Renaming ‘Chemosensory’ Proteins (CSPs): ‘Lipoid-Binding Proteins’ — Molecular Nomenclature, Structure, Expression, Evolution, and Intracellular Functions

1. Introduction

A few years ago, there was a case regarding the reevaluation and/or renaming of allatostatins due to their pleiotropic properties, which go beyond their inhibition of juvenile hormone (JH) biosynthesis in the corpora allata [1]. This is another instance of an invertebrate peptide family being named for a highly contested function (e.g. chemosensing), with the additional information that several cutting-edge investigations, fresh findings regarding evolution, molecule expression, molecule localization, and binding characteristics, as well as two decades more of documented research, strongly imply a different function that goes well beyond insect olfaction [2].

The term “CSPs” stands for “Chemosensory Proteins”. They typically refer to small, water-soluble binding proteins, also known as odor-binding proteins (OBPs), that are strongly thought to mediate the recognition of odor molecules, odorants, and ligands to olfactory receptors (ORs), at the periphery of sensory dendrites in the insect sensillum [3,4,5,6]. According to Lartigue et al. [7], CSPs are composed of six α-helices with an appropriate molecular weight of 10-12 kDa (or 110-120 amino acid residues), four cysteines that form two tiny loops, two nearby disulfide bridges, and a globular “prism-like” functional structure. Four CSP structures have so far been identified in locusts (Schistocerca gregaria) and moths (Mamestra brassicae, Bombyx mori, and Spodoptera litura) [7,8,9,10,11]. These structures can breathe’ or modify their conformation in certain ways in response to ligand binding [12,13]. However, RNA mutations and mutations of the peptide in the ribosome are probably what give the CSP protein molecule family its versatility for a variety of biological functions [14,15]. Because of the CSP structure’s extreme flexibility, multifunctional features are heavily supported. RNA editing and/or post-translational changes, which were found in the silkworm moth B. mori (Bommo), are additional features of CSPs supporting multifunction [12,13,14,15].

CSP molecules are present in insects at all steps of their life cycles, from eggs and larvae through nymphs and adults [16,17,18,19,20]. They are mostly expressed in the antennae, mouth, pedipalps, and legs of locusts, and they have been connected to phase shift (phenotypic plasticity) in those insects [21,22]. However, following exposure to avermectin pesticide molecules, nearly all CSPs are up regulated in the majority of insect body tissues, especially in the gut, and fat body, which is crucial for highlighting a role in insecticide resistance as well as development, as opposed to a role in smell [23]. The issue is that, although CSP expression in non-sensory organs and during early development is well understood, research on the functional role of these proteins has been largely and obstinately conducted using competitive fluorescent binding essays, recombinant proteins testing, and a set of semio-chemicals, as described by Ban et al. [24]. Many times, the functional significance of the ligand molecules tested (usually far too few) was unknown, not required for the chemical communication of the insect under study, or highly disputed. One such example is oleamide, which is well known for being both a strong inhibitor of salivary mandibular gland branching morphogenesis and a potential CSP ligand [24]. Where and when the protein molecule is expressed should be a major factor in defining the true functional ligand. Similar findings apply to OBPs from Aedes aegypti mosquitoes, which are expressed throughout the insect body, including the antennae, legs, and abdomen, but are only partially identified by means of a small number of randomly selected semiochemicals [25]. There is serious uncertainty regarding the function of the protein because this ‘molecular’ study did not analyze any of the chemicals found in Ae. aegypti, ranging from eggs, pupae and nymphs to adults, in all many different tissues [26].

It is currently widely known that and elevated load of CSPs (‘pherokines’) is observed in hemolymph and in all insect tissues subsequent to chemical, microbial, or viral infection [23,27,28,29]. Liu et al. have discussed the specific function of ‘CSPs’ in lipid transport in relation to insecticide resistance using the sweetpotato whitefly Bemisia tabaci (Bemta) as a model study [29,30,31]. Insecticide-mediated upregulation and interaction of the protein (Bemta-CSP1) with long chain C18-fat lipid (C18:2, linoleic acid, LA) have been demonstrated by Liu et al. [30,31], suggesting that CSP plays a metabolic role in insect immunity and defense within the cell rather than through olfaction or chemical communication. Similar to the majority of CSPs, Bemta-CSP1 associated with LA is expressed in a wide range of insect body tissues [30,31]. LA has never been found in insect fingerprints, which is proof that CSPs are not involved in pheromone chemical communication.

The topic of this review is whether the biological function of “CSP” can be expanded in view of their tissue-expression and the fact that insect organisms inherit these molecules for the purpose of binding DNA and fatty acid lipids. We begin by rethinking the idea of the biological function of the molecules based on transcription initiation factor (TIF), mucin, and CSP analogies. We show that one of the central tenets of the literature—the role of these molecules in chemosensing—is simply accepted without any supporting data or logic approach. We can offer a new definition of the biological function of CSP by highlighting its unique relationship to lipids, intracellular events, DNA regulation, and the particular significance of this cell concept in biology. This is accomplished by examining several distinctions and facts, such as their extensive distribution across tissues, their pervasive expression during growth and development, their reaction to stress, and their strong resemblance to the N-terminus of the nuclear, endoplasmic reticulum, ribosome, mitochondrial, cytoskeleton, and plasma membrane proteins, and thus their localization within the cells in numerous different compartments, as well as the novel insights and lessons that can be derived from them.

2. Primary Sequence Variation in Various Phenotypes of Insects

The evolution of the CSP family, particular variations in the molecule, and ultimately the emergence of a new organismal phenotype have all been linked to RNA editing, post-translational modification, and retrotransposition of RNA mutations [14,32,33,34]. The presence of recoding at the level of molecule synthesis in the CSP family is clearly supported by the inclusion of a Glycine residue next to a Cysteine at certain positions, amino acid inversion, and specific motif insertion in protein primary sequence [34,35,36].

By comparing the CSP primary sequences from Acypi, Apime, Aedae, Anoga, Bommo, Culpi, Droso, Nasvi, Pedhu, and Trica, it may be possible to determine whether these mutations can result in the emergence of a new function for the molecule and a new phenotype for the insect organism (Figure 1 & Table S1) [20,24,31,37,38]. Acypi sequences contrast with those for Bommo and Pedhu (Table S1). While Pedhu-CSPs show high identity to those from Diptera, Hymenoptera, and Coleoptera, those from Acipy show more identity to Hemipterans. On the other hand, Bommo-CSPs exhibit the highest degree of similarity to their molecular orthologs in the kingdom of bacteria and many different types of moth lepidopterans (Table S1). Specifically, it is interesting to note that Bommo-CSP5*, Bommo-CSP10, Bommo-CSP11, Bommo-CSP14, Bommo-CSP16*, Bommo-CSP17 and Bommo-CSP18* are similar to Allergen Tha p 1 and PAN-1-like protein molecules (Table S1). Allergen Tha p 1 is connected to the molecular sequence of the three truncated CSPs (*) or pseudogenes. In addition to the findings in Bemta [30,31], this could imply that the ancestral function of the protein molecule family is more closely linked to the immune system’s activation during growth or within the cell’s defense mechanism than it is to smell. Tha p 1 is isoallergen variant (Allergen Nomenclature; 15-kDa IgE-binding protein). PAN-1 is a protein that is both transmembrane and cytoplasmic in nature, and essential for developmental events that take place throughout the mid to late larval stages as well as for early larval ecdysis and the execution of the molting cycle during the adult molt. It is an essential control point for the development of larvae and the progression of several tissues during the transition from the larval to adult state. PAN-1 and Tha p 1 molecules are unrelated to chemosensing, and it is very likely that the proteins that are associated with these molecules are unrelated to chemosensing as well (see Table S1).

