Tetrodotoxins in Tissues and Cells of Different Body Regions of Ribbon Worms Kulikovia alborostrata and K. manchenkoi from Spokoynaya Bay, Sea of Japan

Nemerteans, or ribbon worms, possess tetrodotoxin and its analogues (TTXs), neurotoxins of bacterial origin, which they presumably use for capturing prey and self-defense. Most TTXs-containing nemertean species have low levels of these toxins and, therefore, have usually been neglected in studies of TTXs functions and accumulation. In the present study, Kulikovia alborostrata and K. manchenkoi, two closely related species, were analyzed for TTXs distribution in the body using the HPLC–MS/MS and fluorescence microscopy methods. The abundance of TTXs-positive cells was determined in the proboscis, integument, and digestive system epithelium. As a result, six TTXs-positive cell types were identified in each species; however, only four were common. Moreover, the proportions of the toxins in different body parts were estimated. According to the HPLC–MS/MS analysis, the TTXs concentrations in K. alborostrata varied from 0.91 ng/g in the proboscis to 5.52 ng/g in the precerebral region; in K. manchenkoi, the concentrations ranged from 7.47 ng/g in the proboscis to 72.32 ng/g in the posterior body region. The differences observed between the two nemerteans in the distribution of the TTXs were consistent with the differences in the localization of TTXs-positive cells. In addition, TTXs-positive glandular cell types were found in the intestine and characterized for the first time. TTXs in the new cell types were assumed to play a unique physiological role for nemerteans.


Introduction
Nemertea is a phylum of mostly marine worms comprising approximately 1300 species [1,2].These are soft-body, predominantly carnivorous worms that use various toxins presumably to defend themselves against predators and/or for hunting [3][4][5].Tetrodotoxin (TTX), a voltage-gated sodium channel-blocking neurotoxin, is widespread across all classes of nemerteans [6,7].TTX and its analogues (TTXs) are exogenous toxins found in numerous marine and terrestrial taxa.These are hypothetically accumulated through the food web [8,9] and/or directly from TTX-producing bacteria inhabiting the host's digestive system [10,11].The concentration of TTXs varies both within and between nemertean species [6,7].While toxin concentrations in some nemerteans reach extremely high levels, other TTX-bearing species contain trace amounts of TTXs.

HPLC-MS/MS
The study of TTX and its analogues in different body regions (Figure 1) of K. alborostrata and K. manchenkoi showed 5,6,11-trideoxyTTX as the only toxin whose concentration was above the limit of quantification (LoQ).In several body regions of both species, TTX and 5-deoxyTTX were found at concentrations below the LoQ (Tables 1 and 2; Figure S1).

Morphological and Immunohistochemical Studies
The fluorescent microscopy with anti-TTX antibodies revealed TTXs-like immunoreactivity in all six regions of K. alborostrata and K. manchenkoi: the proboscis, precerebral, mouth, anterior body, middle body, and posterior body regions.Both studied nemertean species showed immunofluorescent labels in the integument, intestine epithelium, and   For the quantification of toxins, we used pooled samples of the same tissue regions from 25 specimens of K. alborostrata and 9 specimens of K. manchenkoi because the size of the tissues in some of the specimens was inadequate for estimating the toxin content.Thus, the mean weight of K. alborostrata specimens was 0.19 ± 0.13 g, and that of K. manchenkoi was 0.42 ± 0.32 g.The analyzed body parts constituted from 1 to 31% of the whole-body weight in the case of K. alborostrata and from 2 to 30% of the whole-body weight in the case of K. manchenkoi.The weights of the pooled samples are presented in Tables 1 and 2. The analysis results were expressed as ng of toxin per 1 g of tissue of the respective body part (Tables 1 and 2) to compare the contribution of the body part to the whole organism's toxicity.
Although the number of the pooled K. alborostrata samples was 2.5-fold larger than that of the pooled K. manchenkoi samples, the total 5,6,11-trideoxyTTX quantity in the K. alborostrata samples was 10-fold lower than in K. manchenkoi: 9.34 vs. 108.21ng, respectively (Tables 1 and 2).

Morphological and Immunohistochemical Studies
The fluorescent microscopy with anti-TTX antibodies revealed TTXs-like immunoreactivity in all six regions of K. alborostrata and K. manchenkoi: the proboscis, precerebral, mouth, anterior body, middle body, and posterior body regions.Both studied nemertean species showed immunofluorescent labels in the integument, intestine epithelium, and musculature and glandular epithelium of the proboscis (Table 3, Figure 2).

