Leucokinins: Multifunctional Neuropeptides and Hormones in Insects and Other Invertebrates

Leucokinins (LKs) constitute a neuropeptide family first discovered in a cockroach and later identified in numerous insects and several other invertebrates. The LK receptors are only distantly related to other known receptors. Among insects, there are many examples of species where genes encoding LKs and their receptors are absent. Furthermore, genomics has revealed that LK signaling is lacking in several of the invertebrate phyla and in vertebrates. In insects, the number and complexity of LK-expressing neurons vary, from the simple pattern in the Drosophila larva where the entire CNS has 20 neurons of 3 main types, to cockroaches with about 250 neurons of many different types. Common to all studied insects is the presence or 1–3 pairs of LK-expressing neurosecretory cells in each abdominal neuromere of the ventral nerve cord, that, at least in some insects, regulate secretion in Malpighian tubules. This review summarizes the diverse functional roles of LK signaling in insects, as well as other arthropods and mollusks. These functions include regulation of ion and water homeostasis, feeding, sleep–metabolism interactions, state-dependent memory formation, as well as modulation of gustatory sensitivity and nociception. Other functions are implied by the neuronal distribution of LK, but remain to be investigated.


Introduction
Neuropeptide signaling regulates major aspects of development, growth, reproduction, physiology, and behavior of animals. A large number of structurally diverse peptides have been identified that act on different types of receptors as co-transmitters, neuromodulators, and hormones [1][2][3][4][5][6]. In insects, one of the peptides that has attracted substantial attention recently is leucokinin (LK), although it was discovered in a cockroach more than 30 years ago [7]. We thus decided that it is timely to review what we know about LK signaling in insects and other invertebrates.
Like many other well-known insect neuropeptides, LKs were first identified from extract of the head of the Madeira cockroach Leucophaea maderae (now Rhyparobia maderae) by assaying purified fractions for their activity on hindgut contractions of this animal (see [7][8][9]). Altogether, eight LKs (sequence-related paracopies) were identified in L. maderae, which share the C-terminus pentapeptide FXSWGamide [7,10]. Apart from stimulatory action on muscles, another early function assigned to LKs was a role as a diuretic factor that increases secretion in the Malpighian (renal) tubules of various insects [11][12][13][14]. As we shall see in later sections, we now know that LKs have truly pleiotropic functions as neuromodulators and hormones in insect development, physiology, and behavior.
In earlier studies, these peptides were named kinins with a species prefix, such as achetakinins, muscakinins, and lymnokinins, and only later the original name leucokinin was adopted more generally for peptides with the generic C-terminus pentapeptide. Thus, we will use LK here, except when some species-specific aspect is discussed and a speciesspecific name has been assigned. Early on, it was suggested that the LKs are ancestrally related to the vertebrate tachykinins due to some rather minor amino acid sequence
Outside arthropods, multiple LK paracopies are also known. In the annelid worm Urechis unicinctus, eight paracopies of LKs have been identified [56], and the largest number of LKs was found in the LK precursor of the marine slug Aplysia californica with 30 ( Figure 3) [22]. In some species, such as Frankliniella, Rhodnius, the bed bug Cimex lectularius, and Aplysia, the prepro-LK can give rise to additional non-LK peptides, resulting in a total of about 60 peptides in Aplysia [21,22,52,57]. Of note is that, to our knowledge, none of these non-LK peptides have been studied further in any organism (and very few have been verified by mass spectrometry). There are few cases of a species having more than one LK precursor; one is the squid Sepia officinalis with two prepro-LKs [58] ( Figure  3).  [55], and Zeng et al. 2020 [20], respectively.
Outside arthropods, multiple LK paracopies are also known. In the annelid worm Urechis unicinctus, eight paracopies of LKs have been identified [56], and the largest number of LKs was found in the LK precursor of the marine slug Aplysia californica with 30 ( Figure 3) [22]. In some species, such as Frankliniella, Rhodnius, the bed bug Cimex lectularius, and Aplysia, the prepro-LK can give rise to additional non-LK peptides, resulting in a total of about 60 peptides in Aplysia [21,22,52,57]. Of note is that, to our knowledge, none of these non-LK peptides have been studied further in any organism (and very few have been verified by mass spectrometry). There are few cases of a species having more than one LK precursor; one is the squid Sepia officinalis with two prepro-LKs [58] (Figure 3).   [22], squid from [58], polychaete worm from [15], and cattle fever tick from [59].
A striking feature is that in many invertebrate species whose genomes have been sequenced, LK precursors have not been found. Actually, to our knowledge, only arthropods, tardigrades, annelids, and mollusks have thus far been shown to produce LK Except for Aplysia, red boxes represent leucokinins, and signal peptides are indicated by blue boxes. Primary sequence data of Aplysia are from [22], squid from [58], polychaete worm from [15], and cattle fever tick from [59].
A striking feature is that in many invertebrate species whose genomes have been sequenced, LK precursors have not been found. Actually, to our knowledge, only arthropods, tardigrades, annelids, and mollusks have thus far been shown to produce LK precursors. Even among insects, not all species have LKs. For instance, in the order Coleoptera (beetles), 34 species have been analyzed, and only in four, Pogonus chalceus, Gyrinus marinus, Carabus violaceus, and Carabus problematicus (all in suborder Adephaga), LK precursors were found [60][61][62]. Thus, no LK precursor was detected in the "model Coleopteran" Tribolium castaneum. LK precursors and receptors are missing also in, for example, some parasitic wasps (e.g., Nasonia vitripennis), but not in all [63,64], and were not found in any ant species analyzed to date [65][66][67]. An LK precursor is also missing in the phyllopod crustacean Daphnia [68], although they are present in decapod crustaceans (see [69]). It can be noted that an LK-like peptide sequence (Nlp43: KQFYAWAamide) has been identified in nematodes such as C. elegans [70] (see Figure 1), but it is not derived from a canonical LK precursor and no LKR could be found [3,15]. Furthermore, LK signaling components are not found in cnidarians (see [71,72]) or flatworms (Platyhelminthes) [73]. In lower bilaterians, such as species of Xenoturbella and Nemertodermatid worms (both phylum Xenacoelomorpha), orthologs of LK-type receptors were detected by bioinformatics, but no LK peptides [74]. Finally, in some species, such as honey bees, LK precursors have been identified that could generate three LKs, but the cleaved peptide products could not be detected by mass spectrometry [65]. However, since orthologs of LKRs have been identified in five sequenced bee genomes, it is likely that LK signaling is present in these hymenopterans, but not in ants [65,75]. In support of the importance of LK signaling in honeybees, a recent paper showed that the lkr gene influences labor division in foraging for pollen and nectar in the Asian honeybee (Apis cerana) [76].
As noted above, the LKRs identified seem to have no vertebrate orthologs and are found only in the invertebrate species where LK precursors have been detected (Figure 4), possibly with the exception of Xenacoelomorphs mentioned above. Only a few LKRs have been characterized by ligand activation (Figure 4). Thus, LK signaling is not universally present among invertebrates, in contrast to several other more widespread neuropeptides, such as adipokinetic hormone (AKH)/GnRH, neuropeptide F, and insulin-like peptides (see [1][2][3]15]), and this begs the question as to whether some other neuropeptide system has taken over LK functions. It is also interesting to note the large differences in number of paracopies in the different LK precursors, ranging from 1 to about 30. Amino acid sequences of full-length receptors were used for the analysis. Sequences were aligned using the Clustal X. Maximum likelihood trees were constructed by MEGA X software. The numbers at the nodes of the branches represent the percentage bootstrap support (1000 replications) for each branch. Receptors that have been functionally characterized are indicated by a red symbol after the species name. Sequences used to generate the phylogeny are provided in Supplementary Material Text File S1.