It is noteworthy that, depending on the species of insect, the molecule’s amino acid content between Cys29-Cys37 and Cys56-Cys59 varies from 6-8 to 18-19 (Figure 1). For Culpi-CPIJ002628, Nasvi-NV16080, and Locmi-CSPs an amino acid insertion (two residues) between Cys29-Cys37 has been found [see [16,20,31,39]]. For Nasvi NV16076 and NV16077, Apime-GB17875, and Acypi000345, an amino acid insertion (one residue) between Cys56-Cys59 has been found ([31] and Figure 1). Specifically, amino acid mutation (insertion or deletion) is observed in various regions of the CSP molecule in the jewel wasp phenotype Nasvi (Figure 1). In contrast, the intercysteine gaps in Droso, Anoga, Aedae, Bommo, Trica, and Pedhu are strictly preserved, which indicates that the CSP protein molecule’s degree of mutations is highly dependent on the phenotype of the insect (Figure 1). We found that, in order to maintain the stability of the molecule, all CSPs appear to have four cysteines (4C) and two disulfide bridges. The length and composition of the space between the cysteine residues vary, though. This definitely gives the molecule a new function and could help a new insect phenotype emerge (Figure 1).

3. Existence of “CSPs” in Phenotypes of Bacteria

It should be discussed that mutations on CSPs might have had an impact on not only the evolution of insects but also the evolution of cells overall. These CSP molecules are not an insect’s apnage. They are also expressed in a wide range of organisms, including many species of arthropods, crustaceans, shrimp, crab, lobster, and copepods [34,40]. They don’t, however, only exist in arthropod species. The fact that they are widely expressed at the level of the bacterial superkingdom indicates that they exist also in prokaryotes as well [15,31,34,38,41] (also see WP_149730592 in multi-species). Prokaryote CSPs are twins or ‘identical twins’ to insect CSPs [34,38,41], which is significant when discussing the function of these molecules. They have been reported on bacterial species, including Gammaproteobacteria Lysobacter (Xanthomonadaceae) and Escherichia coli (E. coli), Acinetobacter baumannii (coccobacillus), Macrococcus caseolyticus (formerly Staphylococcus), the filamentous actinomycete Kitasatospora griseola, the Actinobacteria genus in the families Enterobacteriaceae, Nocardioidaceae, Pseudonocardiaceae (Solhabitans fulvus), and Streptomycetaceae [31,34,38,41]. According to RNA and genomic reports, CSP molecules are also found in Firmicutes, Aeromonadales, Alteromonadales, Eubacteriales (Clostridium perfringens), and Hyphomicrobiales (MDK0835621, MDK0841570; see [34]). These findings from oxidase-positive nonendospore forming, motile, nonmotile, and spore-forming bacteria cast doubt on a chemosensory role for CSPs. Not every bacterium uses quorum sensing. These microbes are recognized as typical digestive tract bacteria, primary prokaryotic secondary metabolites, multi-drug resistant opportunistic pathogens, highly positive cytochrome c oxidase reactions, and multi-species symbionts in insects, and plants, but rather not for their olfactory acuity. Acinetobacter species are significant soil and aquatic microbes that aid in the mineralization of molecules like aromatic compounds (benzene). The smallest free-living prokaryotic cell (0.013 μm³) with very low GC (33%) is found in marine Actinobacteria (Candidatus actinomarinidae) [42]. Given that their geographic distribution is similar to that of picocyanobacteria, there appears to be a strong relationship between the Candidatus and picocyanobacteria microbial groups. Based on the existing literature, it appears more likely that these two types of microorganisms exchange or share molecular modules and toxin-antitoxin systems rather than pheromones [43,44]. Clostridium perfringens (also known as C. welchii, or Bacillus welchii) is known for α-toxin, the toxin involved in clostridial myonecrosis. Nevertheless, other bacteria also produce volatile organic compounds (VOCs), including formaldehyde, methyl mercaptan, isopentanol, and trimethylamine [45]. These VOCs can act as biological indicators of the presence of certain pathogens because humans (and industrial animals) do not produce them. Even better, some VOCs have been found to be useful in the identification of specific bacterial species: isovalefor bacterial identification have even been identified: methanol, pentanol, ethyl acetate, and indole for E. coli [45]. This is not the case with volatile carboxylic acids (VACs), pyrazines, chemosensory signals, aggregation odors, cohesion, or sex pheromones, which have infrequently been connected to associations between microbes and/or insect-microbe interactions [46,47]. Although studying the diversity and binding characteristics of ‘CSP’ proteins in axenic insects—which do not have bacterial populations in their digestive systems—would be interesting, it is highly unlikely that fecal bacterial CSP molecules play a role in odorant pheromone signal transmission: 1) for all CSPs, there is no evidence of specific binding to VACs, VOCs, E. coli odors or insect cohesion pheromones, 2) numerous bacteria produce CSPs that are strictly identical to those found in B. mori, however, the Bommo-CSPs are all widely distributed throughout the entire body of the insect, not just found in its sense organs. They experience a striking up-regulation in response to pesticide drug exposure [24].

4. Expression Profiling in Development, Organisms, and Tissues

Referring to a whole family of proteins whose primary function is supposed to be chemical detection (i.e., proteins carrying odor molecules to olfactory receptors) as “chemosensory proteins” is highly incongruous, given the striking similarities between insect and bacterial CSPs and the fact that all CSP molecules are expressed outside of the sensory organ paradigm. To call these proteins “chemosensory”—that is, proteins that carry smells to olfactory neurons— would be incredibly inconsistent [24,31].

‘CSPs’ are found in phyllosoma, in larval and adult stages, and in the adult stage, in many different non-sensory organs of many different crustacean species, including Antarctic copepod, crab, crayfish, lobster, salmon louse, prawn, shrimp, and water flea [34]. This situation is pretty similar to how insect CSPs are being described right now. Insect gland and venom can also contain CSPs, in addition to the sensillum [5,14,48]. In moths, sixteen CSP molecules appear to be expressed in the sex pheromone gland of a species that uses only one sex pheromone compound, bombykol [14,24,36,49]. However, in addition to the female moth pheromone gland, CSP-expressing tissues and secretions also include the antennal branches, mouth, mandibles, salivae, proboscis, cephalic capsula, eyes, head, thorax, abdomen, epidermis, fat body, gut, wings and legs, i.e., a variety of reproductive and non-reproductive, sensory and non-sensory fluids, but in particular the majority of covering tissues and metabolic tissues [24,31,50,51,52,53]. Most CSPs are expressed in enveloping and metabolic tissues, so there is little clear function assigned to a single tissue or organ such as proboscis or antennae. Expressed Sequence Tag (EST)-base data analysis in Pedhu and Acipy phenotypes (Figure 2) reveals distribution of CSP-RNAs throughout the entire body, in the antennae but also in the head, thorax, and abdomen, similar to Apime, Bemta, Droso, Trica, and Bommo [20,24,30,31,37,38]. Six transcripts from first instar Pedhu larvae and engorged adults were used to identify the CSPs in the body louse [38,54,55]. According to research from The International Aphid Genomics Consortium (2010), Acipy-CSPs are also found in a variety of stages, including the head and the antennae of third instar nymphs, and in winged and wingless parthenogenetic females that have either received an ampicillin treatment or have been inoculated with bacteria for removal of pathogenic agents [56]. Finding out how CSP molecules are expressed in multiple different tissues can provide information on how they function. By definition, the insect EST database, which contains information about the relationships of molecules and tissues of origin, contains more than 30,000 messenger RNA sequences from n tissue libraries [57,58]. We ”dissected” Apime, Aedae, Anoga, Bommo, Culpi, Droso, Nasvi, and Trica using Flybase and specific databases such as VectorBase or KAIKO/SilkDB [20,24,31,37]. The same methodology was utilized to analyze Acypi- and Pedhu-CSPs from Flybase and VectorBase (Figure 2). EST-cDNA libraries from ten different insect phenotypes were analyzed, and the results corroborated Northern blot, Western blot, PCR, and enhanced real-time PCR results when multiple tissues were analyzed in the same combined experiment (not just reporting one clone from a single organ or tissue): CSP is widely distributed throughout the insect body [20,24,31,37] (Figure 2).