Integument
The integument of both K. alborostrata and K. manchenkoi had a pseudostratified ciliary epithelium (epidermis) resting on the subepidermal extracellular matrix (ECM) and cutis (Figures 3 and 4A).The epidermis thickness was uniform all over the nemertean's body (40 and 54 µm in K. alborostrata and K. manchenkoi, respectively), while the cutis thickness decreased from the precerebral to the posterior regions (from 320 to 20 µm in K. alborostrata and from 340 to 20 µm in K. manchenkoi).The epidermis was mainly composed of ciliated and serous glandular cells (Figure 3A).The cutis of K. alborostrata and K. manchenkoi included cutis musculature, nerve fibers, and subepidermal (cutis) glandular cells (Figure 4A).Bodies of subepidermal glandular cells were large and irregular in shape.The apical parts of these cells formed long necks that extended through the pores of the subepidermal ECM and opened at the epidermal surface, forming papilla (Figures 3C, 4B and 5A).Four types of cutis glandular cells (gc1-gc4) were identified in both K. alborostrata and K. manchenkoi (Table 3).All cutis glandular cell types were evenly distributed throughout the integument (Table 3, Figure 2).ary epithelium (epidermis) resting on the subepidermal extracellular matrix (ECM) and cutis (Figures 3 and 4A).The epidermis thickness was uniform all over the nemertean's body (40 and 54 µ m in K. alborostrata and K. manchenkoi, respectively), while the cutis thickness decreased from the precerebral to the posterior regions (from 320 to 20 µ m in K. alborostrata and from 340 to 20 µ m in K. manchenkoi).The epidermis was mainly composed of ciliated and serous glandular cells (Figure 3A).The cutis of K. alborostrata and K. manchenkoi included cutis musculature, nerve fibers, and subepidermal (cutis) glandular cells (Figure 4A).Bodies of subepidermal glandular cells were large and irregular in shape.The apical parts of these cells formed long necks that extended through the pores of the subepidermal ECM and opened at the epidermal surface, forming papilla (Figures 3C, 4B,  and 5A).Four types of cutis glandular cells (gc1-gc4) were identified in both K. alborostrata and K. manchenkoi (Table 3).All cutis glandular cell types were evenly distributed throughout the integument (Table 3, Figure 2).As the immunofluorescence images show, in the integument of both nemertean species, TTXs were localized in several types of cutis glandular cells.In both species, high- As the immunofluorescence images show, in the integument of both nemertean species, TTXs were localized in several types of cutis glandular cells.In both species, high-intensity TTXs labeling was observed in the secretory granules of gc4; TTXs-positive secretory granules filled both bodies and necks of the cells (Figures 4C and 5B).In K. alborostrata, weak TTXs labeling was revealed in the secretory granules of gc1; TTXs-positive secretory granules were detected in the cell bodies only (Figure 4D).TTXs-positive gc1 was found in the precerebral and mouth regions; the same cells localized in the rest of the body regions were not TTXs-positive (Figure 2).intensity TTXs labeling was observed in the secretory granules of gc4; TTXs-positive secretory granules filled both bodies and necks of the cells (Figures 4C and 5B).In K alborostrata, weak TTXs labeling was revealed in the secretory granules of gc1; TTXs-positive secretory granules were detected in the cell bodies only (Figure 4D).TTXs-positive gc1 was found in the precerebral and mouth regions; the same cells localized in the rest of the body regions were not TTXs-positive (Figure 2).

Digestive System
The digestive system in heteronemerteans, including K. alborostrata and K. manchenkoi studied here, consists of a mouth, a wide buccal cavity, a foregut, an intestine, and an anus [29][30][31][32].The epithelia of the buccal cavity, the foregut, and the intestine of the middle and posterior body regions of K. alborostrata and K. manchenkoi were examined.Both the buccal cavity and the foregut epithelia were composed of non-phagocytic enterocytes and several types of glandular cells; the intestine was composed of phagocytic enterocytes and several types of glandular cells (Figure 3A).Nine types of glandular cells were found in the epithelium of the digestive system in K. alborostrata and K. manchenkoi; the cell types of both nemerteans were numbered sequentially upward (Table 3).
In the digestive system of K. alborostrata, there were three types of glandular cells in the buccal cavity epithelium (g1, g2, and g7) (Figure 6A), six types of glandular cells in the foregut (g1-g6) (Figure 6B), and two types of glandular cells in the intestine (g8 and g11) (Figure 6D; Table 3).Secretory granules of g6 (Figure 6C) and g11 (Figure 6E) intensity TTXs labeling was observed in the secretory granules of gc4; TTXs-positive secretory granules filled both bodies and necks of the cells (Figures 4C and 5B).In K alborostrata, weak TTXs labeling was revealed in the secretory granules of gc1; TTXs-positive secretory granules were detected in the cell bodies only (Figure 4D).TTXs-positive gc1 was found in the precerebral and mouth regions; the same cells localized in the rest of the body regions were not TTXs-positive (Figure 2).