LK Expression Is in Diverse Types of Neurons in the Cockroach L. maderae (R. maderae)
The distribution of a given neuropeptide in neurons and other cells can provide some initial hints as to whether its functions are diverse or not. Thus, some peptides are present in very small sets of uniform neurons (e.g., SIFamide or eclosion hormone), suggesting few and/or orchestrating functions, and others in large populations of diverse types (such as short neuropeptide F (sNPF) and tachykinins), indicating multiple diverse functions [1,77]. Functional analysis has verified that some neuropeptides are utilized by neurons (and/or other cells) to globally orchestrate development, physiology, or behavior, and others play multiple distributed roles that are more localized and circuit-specific [1,77]. The latter type of peptide action may be in the form of cotransmission, together with other neurotransmitters or neuromodulators [78][79][80]. Therefore, what does the distribution of LKs in different insects tell us about their functions?
The distribution of LKs was first analyzed in L. maderae, using antisera to LK-I that recognized the eight isoforms known at the time [26,27,32]. In this cockroach, the number and diversity in cell types expressing LKs is large, suggesting a wide range of functions for this set of peptides ( Figure 5A-C). Thus, we used these old LK immunohistochemical data to illustrate a peptidergic system quite different from that in Drosophila (see Figure  5D) and some other insects, such as locusts ( Figure 6A). Amino acid sequences of full-length receptors were used for the analysis. Sequences were aligned using the Clustal X. Maximum likelihood trees were constructed by MEGA X software. The numbers at the nodes of the branches represent the percentage bootstrap support (1000 replications) for each branch. Receptors that have been functionally characterized are indicated by a red symbol after the species name. Sequences used to generate the phylogeny are provided in Supplementary Material Text File S1.