5. The Evolution and Diversity of CSP Genes

A function in chemical communication is also at odds with the diversity, evolution, quantity, and number of CSP genes. The 4-8 CSPs found in Apime, Anoga, Droso, Phedu, and Nasvi show that insects generally have very few “CSP”-coding genes [6,20,31,34,38,59,60]. Does this imply that these insects have poor sense of smell or that they don't communicate chemically with one another? Instead, the small number of CSPs disproves their potential for chemical communication (4 in Droso). For chemical communication, pheromones, and mate recognition, flies and many other arthropods are known to use a complex mixture of long chain epicuticular hydrocarbons [61]. Phenotypes induced by RNA interference suggest a role for CSP in Apime brain integument development rather than a role in chemosensing [62]. Bees, lice, and wasps only maintain 6-8 CSP genes, making it impossible for them to distinguish a variety of scents or complex chemical cuticular if CSP molecules only have one distinct function in chemosensing [31]. According to Liu et al. (2020), CSPs are distinctly arranged in pairs of duplicates on individual chromosomes in bees. When examining Acypi-CSPs (Acypi000094-Acypi009116, Acypi000093-Acypi002311, and Acypi000096-Acypi003368), we find a similar distribution of duplication on particular scaffolds (Figure 3). CSPs may therefore cooperate to cause cellular function. These Acypi-CSPs resemble DNA sequences that have been copied invertedly (Figure 3). They are pointed in different directions and have a junction between them. As such, it is expected that they will raise translocation rates [63]. The most prevalent type of chromosome rearrangement, inverted duplications, also known as foldback inversions, is frequently seen in the emergence of new phenotypes, including particular developmental features in the brain. Chromosomal rearrangement and the degree of synteny play a role in pesticide resistance in aphids, which has long been known [64,65]. As seen in Acypi, chromosome rearrangements are undoubtedly the most dramatic type of mutation, frequently resulting in rapid evolution, phenotype adaptation, and speciation, independent of the development of the odor pheromone system [66].

The Acypi-CSPs are composed of two exons separated by a single, variable-length intron, just like in most other insect phenotypes (Figure 3) [15,24,31,34,37,38,67]. A few nucleotides following the start codon that codes for the amino acid methionine, one more intron is inserted in Acypi-005842 and genes like Trica-AAJJ1196A, Bommo-CSP19, Apime-GB19453 and Pedhu-594410. This intron (phase 0 intron) is inserted after the third base and does not disrupt the codon. This demonstrates the tight regulation of the splicing of the signal peptide region and supports the notion put forth by Blobel (2000) that the functional significance of a signal peptide molecule is directly related to its length [68]. The first six amino acids of CSPs could be the needle tip that breaks through the cellular or subcellular membrane. Particular molecular protein partners from the Signal Recognition Particle complex may depend on the other amino acid residues for their proper localization and/or transit within the cell system [69,70]. This renders the “CSP” molecule more elucidable, possibly as an intracellular protein in a multimolecular complex as well as a secreted protein (in lymph and hemolymph).

The fact that the introns in more than 70 CSPs and all other single-intron CSP genes vary in length but are always found in the same place or boundary (Lysine 45) in aphids, bees, beetles, flies, lice, mosquitoes, moths, wasps, and whiteflies suggests that all these genes share a very ancient common ancestor [15,20,24,30,31,34,37,38,67] (Figure 3). The Ordovician, when terrestrial plants first appeared, is thought to be when the class of insects first appeared on Earth 480 million years ago. Overall, the current findings would be compatible with a shared heritage for the CSPs of all insect species, including those belonging to the orders Coleoptera, Diptera, Homoptera, Hymenoptera, Lepidoptera, and Neoptera. There is an extremely long history of the CSP molecule. After that, every group or order eventually started to exhibit some unique patterns, maybe as a result of the appearance of unique phenotypic and/or functional traits [31].

6. Ancestral Functions and Lipoid-Binding Properties

This protein molecule family is extremely common due to its presence in prokaryotes, binding to LA, and widely distributed profiling of gene expression, in addition to its highly conserved gene structure. This strongly suggests that it plays a fundamental role in the transport, uptake, lipid exchange, and lipid metabolism of long chain fatty acids (FAs) that are essential for development, flight, pheromonogenesis, reproduction, and immune responses [31].

Nomura et al. (1982) described the first member of this small soluble protein family as an up-regulated factor (p10) in the regenerating legs of Peram [71]. The same protein molecule (called p10) was found in the antennae and legs of Peram at the adult sexually mature stage, with some differences between males and females. This finding in the cockroach rather suggests a “chemodevol” function for this protein, contributing to tissue development and recognition of sex-specific signals like plant odors and/or odor sexual pheromones [4]. One (polyclonal) antibody against “CSP” protein labeled the antennal sensillum in immunocytochemistry experiments, but the labeling was diffused to the cuticle and supporting cells as well as sensory structures [5,72]. Serums containing polyclonal antibodies are combinations of antibodies with distinct specificities. The fact that polyclonals are far less specific than monoclonals is one of their drawbacks. Significant heterogeneity within the antibody pool, non-specific interactions with the molecule, and an increased risk of cross-reactivity (false positives) are present. For a rabbit to produce the “correct” antibody, multiple vaccinations against the same molecule are typically required. It is necessary to determine which sensilla express CSPs, how signals move from internal heating of supporting cells to sensory dendrites submerged in sensory lymph, and/or how these extracellular CSPs can arise from an intracellular one using monoclonals. It is primarily necessary to clarify the labeled sensilla’s purpose. Coeloconica sensilla, peg-in-pit sensilla, thermoreceptor, hygroreceptor, infrared receptor, and acoustic receptor all have dendritic branchings similar to that of chemosensory pheromone sensilla [73].

The vast majority of CSP molecules is expressed outside of the olfactory paradigm and takes part in the moth response to avermectins, as reported by Xuan et al. [24]. According to Loftus et al. [74], Verjovski-Almeida et al. [75], Noriega et al. [76], Nene et al. [77], CSPs are also highly prevalent in Plasmodium gallinaceum, Brugua malayi, and Dengue virus infections in Aedae, and bacterial infections in Culpi. The abundance of CSP genes in moths and mosquitoes is probably related to toxin, insecticide, viral, or microbial resistance rather than chemosensing [20,24]. Interestingly, the Dengue fever mosquito, Aedae, has another use for CSPs. These molecules are found in both adult Aedes and larvae, but they are primarily concentrated in the adult's fat bodies, corpora allata (endocrine sources of JH), and salivary glands [20]. This suggests that, at least in Ae. aegypti, “CSPs” are involved in the binding, transport, and/or biosynthesis of JH.

