur e fr om whic h fr esh spor es ar e r eleased, initiating a ne w infection cycle (de Bekker et al. 2015 ).Fossil evidence dating back to 48 million years suggests that this manipulated behavior of ants can be traced back to the Eocene (Hughes et al 2011b ).Certain aspects of the Ophiocordyceps -ant infection cycle remain unclear due to the uniqueness and complexity of this model system (de Bekker 2019 ).Ne v ertheless, it has been suggested that enterotoxins, aflatrem, and other potential fungal effectors that disrupt ant feeding behaviors could be linked to differ entiall y expr essed genes associated with circadian rhythms, cloc k-contr olled genes, odor detection pathways (odorant receptors, OBPs), and neurotransmitter signaling (kynurenic acid, biogenic monoamines, and dopamine) (Will et al. 2020, de Bekker et al. 2021 ), and many of these genes ma y ha ve dual functions in both beha vior and immunity.

Concluding remarks
Current and future directions linking fungal infection stages with specific olfactory or immune components that may then function/elicit both behavioral and immunological responses are likely to yield important insights into lar gel y unexplor ed aspects of the infection process.Although each stage appears to have distinct behavioral and immune-related responses, the links between these have only begun to be elaborated.In the longer term, these studies are likely to shed important fundamental insights into the biology of the interaction between these organisms and can help impr ov e str ategies for exploiting entomopathogenic fungi in pest contr ol.Suc h a pplications, either as an alternativ e to chemical pesticides and/or as part of integrated pest management practices, can be useful by targeting specific innate or behavioral immune pathways in host-specific ways that would help minimize nontar get effects, r esulting in mor e envir onmentall y friendl y and effective means of eliminating noxious insect pests.

Figure 2 .
Figure2.Fungal and host factors that mediate insect behavioral and immune responses.Before and during the cuticle contact/adhesion stage, fungal released VOCs act as immune/behavioral elicitors on host olfactory proteins; fungal hydrophobins , adhesins , and cell wall components mediate attachment, and the insect cuticle acts as the initial antimicrobial barrier through the production of hydrocarbons, melaninization, and/or antimicrobial peptides (AMPs).As spores germinate and penetrate the host cuticle, fungal-produced cuticle-degrading enzymes hydrolyze host chitin and sclerotized protein cuticle.Within the hemocoel, fungal secondary metabolites , miRNAs , effectors , enzymes , and cell wall components are released that may be recognized and affect immune/behavioral responses through action on host (PRPs), Toll/Imd other immune pathwa ys , and/or ligand binding proteins (chemosensory and odorant binding proteins/receptors) to modulate insect behavior or immunity.

Figure 3 .
Figure 3. Outline of patterns of cross-talk between behavior and immunity.The behavioral responses of insects induced by fungal infection at differ ent sta ges of infection can include: a voidance , self/intr a gr ooming, molting, fe v er, ele v ation seeking sanitation and hygiene beha viors , and corpse mana gement.Man y of these processes are linked to ligand binding/olfactory proteins in the antennae, hemolymph or other tissues, and to neur or eceptors or resident immune proteins in the CNS.Insect immune responses, including melanization, clotting, production of immune effectors (AMPs) hemocyte activ ation, pha gocytosis, nodulation, and encapsulation can be induced by fungal VOCs, cell wall components, and/or metabolites, which act via humoral immune proteins, ligand binding proteins (CSPs and/or OBPs) in the hemolymph and fat body, cellular immune proteins, and receptors in hemocytes and CNS.