LK Expression Is in Diverse Types of Neurons in the Cockroach L. maderae (R. maderae)
The distribution of a given neuropeptide in neurons and other cells can provide some initial hints as to whether its functions are diverse or not. Thus, some peptides are present in very small sets of uniform neurons (e.g., SIFamide or eclosion hormone), suggesting few and/or orchestrating functions, and others in large populations of diverse types (such as short neuropeptide F (sNPF) and tachykinins), indicating multiple diverse functions [1,77]. Functional analysis has verified that some neuropeptides are utilized by neurons (and/or other cells) to globally orchestrate development, physiology, or behavior, and others play multiple distributed roles that are more localized and circuit-specific [1,77]. The latter type of peptide action may be in the form of cotransmission, together with other neurotransmitters or neuromodulators [78][79][80]. Therefore, what does the distribution of LKs in different insects tell us about their functions?
The distribution of LKs was first analyzed in L. maderae, using antisera to LK-I that recognized the eight isoforms known at the time [26,27,32]. In this cockroach, the number and diversity in cell types expressing LKs is large, suggesting a wide range of functions for this set of peptides ( Figure 5A-C). Thus, we used these old LK immunohistochemical data to illustrate a peptidergic system quite different from that in Drosophila (see Figure 5D) and some other insects, such as locusts ( Figure 6A).    Panel A is altered from [34] with SIF neurons added [83], B is from [86], and C is altered from [87]. All figures used with permission from publishers.
There are about 160 LK neurons with cell bodies in the protocerebrum of the brain ( Figure 5A), some in bilateral clusters and others occurring in bilateral pairs distributed in different regions [27]. No cell bodies were detected in deuto-and tritocerebrum, and only a small set of weakly immunoreactive neurons were detected in the fused subesophageal ganglion. In each of the two lateral neurosecretory cell (LNC) groups there LK cell bodies are predominantly found in protocerebrum (Protoc), including the optic lobes (OL) and accessory medulla (aMe; pacemaker region of clock), but some are in tritocerebrum (Tritoc). Neuronal process from LK neurons (not shown) are in the central body, optic lobe, and antennal lobe (AL), and less delineated neuropils are shown in all three brain neuromeres. A group of four neurons (SIFamide producing (SIFa)) in the pars intercerebralis coexpress SIFamide and LK. These SIFa neurons are known to send processes throughout the brain and ventral nerve cord [83,84] [34] with SIF neurons added [83], B is from [86], and C is altered from [87]. All figures used with permission from publishers.
There are about 160 LK neurons with cell bodies in the protocerebrum of the brain ( Figure 5A), some in bilateral clusters and others occurring in bilateral pairs distributed in different regions [27]. No cell bodies were detected in deuto-and tritocerebrum, and only a small set of weakly immunoreactive neurons were detected in the fused subesophageal ganglion. In each of the two lateral neurosecretory cell (LNC) groups there were six LK cells, and in the median neurosecretory cell group (MNC), about 100 were found ( Figure 5A).
Both the LNCs and MNCs send LK-immunolabeled axons to the neurohemal area of the corpora cardiaca, suggesting that the LKs can be released as hormones into the circulation. Radioimmunoassay analysis of HPLC-separated corpora cardiaca extracts suggested that all eight LKs known at the time are present in this tissue [32]. Furthermore, it was indeed shown by radioimmunoassay (RIA) that release of LKs can be triggered in vitro from the corpora cardiaca of both cockroach [88] and cricket [89]. Furthermore, in the bug Rhodnius, RIA of hemolymph demonstrated both LK and diuretic hormone (DH44) release after feeding, suggesting a postprandial hormonal role of LK [90].
In contrast to Drosophila where one pair of LK interneurons is seen in the brain and one pair in the subesophageal zone (SEZ) (Figure 5D), the cockroach brain has a complex set of interneurons ( Figure 5A-C). Different LK neurons, originating in the protocerebrum, send processes to the central body; optic lobe (medulla and lobula); antennal lobes; and to various neuropil regions in the proto-, deuto-, and tritocerebrum. Two pairs of large LKimmunoreactive descending neurons (DNs) send axons throughout the ventral nerve cord, finally ending in the terminal abdominal ganglion. These pairs of DNs have collateral arborizations ipsilaterally in most of the glomeruli of the antennal lobe and posterior deutocerebrum ( Figure 5A,B). A small set of branches from the DNs innervates the calyces of the mushroom bodies [81] ( Figure 5B). The group of LK neurons associated with the medulla [27] has been described in more detail as part of the accessory medulla complex that is a pacemaker region of the circadian clock [91][92][93]. Some of these LK neurons colocalize pigment-dispersing factor (PDF), which is one of the major neuromodulators of the clock in L. maderae and Drosophila [91,93,94].
Similar to Drosophila and other studied insects, each abdominal ganglion has sets of neurosecretory cells (ABLKs; abdominal ganglion LK neurons) expressing LK. However, instead of one pair of ABLKs in each ganglion/neuromere, as seen in Drosophila and some other dipteran flies [28], L. maderae has two pairs [27]. Two pairs of ABLKs are also seen in, e.g., crickets, crane flies, moths, and mosquitos, whereas there are three pairs in the first four abdominal ganglia of locusts and two in the following ganglia ( Figure 6B) and up to 10 pairs per ganglion in dragonflies [29,30,95,96]. In cockroaches and locusts, these ABLKs send varicose axons to the lateral heart nerves and transverse nerves, where neurohemal areas (perivisceral organs) are formed; moreover, spiracles receive LK axon terminations [27,30,97]. Although LK was originally isolated by means of its activity on hindgut contractions, no LK innervation of this tissue was detected [27], suggesting that this myotropic action is mediated by hormonal LK. Another difference to Drosophila is that the cockroach thoracic ganglia each have at least two pairs of LK-expressing interneurons that arborize widely in the lateral portions of the ganglia [27,30].
As in Drosophila, there are no LK expressing enteroendocrine cells (EECs) in the L. maderae intestine. However, there are bi-or multipolar LK neurons in the posterior midgut with ascending axons running via the esophageal nerve to end with arborizations in the frontal ganglion and tritocerebrum [27]. These might be proprioceptive cells that signal gut distension to the frontal ganglion and other feeding circuits. Additionally, LK-immunoreactive axons from the retrocerebral complex (in particular the frontal and hypocerebral ganglia) were found to innervate the pharynx and esophagus [27]. Mapping of LK neurons in the brain of the cockroach Nauphoeta cinerea revealed a similar set of neuron types [30].
Thus, taken together, the cockroach LK neurons are more diverse than those in Drosophila ( Figure 5A-D) and seem to underlie distributed functions in different brain/ ganglion regions. Such functions may include neuromodulation in the olfactory system, visual system, central complex, mushroom bodies, circadian clock, tritocerebral neuropil, circuits of the thoracic ganglia, and the frontal ganglion (regulation of feeding) [27]. The two pairs of protocerebral descending LK neurons ( Figure 5A,B), which span the entire ventral nerve cord, may provide a pathway for linking protocerebral and olfactory systems to regulate ganglionic activity. In addition, there are three types of neurosecretory cells producing LKs, namely, LNCs, MNCs, and the ABLKs, which probably release LKs into the circulation to target peripheral organs such as Malpighian tubules, heart, and visceral muscle. Furthermore, peripheral cells were found in the intestine of L. maderae that may be proprioceptors.
Unfortunately, there are no data on any functions of LKs in cockroaches, except the stimulatory activity on the hindgut muscle in vitro [7,9]. Thus, we can only speculate that LK signaling in the cockroach is functionally more diverse than in Drosophila with its four neurons in the brain/SEZ and 22 ABLKs. The four brain/SEZ neurons of Drosophila ( Figure 5D) do not seem to have any obvious analogs in the cockroach brain, but there are three bilateral pairs of L. maderae LK neurons that could play roles similar to the pair of LHLKs (one is labeled LHn in Figure 5A,C). The SELKs are descending neurons in Drosophila with cell bodies and processes in the SEZ [33,51], whereas the cockroach descending neurons originate in the protocerebrum and innervate the antennal lobes on their descent ( Figure 5A,B). The LK-expressing LNCs of L. maderae may be analogous to the ALKs of Drosophila ( Figure 5D). These Drosophila ALK neurons can be seen in several Lk-Gal4 lines, but only in early larvae do they consistently label with antisera to LK [33,51]. These Drosophila neurons were first described as LNCs expressing ion transport peptide (ITP), a peptide that may act in regulation of thirst and hunger and probably also plays a role in ion transport in the intestine [98,99]. The Drosophila ALKs were also shown to express tachykinins (TKs) and short neuropeptide F (sNPF), and these peptides were found to regulate metabolic and desiccation stress responses [82]. It is not known whether the L. maderae LNCs express further neuropeptides, but possibly their functional roles are similar to those of Drosophila. On the basis of the anatomy and distribution of the cockroach LK neurons, one could speculate that some of the other LK functions determined in Drosophila also apply to L. maderae-roles in the circadian clock output and sleep, in feeding, and in regulation of water and ion homeostasis (see [17,51,[100][101][102][103]).