The widespread tissue and developmental expression of CSPs, as well as the fact that CSP is present in other arthropod species, such as copepods, shrimps, and water fleas in addition to insects strongly suggest the function of CSP molecule in relation to juvenoids [34]. This role would be similar to that of the OBPs in the hemolymphatic transport of the JH molecule, according to Kim et al. [78]. It is well known that JH regulates the developmental processes in arthropods [79]. Therefore, “JH-related proteins”, or “JHRPs” would be a better name for these proteins than CSPs, or SAPs (“Sensory Appendage Proteins”). There are no sensory appendages on corpora allata. Nearly every facet of physiology in both systems is regulated by JH function in both larvae and adults. Caste differentiation, pheromone production, development, male genital morphogenesis, female ovarian egg production, social and mating behaviors, and immune function are all regulated by JH [80]. Specifically, it has been shown that JH controls neuronal plasticity linked to brain structure, neurogenesis, and behavioral maturation; this is comparable to the pattern of CSP expression observed in arthropods [31,34,81,82] (see Figure 2). According to transcriptomic studies from Bian et al. [83], even the prothoracic glands (a source for ecdysone) express CSP, which is incompatible with a function in olfaction.

An important observation in this analysis is that bacteria and prokaryotic cells share the JH relationship and CSPs. Phurelipids are secondary metabolites that bacteria produce. Phurelipids and juvenoids have a similar molecular structure. Not only did JH and other phurelipids hinder insects' immune responses, but they also prevented pupae from developing into adults [84]. These findings strongly suggest that “CSP” molecules serve a variety of functions in the immune defense system of microbes and arthropods, ranging from the binding of a wide range of exogenous foreign toxic insecticide chemicals to the intracellular transport of endogenous fuel lipids, FAs, and hormones [24,30,31,37,38]. This is accurate, unless it can be shown that CSPs are a component of the microbe bacterial olfactory hoedonics. Sifting out a subset of the CSP family that is unique to antennae or olfaction is challenging because most of the molecules are produced in the gut (hindgut) and fat body—which are believed to be the arthropod body's main organs for storing lipids and FAs as energy—. These fats and FAs are released into the hemolymph through lipolysis, allowing other organs to use them as fuel for growth, development, regeneration, and defense against infectious viruses and pesticide chemicals [15,24,27,28,29,30,31,37].

The existence of CSPs in bacteria and their crucial roles in the development of honeybee heads, shrimp molt, arthropod general immunity, JH, pheromone synthesis, and/or behavioral changes in specific phenotypes are consistent with a role for CSP molecules in lipid transport [20,21,22,23,24,25,26,27,28,29,30,31,32,33,34].

Ozaki et al. [85] state that “CSP molecules” mediate the identification of chemical signatures composed of cuticular lipid FA hydrocarbons, like those found in ants; among other organisms. It is currently unknown how each of these “CSPs” connected to FAs functions in chemical communication, development, and/or other physiological processes. Whiteflies have demonstrated that FAs such as LA bind to the CSP’s functional structure, but LA (C18:2) does not involve chemical communication [30]. Like crustaceans, most insects are unable to produce LA on their own. The body’s various cells need to use uptake and transport mechanisms like CSPs to use it as fuel. Insecticide chemical compounds (cinnamaldehydes) generated from plant oils, such as “dangerous” toxic chemicals, can interact directly with other functional CSP structures, as has been shown in whiteflies [30]. Therefore, two more points are essential to understand the role of CSP molecules: 1) CSPs appear to play a variety of biological roles and are expressed in both bacteria and arthropods; 2) CSP molecules in particular can activate specific innate immune pathways when injected into the phloem of plants [86,87].

It is not a sensory lymph, phloem. Phloem is the vascular tissue that carries and distributes water, carbohydrates, and other soluble organic substances to the various sections of the plants. The phloem transports nutrients and food that the leaves produce through photosynthesis (photosynthates) to all other parts of the plant. It functions structurally in the body of the plant and serves as a pathway for numerous hormones and other signaling chemicals. The three cell types that typically comprise this tissue are sieve elements, parenchyma, and sclerenchyma. It is through these tissues that CSP molecules, like Myzpe Mp10, can trigger an immune response instead of activating OR [86,87]. It becomes significant to note that in this case, Mp10 shares a great deal of similarities with other proteins, including neural Wiskott-Aldrich Syndrome (WAS/WASL)-like proteins, actin skeleton regulatory protein (ASRP), Arp2/3 complex (Actin Related Protein 2/3 complex) activator (actin filament binding proteins and WASPs), peplos-cell cross-linker, stress response initiator, cell wall/envelope protein (CWP), Mucin, Extensin, PAN domains (these versatile domains mediate interactions between proteins and carbohydrates, fulfilling a variety of biological roles), splicing regulator, Rho GTPase (Rho) activator, Serine/Threonine-protein kinase from Social amoeba (SamkC), transcription initiation factor (TIF), nuclear pore complex protein (NPCP), Sec31, UL36 tegument protein, and many others (30.71-45.13% identity; Figure 4, Figures S1 and S2 and Table S2). These findings raise very interesting questions about the possible roles of “CSPs” in the cytoplasmic membrane and cell surface, as well as, in the cell cytoplasm and nucleus, RNA/DNA binding, RNA/DNA regulation, interactions with multiple molecular and genetic components, transcription control, splicing regulation, and activation of specific protein complexes.

7. CSPs’ Intracellular Functions (Gene Regulation to Stress Response)

The fact that the Myzpe CSP Mp10 and many other “intracellular” regulatory elements are related cast doubt on the claim that CSPs are “chemosensory” molecules (Table S2). Mp10 appears to even be expressed by viruses when compared to some peplos proteins and outer surface membrane proteins (see Table S2).

7.1. Evolutionary Evidence Derived from Amino Acid Sequence Phylogenetic Analysis

A preliminary phylogenetic analysis in IQ-Tree found some connections between CSPs, CWP, Rho, SamkC, Sec31, SamkC, TIF, SamkC, WAS/WASL, DNA-binding proteins (DBPs), DNA-regulatory proteins (DRPs), and several RNA-binding proteins (RBPs) sequences molecular sequences (Tables S3–S4; [38]). Transcriptional/cell division repressor, helix-turn-helix, and DRP from the Xenobiotic Response Element (XRE) family of transcriptional regulators, branched with Bommo-CSP16, Bommo-CSP8, and Bommo-CSP9 and their bacterial counterparts, whereas another orthology group included Trica-CSP AAJJ0012J and Ruminococcus DRP WP_044998036 (nucleotide binding; see Table S3 and [38]). This implied that some CSPs have evolved, at least in Bommo and Trica, to perform functions associated with nucleotide binding, transcription, translation, DNA/RNA templates, DNA/RNA control, and/or intracellular gene expression mechanisms. The XRE family of proteins, which is extensively present in bacteria and eukaryotes, is involved in many aspects of controlling cellular metabolism. Under normal conditions, XRE transcriptional factors bind to the promoter region of the gene and function as repressors.

A phylogenetic examination of Bommo-CSPs, Trica-AAJJ0012J, BemtaCSP1, Myzpe-Mp10, its derivative sequences, Mucin-like sequences, XREs, and several RBPs sequences in PAUP (*10Altivec) provided further evidence for this (see Tables S2–S4 and Figure 4, Figures S1 and S2). A neighbour-joining tree (BioNJ study) showed that Mp10 did not join the Bommo-CSPs, but instead seemed to be much more closely connected to the Mucins group (G1; Figure S1A), albeit not clearly forming an orthologous group (Figure S1B). In G1, the “CSPs” from E. balteatus, EbalDDB_G0285119X1 and CSP3 (QIS77910), linked with Bommo-CSP10 and showed high similarity to Rho-activator isoform X3 (Table S3; Figure S1A). The Trica-CSP AAJJ0012J molecule clustered with Allergen Tha p 1, and associated RBPs instead of BommoCSPs. The marmalade hoverfly Epiba-CSP4 (QIS77191) clearly deviated from the CSPs groups (see G2, Figure S1A). Only the G1 “EbalCSP”-group maintained a relatively high bootstrap value (94%) in the Jackknife analysis (bootstrapping calculation over 1000 repeats), strongly demonstrating the branching of “CSPs” with Rho GTPase enzyme. A common branch with a significant bootstrap value (57%) was formed by the sequences combining CSP, CWP, LRR, Mucin, SamkC, TIF, and WAS/WASL. Mp10 slipped off of this group, but AglaCWPX3 drew it in (Table S3; Figure S1B). The papilionid IpodCSP (CAH2042437), DBPs, RBPs, and TIFs were drawn at the bottom of the tree in this analysis using Bommo-CSP2 and bacterial counterparts as the reference outgroup (Figure S1B). This strongly implied that there were significant and expected relationships between “CSPs” and all of these various intracellular proteins, mucins, Rho-activators, and translational regulatory factors.