Distribution of LK in Other Invertebrates: What Can Comparative Studies Teach Us?
In the previous section, we described the LK neurons of the cockroach L. maderae with some comparative comments on Drosophila, two insects that highlight two extremes in terms of number and diversity of LK neurons. Here, we briefly summarize findings of interest in other invertebrates and discuss coexpression of LK and other peptides.

LK in Neurons of the Brain of Other Insects
LK distribution has also been described in the brains of several other insects, including the blood-sucking bug Rhodnius prolixus, the locusts Locusta migratoria and Schistocerca gregaria, the cricket Acheta domesticus, and the mosquito Aedes aegypti [30,31,34], which is summarized in Table 1. As an example, we show LK neurons in L. migratoria ( Figure 6A), where some interesting features differ from Drosophila and Leucophaea.
Notes: x, present; −, not present; no annotation, not clear whether present or not (no statement is provided in papers). Acronyms: CB, central body; AL, antennal lobe; OL, optic lobe; LK, lateral horn; TC, tritocerebrum; SEZ subesophageal zone; DNs, descending neurons; LNC, lateral neurosecretory cells; MNC, median neurosecretory cells. 1 The majority of the LK cell bodies are in the protocerebrum and subesophageal zone (SEZ), but processes innervate neuropils in other brain regions. 2 In ALK neurons (LNCs), the LK expression is strong in larvae and weak and variable in adults. 3 The description of distribution of LK neurons and their processes is not detailed.
The distribution of various neuropeptides has been extensively investigated in the locust brain, some in exquisite detail (see [104,105]), whereas the LK distribution has received more superficial attention. In the brain of L. migratoria, about 140 LK immunoreactive neurons were detected [34] (Figure 6A). Their cell bodies are primarily located in the protocerebrum, but about 5-6 pairs were detected in the tritocerebrum. No clear-cut neurosecretory cells were seen in the brain, but LK-expressing interneurons are associated with the optic lobe and the accessory medulla (pacemaker center of the clock), the central body, and antennal lobe [34,105]. As in the L. maderae brain, two pairs of descending LK neurons innervate the antennal lobes on their way to the ventral ganglia in S. gregaria [34,106]. There is an additional pair of larger tritocerebral descending neurons in L. migratoria [34] ( Figure 6A). Distinct LK immunolabeled processes can be seen in protocerebral neuropils such as the upper and lower divisions of the central body, the median and lateral accessory lobes of the central complex, and the protocerebral bridge, but not in the mushroom bodies. In the optic lobes, specifically the most basal portion of the lamina, different layers of the medulla (including the accessory medulla) and lobula contain LK fibers. A supply of immunoreactive fibers can also be seen in the glomeruli of the antennal lobe and many of the non-glomerular neuropils of proto-, deuto-, and tritocerebrum contain diffusely arborizing LK fibers.
An interesting finding is that in S. gregaria a set of four SIFamide-expressing neurons in the pars intercerebralis of the brain colocalize LK [83] (see Figure 6A). As in Drosophila, the processes from these SIFamide neurons innervate most neuropil regions of the brain and ventral nerve cord [83,85]. The LK expression in these neurons is weak in adult locusts, but nevertheless suggests that LK may play a role in the signaling of these SIFamide neurons. The homolog SIFamide neurons in Drosophila are known in to orchestrate feeding, sleep, and mating in a nutritional state-dependent fashion [85,107,108]. Another interesting aspect of these SIFamide neurons in the locust is that they are identical to the LK-expressing primary commissure pioneer neurons (PCPs) that lay down an early axonal tract (commissure) in the brain of the locust embryo [83,84]. Since the LK immunolabeling was found stronger in the SIFamide neurons in younger stages than in the adult [84], it is suggestive that LK plays a role of during neuronal development and axonal pathfinding in the brain.
In the brains of the cricket Acheta domesticus and the mosquito Aedes aegypti, the distribution of LK neurons is similar to that in L. maderae, with both LNCs and MNCs and their axon terminations in the corpora cardiaca expressing the peptide, but other interneurons were not described in enough detail for comparisons to be made [30]. The same authors found that there are no LK-immunoreactive neurons in the brain of the honeybee Apis mellifera, but only neurosecretory cells in the abdominal ganglia [30].
Finally, in the brain of the blood-sucking bug Rhodnius prolixus, about 180 pairs of LKimmunoreactive neurons were detected, 30 pairs of which were more strongly labeled [31]. These were later confirmed by in situ hybridization [109]. Processes of LK interneurons were seen widely in brain neuropils. In starved specimens, a set of MNCs and their processes in the corpora cardiaca could be detected with LK antiserum [31], suggesting LK expression is dependent on nutritional state and that this peptide plays a role as a systemic hormone. Injection of a biostable analog of an LK displayed decreased intake of blood in a feeding assay [110]. Furthermore, RIA of hemolymph demonstrated that LK is released after feeding [90]. In R. prolixus, LK does not display diuretic activity in the Malpighian tubules or anterior midgut (in contrast to, e.g., DH44), but it decreases the resistance and transepithelial voltage of the epithelium and also increases the frequency of contractions in the anterior midgut [31,111]. LK also induces contraction in the R. prolixus hindgut [109,110]. R. prolixus is the only insect that has thus far displayed LK-producing enteroendocrine cells in the midgut [31].