Focusing on Mp10 (referred to as a very typical “CSP”, 153 amino acids, 17.2 kDa, “consensus residues”, four cysteines pattern, whole body expression), we found that this specific protein sequence (XP_022173691) constructs a relevant hierarchical clustering UPGMA (unweighted pair group method with arithmetic mean) analysis and a phylogenetic tree supported by a high bootstrap value when compared to Allergen, Mucin, TIF, NPCP, and ASRP amino acid sequences (Figure 4). It's interesting to note that Mp10 molecule does not cluster with other “CSPs” on the UPGMA tree. It belongs to a large group that includes all Mucin taxa and TIF. It shares a close relationship with Rho-activator (XM_056065396; Figure 4A). The Rho families of small GTP-binding proteins (20-30 kDa) are not extracellular molecules. These are intracellular proteins that control the actin cytoskeleton-related Rho-GTPase signaling pathways. They act as molecular switches that control a variety of cellular processes, including gene transcription and cytoskeleton-related events. RhoGAPs, one of the main classes of Rho-regulators, are found in all eukaryotes (within the cell) and have been shown to regulate a variety of cellular functions, such as the organization of the cytoskeleton, growth, differentiation, neuronal development, and synaptic functions [88]. They have nothing to do with smell; they don't trigger transmembrane chemosensory receptors.

As for the other “CSPs”, they belong to either the ASRP group (see the position of BAY56819, KAI5642933, and Bommo-CSP10, in the UPGMA analysis on Figure 4A) or the Allergen group (which looked to be the most ancestral molecule): XP_002432595, ALC42649, KAG5343447, XP_002092928, EDW02527, OIC81003, and OIC85870 (bacterial proteins). Significant outer envelope proteins, RickA-like (Arp2/3), and particular nuclear nucleoside kinase (NNK) are found in the ASRP group, while IgE-BPs, pherokines (fly hemolymph CSP proteins), acid trehalase (AHX71992, involved in intracellular trehalose mobilization during postdiauxic growth and severe saline stress in yeasts), Cell Wall-Anchored (CWA-3, XP_018563025), and TIF sequence (XP_044745729) are found in the Allergen group (Figure 4A). RickA is the protein from rickettsiae bacteria, which are carried by ticks and lice. It is involved in bacterial host cell binding and infection as well as the actin-based motility of bacterial cells. It also triggers host cell factors related to the cytoskeleton [89]. CWA-3 molecule is a component of the cell wall integrity-signaling pathway, which is regulated by small proteins like GTP-BP Rho1. Controlling gene expression and coordinating periodic modifications to the cell wall are the primary functions of CWA molecule during the cell cycle and in response to various forms of cell stress [90]. NNKs (or nucleoside diphosphate kinases, NDPKs) catalyze the transfer of the terminal phosphate (P) from a donor triphosphate (TP) to an acceptor diphosphate (DP). Arp2/3 is a ubiquitous and essential component of the actin skeleton found in eukaryotic cells. It nucleates actin filaments, caps their sharp ends, and cross-links them to form orthogonal networks. These molecules form a large group that is associated with Mp10, a protein classified as a “chemosensory protein”. The functions of these proteins are not as closely related to smell as they are to the cytoskeleton, actin filaments, transcription of genes, phosphate transfer reaction/exchange, phosphorylation, mitochondrial energy production, TP-DP conversion, and cell regulation (see Figure 5).

Mp10, “CSP”, Allergen, TIF, Mucin, NPCP, and ASRP have a very distant common ancestral origin according to the topology of the UPGMA tree, which is based on the assumptions of a common root and constant evolutionary rates for all lineages (i.e., it takes the "Molecular Clock Hypothesis" [91] to account for mutation rates). They are the outcome of a series of duplication events that produced Allergen, which includes CSP and TIF proteins, prior to Mp10, Mucin, NPCP, and ASRP (Figure 4A). Mp10 (and Rho activator) emerged later in a sequence of duplication events that produced a wide range of Mucin variants, particularly in the mosquito genera Aedes, Anopheles, Culex, Uranotearia, and Wyeomyia (Figure 4A). The high level of duplication and variation observed in Culpi- and Aedae-CSPs makes this point noteworthy [20]. During its evolutionary history, the molecule protein gene family that includes Mp10, CSP, Rho, Allergen, TIF, MUCIN, NPCP, and ASRP appears to have undergone multiple duplications. Some of these duplications are specific to particular taxonomic lineages, like mosquitoes for the long Mucin precursor proteins needed for growth, development, digestion, oviposition, and control of viral infection [92,93]. Other gene duplications are more ancient and common to all lineages (see Figure 4A). This is accurate for the Allergen group, which comprises Trica, ants, damselflies (Ischnura forktails) flies, garden whites (pierids), ladybirds, lice, neodiprions, parasitoid wasps, and tuberworm moths. This also holds true for the ASRP group, which comprises taxa from multiple families of moths and butterflies (swallowtails, speckled woods, Papilio, Pararge, etc; see Figure 4A & Table S2). The UPGMA tree of amino acid sequences indicates that all of these molecules have a distant common origin that is estimated to be 324–440 Mya (the latest Mississippian–Silurian or Devonian [94]), much earlier than the emergence of the various flying insect species. This is because of the distance between the ASRPs/CSPs and the Allergen molecules.

The relationships between the CSP, Mp10, Rho, Allergen, TIF, Mucin, NPCP, and ASRP proteins were further examined using maximum parsimony analysis (Figure 4B). Strict consensus trees were established using MP and the PAUP4.0b10 (Altivec) program, as detailed in Abraham et al. [95]. CSP, Mp10, Rho, Allergen, TIF, Mucin, NPCP, and ASRP molecules are all widely expressed in different tissues, which is already a striking feature. There have been reports of allergens from the abdomen and thorax clones. It is known that Mucin, PAN-1, DAN4, NPCP, WASP, and the YLP motif are present in all body parts, larvae, and pupae (see Tables S2 and S3). The YLP motif is known to facilitate RNA binding activity, regulate telomere maintenance, and contribute to the decrease in telomerase activity that occurs during stem cell differentiation by attaching to the core promoter of Telomerase Reverse Transcriptase (TERT) and leading to its down-regulation [96]. DAN4 determines cellular morphology and plays a crucial role in maintaining cell integrity during cell growth and division, under stress conditions and upon cell fusion. It is a component of the molecular interactions and enzymatic activities in the cell wall in response to different growth phases and toxic signals from the environment [97]. This is remarkably similar to what it is known about CSP ontogeny, tissue distribution, and response to chemical stress (see Chapter 4). Phylogenetic information and expression analysis are combined to link CSPs to intracellular proteins, cell walls, and gene promoter regions. The insect CSP, Mp10, Rho, Allergen, TIF, Mucin, NPCP, and ASRP molecules are closely related to each other; they formed groups with a high bootstrap value (89-100%), ranging from Pedhu-CSP (XP_002432595) to Uralo-Mucin (XP0055592470) and Eupco-Rho (XM_056065396). Together, the Mucin and NPCP molecules form a group with 99% bootstrap value. Additionally, they attach to ASRP and Rho groups, which may indicate a strong relationship between these molecule familes based on their extremely high bootstrap values (93-99%; Figure 4B). Aside protein molecules implicated in actin microtubule association, cytokinesis, Arp2/3, and NDPK, the ASRP group attaches to CSPs with significantly high bootstrap value (94%; Figure 4B).

Interestingly, when bacterial CSPs (A. baumannii OIC81003 and OIC85870) were used as outgroup in the MP analysis of protein amino acid sequences, they attracted Rho at the base of the tree but not Mp10 (Figure 4B). The Allergen group, especially TIF, and Mp10 remained closer in relationship (Figure 4B). Coleopteran, dipteran, hemipteran, hymenopteran, lepidopteran, phthirapteran, and zygopteran (Odonata, blue-tailed damselfly) Allergens/CSPs and Mp10 protein are related, albeit they do not form a general orthology grouping, in contrast to Mucins, NPCPs, and ASRPs (Figure 4B). This is where the UPGMA tree and the MP tree (Bootstrap Jackknife) in PAUP4.0b10 (Altivec) diverge significantly (Figure 4). Following the evolutionary distances between insect species, Allergens/CSPs, including TIFs, segregated independently on the MP tree. Through nucleocytoplasmic transport regulation, CSPs, NPCPs, and TIFs may serve as a hub for changes in gene expression that are unique to a cell, a tissue, or a phenotype [97]. This implies that, in keeping with the previous description of “CSPs” (see Chapter 5), the NPCP-ASRP molecule family is evolving as a fast, intense, frequent, and high rate, despite its extreme age [98,99].

The Mp10 orthology of the aphid, beetle, dragonfly, fly, louse, mosquito, moth, and sawfly belongs to different groups, ranging from Mucins and NCPCs/ASRPs to Allergens and TIFs, according to our phylogenetic analysis (see Figure 4, Figures S1 and S2), regardless of anything related to olfaction, taste, and/or chemosensing (see Table S2). This suggests that, as a result of multiple local duplications, the Mucin, Allergen, TIF, and CSP families underwent common diversification before the emergence of insects (e.g. the Carboniferous Period of the Paleozoic era 299-359 Mya; Figure 4). Mosquito Mucins were grouped together in accordance with the phylogenetic distances between their genomes. The evolutionary histories of CSP and Mucin proteins are then different. Mp10 orthologs in insects seem to have undergone one to five separate duplication events (see Figure 4). Four consecutive duplication events may have given rise to Mucins: two early duplications (d1 and d2) produced Allergens/CSPs, TIFs, and ASRPs; two late duplications (d3 and d4) produced Mp10 and Mucins. If the protein amino acid tree is the right tree to explain duplication and evolution within these relatively similar molecule families, then all of the molecules in the CSP, Allergen, TIF, Mucin, NPCP, and ASRP groups have the same root (Figure 4, Figures S1 and S2 and Table S2). The following are linked: Mp10, CSPs, pherokines, ejaculatory buld-specific proteins (Ebsps), and a multitude of immune system, actin-related complex, nuclear complex, cell regulation, and cytoskeleton regulation proteins. The molecular sequences of ORs do not correlate in this instance. All of these Allergen-CSP and ASRP-NPCP molecules have a very old origin that dates back to the time before flying insects even existed and even further back to the time of microbes’ prokaryotes’ origin (about 3.5 Bya), even though a fifth duplication that produced DAN4 and NPCP may only be found in Culpi (see Figure 4).

7.2. Molecular Evidence Derived from Amino Acid Sequence Modeling Analysis

The strong relationships observed between CSP, Allergen, TIF, Mucin, NPCP, and ASRP molecules can be explained by the alignment of protein amino acid sequences and structural modeling (Table S2, Figure 4, Figure 5, Figures S2 and S3). Protein amino acid sequence alignment demonstrates that CSP precisely matches Allergen and the N-terminus of TIF, Mucin, NPCP, and ASRP (Figure S2). When compared to 1kx9.1 as template reference (X-ray crystal structure, [7]), it appears that the majority of query molecules fold into a prism of six α-helices using SWISS-MODEL Workspace/ GMQE [100]. This was especially true for pherokine Phk-3, immune response protein, acid trehalase, Allergen Tha p 1, Mp10, Bommo-CSP, and Coccinella septempunctata Cocse-TIF (Figure S3). While prism structure is only seen in certain areas of the molecule, such as the N-terminus, the other proteins studied in the SWISS-Model are all significantly larger molecules (Figure 5 and Figure S3). A long α-helical stretch (7-13 turns) forming the C-terminal tail and a CSP prism at the N-terminal region characterize the majority of representative molecules in the TIF, Mucin, NPCP, and ASRP groups (Figure S3). Swissmodel analysis predicts a transmembrane segment at the C-terminal tail is predicted for these structures (see Danpl-WASP, XP_032519994; Pappo-PgIb, XM_013282121; GMQE 0.64, Sequence Identity 70-100%; Figure 5A,B). The butterfly Danpl-WASP protein’s gene locus and molecular structure are known (A0A212FBN2.1.A AlphaFold DB model of A0A212FBN2_DANPL, LOC11677206), and they do not match the characteristics of a molecule that is categorized as “chemosensory”. Danpl-WASP is a single transmembrane domain-molecule that is 297 amino acids long and 33 kDa in mass. Among the most typical uses of these kinds of molecules are their actin-bound structures and their implications for filament assembly and actin cytoskeleton regulation [101,102]. These bigger molecules, such as WASP, are found in the cytoskeleton, microtubules, integument, cell wall, nuclear pore, cytoplasm, and many other intracellular organelles. Their remarkable long loop that joins the “CSP” structure at the N-terminus and the C-terminal transmembrane domain is what distinguishes them (Figure 5 and Figure S3). To obtain this, the AlphaFold DB model of an uncharacterized protein or proteins categorized as “chemosensory protein” or “putative insect pheromone-binding protein (PBP)” is used as a template. It is strongly suggested that CSP and/or putative insect PBP exists in viruses, at least in herpesviruses that target the Golgi apparatus (HSV-1 and HSV-2), given the presence of the ‘CSP prism’-prominent loop-transmembrane domain in a Palearctic butterfly molecule (CAH2235359, “jg5928” with BLLF1; Figure S3) [103]. “CSP” molecule is associated with the herpes virus’s major outer envelope glycoprotein (see “jg5928”; A0A1V1WC08, GMQE 0.64, Sequence Identity 70.31%, Figure S3). We have found that bacteria possess the same kind of molecular structure as described by Liu et al. [38]—a CSP joined to a larger molecule with transmembrane and glycoprotein domain (Lysobacter capsici WP_096417339, 491 amino acids, 50.6 kDa). Therefore, rather than olfaction, it is more likely that infection (interaction with virus or microbe binding and immune response) is the primary function of CSP.

7.3. Cellular Evidence Derived from Location, Size, Structure, and Expression in Viruses and Microbes

These striking similarities between CSP, Allergen, TIF, Mucin, NPCP, and ASRP molecules suggest that “CSP” molecules function in a multitude of diverse ways inside the intracellular systems of both eukaryotes and prokaryotes (Figure 6).