LK in Neurons of the Nervous System of Other Invertebrates
The only phylum outside arthropods where bona fide LK distribution has been described is in mollusks. LK-expressing neurons in mollusks have been mapped for Lymnaea stagnalis, Helix pomatia, and Aplysia californica [22,44,112].
In the snail Helix, about 700 LK immunoreactive neurons were found in the CNS [112]. Buccal, cerebral, and pedal ganglia, as well as the viscero-parietal-pleural ganglion complex, all express LK in numerous neurons. One giant LK neuron was found in the pedal ganglion. Two major groups of LK neurons in the cerebral ganglia send axons into commissures to other ganglia and into several peripheral nerves [112]. Several peripheral tissues such as buccal mass, oviduct and intestinal muscle, and "skeletal" muscle (of foot, lip, and tentacle) are supplied by varicose LK axons. In addition, bipolar LK neurons were found in the intestine and were shown to send axons into the extensive meshwork of LK fibers seen there. Some groups of LK neurons in the cerebral ganglion coexpress tachykinin immunoreactivity [112]. It is not clear whether any of the LK neurons serve as bona fide neurosecretory cells, but it cannot be excluded that the abundant superficial LK axons in peripheral tissues might release LK into the circulation.
In Aplysia, the majority of the LK neurons were found in the buccal ganglion, which is known to house feeding motor neurons and pattern-generating interneurons [22]. LK neurons were also seen in the cerebral ganglion, where higher-order feeding interneurons are located. These authors found that the buccal motor neuron B48 expresses LK and that application of this peptide ex vivo modulated a parameter of the consummatory feeding behavior [22]. One target of LK action is a central pattern generator element that modulates the duration of the protraction phase of feeding responses. Thus, this Aplysia study provides a mechanistic description of LK modulation of food ingestion, something that is lacking thus far for Drosophila and other insects. However, roles of LK in food consumption and post-feeding physiology have been demonstrated in Drosophila [51,103,113] and are suggestive in Rhodnius [31,111].

Neurosecretory Cells and Hormonal Roles of LK in Invertebrates
One striking conserved feature is that all studied insects have segmental abdominal neurosecretory cells (ABLKs), varying in number between one pair per neuromere in Drosophila ( Figure 6C) and blowflies, to up to 10 pairs in dragonflies [28][29][30][31]96]. Commonly insects have two to three pairs per neuromere/ganglion (see [29,30,97]) ( Figure 6B). These neurosecretory cells have axon terminations associated with peripheral nerves (including lateral heart nerves), perisympathetic organs, and the body wall muscle of the abdomen. Since these abdominal cells are the only LK-expressing neurosecretory cells in several species studied, it is suggestive that these cells release LK as a circulating hormone. Thus, an important function of LKs is as hormones that act systemically, as diuretic factors, and that they are also likely to regulate gut contractions in some species (see [9,11,36,49,50,75,87,114,115]). As mentioned, LK release has been demonstrated in L. maderae, A. domesticus, and R. prolixus [32,89,90]. In several insect species, including Drosophila, Musca domestica, Manduca sexta, and Rhodnius prolixus, the abdominal LK cells coexpress the neuropeptide DH44 [31,87,116,117]. In Rhodnius, the DH44 stimulates secretion in the Malpighian tubules, whereas LK has no direct action on tubules, but may act elsewhere (e.g., anterior midgut and hindgut) to assist in rapid diuresis [31,110,111]. Both LK and DH44 are released after feeding in Rhodnius [90]. In Drosophila, on the other hand, both DH44 and LK stimulate secretion in the tubules, but by acting on different cell types and with different signal pathways downstream the receptors [17,75,87,118,119].
As mentioned above, some insect species possess additional LK-expressing neurosecretory cells systems in the brain. It is not known whether the cells of the brain and the abdominal ganglia (when both exist) play different functional roles, but it is at least likely that LK release in these cell groups are under control by different central neuronal circuits. It is also possible that in the LNCs other neuropeptides are colocalized with LK, as is the case in the Drosophila ALKs with additional TK, ITP, and sNPF [51,82]. For instance, in M. sexta and L. migratoria, sets of LNCs are known to produce ITP [120,121]. Interestingly, the only insect known that has LK-expressing endocrine cells in the midgut is R. prolixus [31]. Thus, LK is a rare peptide in intestinal signaling, in contrast to many other neuropeptides (see [122][123][124]).
In crustaceans, LKs have not yet been detected in the canonical neurosecretory system, the X-organ/sinus gland of the eyestalks, or in the stomatogastric system [125]. However, in pericardial organs of the crab Cancer borealis, varicose LK immunoreactive axons were detected (probably derived from cell bodies in thoracic ganglia), suggesting that hormonal release of LK is possible [126]. Peptides from the pericardial organs are known to act as circulating hormones on circuits of the crab stomatogastric ganglion, and indeed shrimp LK applied to the ganglion has a distinct modulatory action on the pyloric rhythm of the network [126].
Not all arthropods seem to use LKs as hormones. In the spider Cupiennius salei, no LKimmunolabeled neurosecretory cells were detected, and actually the LK interneurons are not segmentally arranged, but the nine pairs of cell bodies are clustered anteriorly in the supraesophageal ganglion [42]. However, in another arachnoid, the tick Rhipicephalus appendiculatus, four pairs of neurosecretory cells located anteriorly in the prothocerebral lobe produce LK [127]. These cells have arborizing axon terminations in neurohemal areas in the neural sheath surrounding the CNS, and colocalize the neuropeptide myosuppressin.
In mollusks, no bona fide neurosecretory cells producing LK have been described, but in the snail H. pomatia, sets of LK neurons clustered in cerebral ganglia have axons running out in several nerve roots to innervate peripheral tissues [112]. These peripheral varicose axons might release LK into the circulation, but further studies are required to verify this.
Although LK has been demonstrated in annelids, such as Urechis unicinctus and Capitella teleta [56,128], there are, as far as we know, no reports on the cellular localization of the peptide. In the parasitic nematode Ascaris suum, LK immunoreactivity was detected in neurons [45], but no LK precursor gene has been identified in nematodes, and thus it is not clear what endogeneous peptide the antiserum recognized.