When it comes to eukaryotes, CSP binds to LA (C18:2) which is made from arachidonic acid (AA, 20:4ω6) and is required for the biosynthesis of many hormones. Neither C18:2 nor 20:4ω6 acts as a mediator in olfaction or chemical communication [see 16]. They are related to the hormones leukotrienes, thromboxanes, and prostaglandins. These three classes of hormones regulate many physiological processes, including innate immune response, ion transport, egg development, and reproduction [104,105,106]. LA and AA phosphorylate a wide range of intracellular proteins (enzymes, pumps, receptors, and so forth), which in turn controls a multitude of signal transduction pathways and cellular processes [see [31]]. CSPs take part in the FA lipid and phosphorylation processes, which influence the biosynthesis of stress responses in the nucleus, mitochondria, Golgi, endoplasmic reticulum (ER), plasmic membrane, ion channels, ion pumps, ion transporters, lysosomes, and ribosomes (see +, Figure 6A). This information is supported by whitefly experiments [30,31]. The “CSPs”, along with LA, AA, FAs, and stress responses, most likely mediate the mechanisms controlling lipid metabolism in the cell cycle at the molecular translational level in the cell ribosome [30,31,107,108]. Moreover, CSP interacts with cytochrome P450 (CYP) enzyme in the ER and the mitochondrial system in response to insecticide exposure (+) and other stress responses. The CSP-CYP interaction is probably important for cellular metabolism, homeostasis, hormonal synthesis, toxin catabolism, and xenobiotic detoxification in response to insecticide exposure [24,109]. The lysosome's functions, including the digestion and breakdown of macromolecules (proteins, lipids, carbohydrates, and nucleic acids), repairs of cell membranes, and defense against pathogens like microbes, bacteria, fungi, and viruses, are probably also all mediated by the CSP-Degradative Enzyme (DE) system (Figure 6A). When food is eaten or absorbed by the cell, the lysosome releases its enzymes, which convert complex molecules—such as sugars, proteins, lipids, FAs, AA, and/or LA brought by CSPs— into energy that the cell needs to survive [110]. After that, the CSPs will probably also need to work with the desaturase enzymes, the ER membrane, and the eversible vesicles that secrete the cuticle, the duct, and the pheromone compound if they are to transport FAs like LA and its two precursor molecules, stearic acid (SA, C:18) and elaidic acid (EA, C:18-1). Lipid droplet formation and pheromonogenesis would strictly require the coordinated action of CSPs, FAs, and ER in the pheromone gland. The sites of metabolism that are likely regulated by CSPs are not the activation of OR on sensory neuron dendrites, but rather the ER membrane of the sex pheromone gland, the oenocytes and the ejaculatory bulb of flies, the abdominal tergites of cockroaches, the coremata (hairpencils) of butterflies and moths, and mandibular gland pheromones [14,30,31,111,112] (Figure 6A).

Here, we find that CSP is linked to the ER, as well as to the endomembranes from the Golgi, mitochondria, and plasma membrane via Rho GTPase signaling complex, Mucin, ASRC, and NPCP (Figure 5 and Figure 6). Together, Rho, Mucin, ASRC, and NPCP are connected to CSPs, allowing these molecules to protect the cell membrane, control membrane interactions, regulate the trafficking of protein precursors and FA lipids between the different cellular organelles, particularly the lysosomes and Golgi system, and control the functions of the genome [113,114]. Furthermore, we find that “CSP” is the N-terminal region of “Gplb”, a protein that resembles the glycoprotein receptor on the surface of human platelets, and as such, it may reside extracellularly to recognize antigens, activate the immune system, and/or recognize JH and promote cell growth (Figure 6A) [115]. We also find that CSP links nucleotide binding, RNA metabolism, and nucleus-wide control of gene expression to splicing factor (SF), TIF, and NDPK [116,117]. As allergens or mucin-like fractions, some soluble fractions of CSP are secreted, bolstering the immune system's resistance against microbial stress, especially in the saliva, gut, and eyes [118]. In the eyes, they mediate substance exchanges (gas, water, etc.) between internal organs and the environment. Additionally, they may coat the insect’s eyes and antennae, enhancing its sensory capabilities. Flying insects have to deal with a variety of foreign, toxic particles, including mold spores, dust, and pollen, in the same way that swimming crabs have to deal with pesticide chemicals, pollutants, varying salinities, and ammonia environments. The existence of CSPs in sensory organs would be explained by this protective role, but not only. According to the reports of Huang et al. [119] and our research presented here (Table S2 and Figure 4, Figure 5, Figure 6, Figures S2 and S3), extracellular CSP-mucin complexes may also be necessary for the adult development of insects and crustaceans. This would account for the presence of CSP, Rho, Mucin, ASRC, and NPCP throughout the various stages of arthropod development (Table S2).

Similar to eukaryotes, bacteria and prokaryotes also require the transport of LA for the phosphorylation of various enzymes (PLC and PKC), ion pumps (K+/Na+), and receptors (microbial rhodopsins) [120]. However, in contrast to eukaryotes, bacteria, fungi, and plants possess soluble intracytoplasmic desaturases [121], and it is plausible that CSP’s interaction with FAs and lipids mediates the activity of this type of biosynthetic enzyme (∆6, ∆9, or ∆12 desaturase) as well as transmembrane desaturase (Figure 6B). Regarding the transport of C:18, C18:1, and C18:2 in cell and molecular stress response, CSPs are intimately associated with the desaturase function [30,31,122,123]. This role is far more focused on supplying the cell with energy than it is on chemosensory receptor complexes (Figure 6). Similar to eukaryotic cells, ribosomes are the targets of biological chemical stress in prokaryotic cells [123]. Desaturase enzymes, FAs, CSPs, and ribosomes seem to be interdependent, especially in situations of biological chemical stress (Figure 6). The different types of desaturase enzymes are differentiated by the specificity of their substrate, which is the location of the double bond on the FA molecule. Nevertheless, there have been reports of desaturases exhibiting multifunctional activities in bacteria, fungi, plants, and insects as well [124,125]. CSP-desaturase coupling may be necessary for substrate switching in lipids and to determine the composition of FAs in glandular cells (female sex pheromone gland), corpora allata, sensory neurons, and/or all other cells in the insect body. Bacterial desaturases, which would need a substrate modification to change the FA content in reaction to environmental stress, may also need CSP [24,30,31,126,127]. The CSP molecule folds in the CWP complex of prokaryotes, bringing it to the cell’s envelope and serving as the main stress-bearing and shape-maintaining element, as opposed to folding in arthropod chemosensory system [128]. Maintaining the shape and structural integrity of cells depends on the integrity of their cell walls, and CSP-CWP may be involved in this process (Figure 6B). Apart from providing a strong and inflexible exoskeleton to ward off damage, CSP-CWP could serve as a point of attachment for proteins that engage with the bacterial milieu. CSP-CWP complexes of microorganisms (and plants) could act as barriers against biotic and abiotic stresses. These functions include shielding the cell from chemicals, drugs, toxins, and other harsh environmental conditions, such as preventing the cell from drying out in high temperature conditions [129]. CSPs may bind to toxin chemicals, expose them to enzymes that degrade them, and/or contribute to the supramolecular agency of the CWP complex because they are found in the N-tail of CWP (see Figure 5 and Figure S2). Rather than using chemosensing, examining these possibilities for the purposes of CSP would be interesting.

Furthermore, we find that CSPs are molecules associated with the Leucine-Rich Repeat protein complex (LRR) present on plasmids (Table S1; Figures 4–6B and S3). Their participation in various protein-protein interactions that activate the plasmid and increase its pathogenicity and virulence may occur there [130,131]. Finally, it can be inferred that CSPs and DNA-dependent RNA polymerases (Dd-Rps) regulate transcription initiation in bacterial cells in a manner similar to that of eukaryotes based on the finding that CSP is linked to the N-terminus of TIF (see Table S2, Figure 4, Figure 5, Figure 6, Figures S2 and S3) [132]. This finding suggests that CSPs affect a gene’s transcription level within a particular cell or tissue. It is rather unclear how this relates to RNA binding and/or RNA polymerase activation, but it is undoubtedly unrelated to a chemosensory function (e.g. ‘CSPs bind odors that activate receptors’).