Specific Roles of LK Signaling in Arthropods
Here, we present a brief summary of the diverse functions of LK signaling in arthropods. Most of the recent work has been performed in Drosophila, but we will describe that only very briefly in Section 4.4.5, since a more detailed review on Drosophila will appear elsewhere. In Table 2, we list known functions of LK signaling in different insects and some other invertebrates.  Notes: 1 The LK expression in SIFamide neurons is stronger during development, but remains throughout development and adult stage. 2 The Asian honeybee Apis cerana.

Myostimulatory Action
LKs act in vitro to increase frequency and amplitude of contractions in the hindgut of L. maderae [7,9] and the housefly Musca domestica [40], and in the anterior midgut and hindgut of the bug R. prolixus [109][110][111], but have no effect on neither hindgut nor oviduct contractions in the locust L. migratoria [36,49].

Diuretic Action
A more widespread action is the stimulatory action of LKs on Malpighian tubules shown in [11,12,14,17,41,46,50,75,97,116]. In the studied insects, LKs activate the LKR, leading to an increase in intracellular calcium, which activates a chloride shunt conductance and water transport across the tubule epithelium [14,118,145,146]. In dipteran insects, such as Drosophila, Anopheles gambiae, and Aedes aegypti, this action is mediated by stellate cells of the tubules, which express the LKR [25,75,118,147]. LK signaling appears secondarily lost in most species of beetles (Coleoptera), and mining of the genome of Triboleum castaneum shows that other signaling systems known to be associated with diuretic functions in insects are greatly expanded [75].

Modulation of Sugar Gustation in the Mosquito Aedes aegypti and Asian Honeybee Apis cerana
In females of the mosquito Aedes aegypti, application of a protease-resistant LK to the mouthparts and proleg tarsi resulted in inhibition of sucrose feeding and induction of an escape behavior, wherein the insect walked or flew away from the food [138]. It was shown that the LKR is expressed in chemosensory cells in proleg tarsi and labellar sensillae, and LK analog applied to mouthparts blocked the electrophysiological response to sugar in chemosensory sensillae. Furthermore, LKR-RNAi (RNA interference) by injection of double-stranded RNA eliminated the inhibitory effect of LK on sugar feeding [138]. This effect of a stable LK analog suggests a promising lead for a feeding deterrent in control of mosquitos as disease vectors [138]. Moreover, in the Asian honeybee A. cerana, sucrosesensing is modulated by LK signaling [76]. Knockdown of the LKR by RNAi decreased the sensitivity to sucrose in a proboscis extension response assay. Furthermore, the Lkr gene influences division of labor in foraging in these bees, and nectar foragers display lower Lkr expression than those foraging for pollen [76].

Feeding and Fecundity in the Cattle Fever Tick
In the cattle fever tick Rhipicephalus microplus, silencing of the LKR by double-stranded RNA injection induced decreased egg production and hatching of eggs laid, and also delayed oviposition [144]. This effect appears to be indirect since the authors did not report expression of the LKR in ovaries but did report expression in the outer muscle layer of the midgut [144]. It was suggested that LK action on the gut affects gut motility and potentially uptake and processing of nutrients, and this in turn affects nutrient availability and fecundity [144]. An inhibitory effect of LKs on release of the digestive enzymes protease and amylase from the midgut was in fact shown in the moth Opisina arenosella [142], and myostimulatory effects of LKs are known in several insects [8,9,111]. It is possible that the LK action in the tick also includes the CNS, which could affect control of feeding and/or hormone release that reduces reproductive output.