8. To Design “CSP”, New Terminology Is Needed: Structure or Function?

Table 1 compiles the history of the various names used to describe this molecule protein family as early as 1992. Even though p10 was found in leg regeneration tissue rather than antennal sensory structures, it changed names three times between 1994 and 2000 (see Table 1). All of the changes ignored the first protein gene family member to be reported in the literature and had the same obvious and unambiguous meaning (chemosensory function, limited to chemosensory tuning). Despite the lack of sufficient evidence, the protein gene family was renamed to exclude any possible developmental functions and to reflect only chemosensory functions [135,136]. It was not even remotely possible to imagine at the time (1992-2000) the relationships between p10, CSPs, mucins, cytoskeleton complexes, genetic elements, nuclear pores, and intracellular processes that we discuss here (see Table S2, Figure 4, Figure 5, Figure 6, Figures S2 and S3).

“CSP” was the fourth revision to the nomenclature for p10, which continues to significantly minimize the developmental role in favor of a chemosensory function. However, the existence of “B-CSPs” (CSPs in bacterial species) raises the question of what potential use these protein molecules may have in chemical detection [31,34,38]. Since there isn’t a single piece of evidence indicating CSP molecules interact with sensory neurons, their precise role in chemosensing or olfaction is still hotly debated. There is no evidence to support the theory that CSPs attach to smells and transfer them to ORs—in insects, crustaceans, or, most definitely, bacteria and viruses.

Since 2003, it has been demonstrated that this family of proteins operates independently of the chemosensory system [6,27]. The term “Pherokines” refers to highly prevalent molecules in flies’ hemolymph that arise from bacterial, viral, or chemical infection ([27,28,29]; Table 1), which presents the concept of p10 or CSP molecule in the insect defense system and immune responses. It was eventually proposed to rename these molecules as “Cuticular Sensory Proteins” (“CSPs”, [138]) in order to highlight their strong expression in lipid-layered chitin cell walls, first immune barriers, the exoskeleton (to which muscles are attached), the epidermis, epicuticles, major outer surfaces, antennae, eyes, legs, and other sensory organs. Thus, a growing body of research has been conducted on this molecule’s protein gene family, reporting functions that go well beyond the sensory branches and the olfactory system. However, none of these studies have addressed the potential role of “CSP” in relation to ARPC, NPCP, mucin, TIF, transcription initiation, RNA, nucleoside kinase, and genetic regulation as our study does (see Table S2 and Figure 4, Figure 5, Figure 6, Figures S1 and S3).

What distinguishes p10, OS-D, A10, SAP, and CSP from one another? The most remarkable characteristic of this family of protein molecules, which is expressed throughout the insect’s body [31], is entirely overlooked by many synonyms (Table 1). It is necessary to rename the entire molecule’s protein gene family, in order to account for all family members across species of arthropods, crustaceans, insects, isopods, and microbes in the bacterial prokaryotic superkingdom [31,34,38]. Additionally, this is required to consider the relationship between CSPs and intracellular mechanisms (Figure 6). Typically, a neuropeptide or hormone molecule is named after the first “physiological” or “pharmacological” effect that is noted for it; however, this naming scheme often proves to be fairly inaccurate. The best course of action is probably to rename all naturally occurring peptides, hormones, carrier proteins, transporters, “CSPs”, and related molecules in a more impartial, methodical and objective manner. The location and long list of functions linked to this molecule protein gene family defy the different labels assigned to “SAP” or “CSP” (Table 1 and Figure 4, Figure 5, Figure 6, Figures S1 and S3). Developing new terms or nomenclature is required, but they cannot be limited to chemosensory or olfactory structures, assume any illusive functions, and/or be used without enough evidence or proof.

When discussing the entire molecule protein gene family, it becomes somewhat inappropriate or rather awkward to use the terms “SAP” and “CSP”, which precisely translate to “Sensory Appendage Protein” and “Chemosensory Protein”, expressed in olfactory cilia, respectively. It is not appropriate to use either term —CSP or SAP— to describe all the genes and protein molecules expressed in the antennae, mouth, and legs, as well as in many non-sensory metabolic cell tissues and hemolymph, in the gut and fat body, and all those that have evolved from organisms ranging from bacteria and viruses to insects and crustaceans [[24,31,34], and this study]. There is no need for additional discussion because of the analysis of genome and EST databases. In line with the molecular expression data obtained in moths [24], ESTs of bacteria, insects, sea crustaceans, and terrestrial arthropods show that none of these “CSP” proteins are specifically tuned to olfactory and/or taste chemosensory organs [20,30,31,34]. Actually, based on our analysis, they resemble general intracellular components more (Table S2 and Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figures S1–S3).

This situation seems to be similar to that of a superfamily of widely distributed, heterogeneous proteins known as “lipocalins”, which are responsible for the transport of small hydrophobic molecules such as lipids and steroids (the term ‘lipocalin’ comes from the Greek lipos, which means fat, and Greek kalyx, which means cup). Lipocalins are diverse and rather poorly conserved throughout evolution [138]. Unlike lipocalins, the “CSP” family describes a group of rather homogeneous, similar gene structures across a wide range of organisms; they share the same intron boundaries, and evolutionary well-conserved molecular structures (a prism or pyramid with a pattern of four cysteines that is always in the same position), and similar tissue profiling (ubiquitous expression; see chapters 1-6). Their binding properties differ somewhat (not only to LA, long FAs, and lipoid chains, but also to cyclic compounds) [30], but the primary distinction between lipocalins and CSPs is that CSPs connect with TIFs, RNAs, Mucins, DNA-regulatory proteins, SamkC, WAS/WASL-like, CWP, cytoskeleton actin-linkers, and other transcriptional factors that have been discussed thus far (Figure 4, Figure 5, Figure 6, Figures S1–S3). It even appears that “CSPs” have undergone natural selection and evolution in viruses prior to becoming phenotypes (Figures 4–5B) [139], which is a stark contrast to lipocalins.

It is probably best to use a new nomenclature to describe the peptide molecule, taking into account intracellular localization and evolution in microbes. It would be appropriate to refer to CSPs and OBPs as “lipoclistins” (from Greek Λίπος lipos=fat and Greek κλειστό kleistó=closed in), as proteins that enclose lipoid ligands, much like lipocalins do; citing Rudolf Alexander Steinbrecht [140]; Max-Planck Institute for Biological Intelligence, Seewiesen, Germany). According to this system of naming based on words and names’ Greek ancestry, following the nomenclature of COVID-19, the term “lip-anoiktins” (perhaps “lip-aniktins”, as oi in modern Greek is pronounced i; derived from Greek words for “fat” and “open”, respectively; open=ανoικτος anoiktos) might be a better way to describe the main structural component of the CSP —an open-air structure—. World Health organization has designated key strains of SARS-CoV-2, the virus that causes COVID-19, with labels that are clear, simple, easy to say, and memorable. The Greek alphabetic names, like “Alpha”, “Beta”, “Gamma”, “Epsilon”, or “Omicron”, will be used going forward. Whatever the nature of the virus, the Latin word “apertus” may be a much more appropriate and straightforward term for the CSP protein (following Rudolf Alexander Steinbrecht [141]).

A generic name such as “arthrolipin” would be inappropriate for CSP molecule since it is not specific to the arthropod family; it can also be found in viruses, bacteria, and prokaryotic cells ([31,34,38], WP_149730592, Figure 5 and Figure S3). Not to be forgotten, in honor of the early literature and the old name, Alejandro P. Rooney (USDA-ARS, Lubbock, Texas, USA) proposed “4CSP” (Four Cys Soluble Proteins) or CSP/4Cys without drawing any broad conclusion regarding their physiological task.