Feeding in Rhodnius prolixus and A. aegypti
In R. prolixus and females of the mosquito Aedes aegypti, protease-resistant LK analogs reduce food intake when injected in the former and applied to the mouthparts and proleg tarsi of the latter [110,138]. Thus, LKs can have anti-feedant activity.

Functional Roles of LK in Drosophila
In recent years, Drosophila studies have employed genetic interventions and have revealed actions of specific LK neurons in the brain, SEZ, and abdominal neuromeres (Table 2, Figure 7). The two LHLK neurons ( Figure 5D) were shown to modulate metabolism-sleep interactions and serve as clock output [100][101][102][103], modulating state-dependent water and sugar-enforced memory [130], and probably food choice [131]. This pair of LHLK neurons also regulates insulin-producing cells, which may contribute to sleep-metabolism effects [51,103]. The abdominal ABLKs (see Figure 6C) regulate water and ionic homeostasis along with associated stress [51,87] and mechanosensory-induced defensive post-mating response in females [132]. Moreover, in Drosophila, LK modulates gustatory neurons, but it is not clear which neurons are responsible [139,141], although the SELKs are in a favorable position. The ABLKs co-express DH44 and specific knockdown of this peptide in ABLKs affect water and ionic homeostasis, as well as feeding [87]. The ALK neurons ( Figure 5D) are likely to signal with LK, ITP, sNPF, and TKs [51,82]. The function of LK in these cells is not yet known, but sNPF and TKs regulate metabolic and ionic stress responses [82], and ITP modulates water and ionic homeostasis, as well as feeding and drinking [98]. As seen in Figure 7, some of the functions of LKs appear conserved between Drosophila and other insects: clock-sleep functions, modulation of gustatory neurons, regulation of water and ion homeostasis, and possibly feeding. well as feeding [87]. The ALK neurons ( Figure 5D) are likely to signal with LK, ITP, sNPF, and TKs [51,82]. The function of LK in these cells is not yet known, but sNPF and TKs regulate metabolic and ionic stress responses [82], and ITP modulates water and ionic homeostasis, as well as feeding and drinking [98]. As seen in Figure 7, some of the functions of LKs appear conserved between Drosophila and other insects: clock-sleep functions, modulation of gustatory neurons, regulation of water and ion homeostasis, and possibly feeding. Figure 7. Summary of LK functions in Drosophila compared to other insects. In insects other than Drosophila, few functions have been explicitly determined (blue boxes), and most are suggested from LK expression (grey or black boxes). Red arrows indicate hormonal signaling, black arrows indicate established functions, and dashed arrows indicate suggested functions. In the mosquito A. aegypti, LK regulates sugar taste in gustatory receptor neurons (GRs) [138]; in the cockroach L. maderae and some other insects, intestinal contractions are regulated by LK; and in many insects, LK acts as a diuretic factor [75,133]. LK-expressing sensory cells in the intestine of L. maderae send axons to the frontal ganglion and brain, suggesting proprioceptive inputs [27]. In L. maderae, LK is present in pacemaker neurons of the clock circuit [27,93], and in In the mosquito A. aegypti, LK regulates sugar taste in gustatory receptor neurons (GRs) [138]; in the cockroach L. maderae and some other insects, intestinal contractions are regulated by LK; and in many insects, LK acts as a diuretic factor [75,133]. LKexpressing sensory cells in the intestine of L. maderae send axons to the frontal ganglion and brain, suggesting proprioceptive inputs [27]. In L. maderae, LK is present in pacemaker neurons of the clock circuit [27,93], and in the locust L. migratoria, LK is expressed in the four widely arborizing SIFamide-producing neurons and in circuits of the central body [34,83]. In several insects, including L. maderae, there are LK-expressing lateral and median neurosecretory cells indicating hormonal LK signaling from the brain [27,30,31]. In Drosophila, genetic interventions have revealed actions of specific neurons in the brain, subesophageal zone (SEZ), and abdominal neuromeres in several functional roles (blue boxes). These are metabolism-sleep interactions [100][101][102][103], food choice [131], water-and sugar-enforced memory [130], food intake, modulation of GRs [139,141], and water and ionic homeostasis [51,87,103]. One set of LK neurons (LHLKs) also regulates insulin-producing cells [51,103]. LK neurons expressing additional peptides contribute to other functions with non-LK peptides (red boxes). These are the ALK neurons that signal with LK, ITP, sNPF, and TKs and regulate metabolic and ionic stress responses (as well as feeding and drinking) [82,98], and ABLKs that also express DH44 and this peptide affect feeding and water balance [87].

Targeting the LK Signaling System with Peptide Analogs to Aim at Pest Control
Neuropeptides regulate many vital processes in the daily life of insects such as development, growth, feeding, reproduction, metabolism, and water and ion homeostasis. These roles, taken together with the high specificity and activity at very low doses, render neuropeptides and their cognate receptors potential leads for the development of eco-friendly insecticidal agents [148][149][150][151][152][153][154][155]. Of the different peptides known, LKs have received considerable attention since the LK/LKRs signaling system seems to have no vertebrate orthologs and it plays a key role in regulation of many vital physiological and behavioral processes in insects, as shown in Section 4.4. In insects, LKs are multifunctional neuropeptides that share a common C-terminal pentapeptide sequence FX 1 X 2 WGamide, where X 1 can be H, N, S, A, or Y and X 2 can be S, P, A, or R (see Figure 1B); this pentapep-tide is also the active core of LKs, facilitating peptide design [40,152,156]. As noted in a previous section, LKs have been identified a wide range of insects (see the DINeR database: http://www.neurostresspep.eu/diner/infosearch), with the exception of most beetles (Coleoptera), all ants, and some wasps (Hymenoptera) [60,[62][63][64][65]67]. Since LKs are rapidly degraded by peptidases, analogs of insect LKs have been synthesized with a modified chemical structure to increase stability [152,156]. Replacement of the X 2 residue of the C-terminal pentapeptide core sequence (FX 1 X 2 WGamide) with an alpha-aminoisobutyric acid (Aib) resulted in resistance to hydrolysis by angiotensin-converting enzyme (ACE) and neprilysin (NEP) [157,158]. A rationale for this is that the X 2 position is the primary site of susceptibility to peptidase cleavage. Incorporation of a second Aib residue adjacent to the secondary peptidase hydrolysis site (N-terminal to the F residue) further enhances biostability [157]. These short LK analogs have activities that are similar to or exceed those of native insect kinins when tested on recombinant LKRs from the southern cattle tick Rhipicephalus microplus and the dengue vector, Aedes aegypti [59,[159][160][161]. Both in tissue bioassays and in recombinant LKR experiments in vitro, it was shown that that the F residue (in position one), W (in position four), and the amidated C-terminus of the LK pentapeptide core are crucial for LK activity [159,160,162]. Some modified biostable insect LK analogs have potential to be used in the integrated pest management because they reduce gain in body weight in corn earworm Helicoverpa zea larvae [157,163] and increase aphid mortality [164][165][166]. A biostable LK mimetic, (analog 1728; K-Aib-1), was shown to inhibit sugar taste receptors and act as a feeding deterrent in Aedes aegypti mosquitoes [138]. Moreover, in the bug R. prolixus, a stable LK analog displayed antifeeding activity after injection [110], and induced increased activity on hindgut contractions [109]. In female ticks, knocking down the expression of the LKR leads to a significant reduction of their reproductive fitness [144]. Hence, the tick LKR might be a promising target for developing more potent analogs. A recent study screened 14 predicted R. microplus LKs (Rhimi-K) and 11 LK analogs containing Aib and found that all of them were full agonists and displayed potent effects on the LKR of R. microplus [59]. These tick LKs and LK mimetics provide putative tools for tick physiology and management. However, the practical exploitation of the insect and tick LKs and LKRs for pest control is still in its early stages. More work is needed to solve the bio-stability, cost of production, and bio-safety of neuropeptide analogs, as well as to find efficient modes of peptide administration to target pest insects.

Conclusions
In this review, we have shown that expression of LKs is variable among invertebrates. Not only is it absent in many taxa, including some insect groups, but also its cellular expression varies between different insect species. Thus, there are 20 LK neurons of 3 major types in the CNS of the Drosophila larva (plus the enigmatic ALKs) and about 250 of multiple types in that of adult L. maderae [27,33]. A conserved feature is, however, the segmentally arranged neurosecretory ABLKs found in all insects studied (see [27,30,31,95]). This suggests that a hormonal role of LKs is a conserved feature among insects, and that a common action is to induce secretion in the Malpighian tubules [50,75] and potentially action on contractility and epithelial transport in the gut [9,111]. Most other functional roles of LK have been studied only in Drosophila, and thus it is not clear at this point to what extent further functions are conserved. However, as seen in Table 2, regulation of taste receptors and feeding, signaling in clock and sleep circuits, as well as gut function may be outputs of LKs in several invertebrate species.
Interestingly, even amongst insects, genes encoding LK and LKR are lacking in many species. Is the lack of LK signaling compensated somehow? A clue can be obtained from looking at diuretic functions in beetles (Coleoptera) where most species have no LK signaling components. In the beetle Tenebrio molitor, genes encoding other diuretic hormones and their receptors (and associated downstream molecules) are upregulated, suggesting that peptide hormones are interchangeable to some extent [75]. This is also emphasized by the fact that LKs are strong diuretic factors in some insect species such as Drosophila and mosquitos, but have no direct action on diuresis in, e.g., Rhodnius [17,31,111]. Regulation of water and ion homeostasis is complex, with several peptide hormones involved [1,50,114,133]. In locusts and Drosophila, colocalized LK and DH44 activate different signaling systems downstream of their receptors, but act synergistically to induce secretion in tubules [87,114]. The interactions between LKs and other diuretic and antidiuretic hormones are not yet known, but it is likely that hormonal regulation of water and ion balance differs between different taxa both in terms of hormones involved and cellular mechanisms.
Moreover, in the CNS, functions of LKs may be carried out by other neuropeptides when LKs have been lost (or never evolved), but what could be the significance of the larger number and diversity of LK neurons in the cockroach brain compared to that of Drosophila? Many neuropeptides act as local neuromodulators, often as cotransmitters of small-molecule neurotransmitters [78][79][80]167]. Thus, it is likely that LK produced in smaller interneurons of L. maderae serve local neuromodulatory/cotransmitter roles, similar to, for instance, TKs and sNPF in Drosophila [16,78,168,169]. In Drosophila, on the other hand, the four LK interneurons in the brain/SEZ have relatively wide arborizations and seem to play roles in orchestration of physiology and behavior. Clearly, we need more experimental data from other insects to be able to understand core functions of LK signaling and to further appreciate how some functions may have diversified during evolution. Finally, as described in the previous section, LKRs have been chosen as candidate targets for development of stable peptide mimetics for use in insect and tick pest control. Perhaps also development of small molecule ligands of LKRs would be useful in this quest to interfere with the vital LK signaling.
Supplementary Materials: The following are available online at https://www.mdpi.com/1422-0 067/22/4/1531/s1: Figure S1. The LK precursor and LK peptides in the cockroach P. americana. Text File S1. Sequences of LKRs in different species used for the cladogram in