Digestive System
The digestive system in heteronemerteans, including K. alborostrata and K. manchenkoi studied here, consists of a mouth, a wide buccal cavity, a foregut, an intestine, and an anus [29][30][31][32].The epithelia of the buccal cavity, the foregut, and the intestine of the middle and posterior body regions of K. alborostrata and K. manchenkoi were examined.Both the buccal cavity and the foregut epithelia were composed of non-phagocytic enterocytes and several types of glandular cells; the intestine was composed of phagocytic enterocytes and several types of glandular cells (Figure 3A).Nine types of glandular cells were found in the epithelium of the digestive system in K. alborostrata and K. manchenkoi; the cell types of both nemerteans were numbered sequentially upward (Table 3).

Digestive System
The digestive system in heteronemerteans, including K. alborostrata and K. manchenkoi studied here, consists of a mouth, a wide buccal cavity, a foregut, an intestine, and an anus [29][30][31][32].The epithelia of the buccal cavity, the foregut, and the intestine of the middle and posterior body regions of K. alborostrata and K. manchenkoi were examined.Both the buccal cavity and the foregut epithelia were composed of non-phagocytic enterocytes and several types of glandular cells; the intestine was composed of phagocytic enterocytes and several types of glandular cells (Figure 3A).Nine types of glandular cells were found in the epithelium of the digestive system in K. alborostrata and K. manchenkoi; the cell types of both nemerteans were numbered sequentially upward (Table 3).
In the digestive system of K. alborostrata, there were three types of glandular cells in the buccal cavity epithelium (g1, g2, and g7) (Figure 6A), six types of glandular cells in the foregut (g1-g6) (Figure 6B), and two types of glandular cells in the intestine (g8 and g11) (Figure 6D; Table 3).Secretory granules of g6 (Figure 6C) and g11 (Figure 6E) demonstrated high-intensity TTX labeling.Secretory granules of g8 (Figure 6E) had weak TTX labeling.It should be noted that g6 was rare and located in the anterior body region only; g11 showed medium occurrence and was located in the middle and posterior body regions; and g8 was the predominant type in the middle and posterior body regions (Figure 2).glandular cells in the intestine (g8-g10) (Figure 7F, Table 3).In the fluorescent microscopy images, an intense TTXs-positive fluorescence was detected in the secretory granules of g6 (Figure 7C,E), g8, and g10 (Figure 7F).A medium intensity of TTXs-positive fluorescence was observed in the secretory granules of g5 (Figure 7E).g6 was rare and located in the mouth and anterior body regions; g8 was the predominant type in the middle and posterior body regions; g10 showed medium occurrence and was located in the middle and posterior body regions; and g5 was the predominant type in the anterior body region (Figure 2).Kulikovia manchenkoi had five types of glandular cells both in the buccal cavity (g1-g4 and g6) (Figure 7A) and in the foregut epithelia (g2-g6) (Figure 7D) and three types of glandular cells in the intestine (g8-g10) (Figure 7F, Table 3).In the fluorescent microscopy images, an intense TTXs-positive fluorescence was detected in the secretory granules of g6 (Figure 7C,E), g8, and g10 (Figure 7F).A medium intensity of TTXs-positive fluorescence was observed in the secretory granules of g5 (Figure 7E).g6 was rare and located in the mouth and anterior body regions; g8 was the predominant type in the middle and posterior body regions; g10 showed medium occurrence and was located in the middle and posterior body regions; and g5 was the predominant type in the anterior body region (Figure 2).

Proboscis
The proboscises in K. alborostrata and K. manchenkoi were composed of the endothelium, three muscle layers, and the glandular epithelium (Figure 3B).In the epithelia of both K. alborostrata and K. manchenkoi, five types of glandular cells (gp1-gp6) were identified (Table 3, Figures 8A,B and 9B,C).In both species, the immunohistochemical studies showed a diffuse, weak intensity of TTX labeling in the cytoplasm of endotheliocytes and proboscis musculature; also, intense TTX labeling was revealed in the secretory granules of gp4 (Figures 2, 8C and 9D).

Proboscis
The proboscises in K. alborostrata and K. manchenkoi were composed of the endothelium, three muscle layers, and the glandular epithelium (Figure 3B).In the epithelia of both K. alborostrata and K. manchenkoi, five types of glandular cells (gp1-gp6) were identified (Table 3, Figure 8A,B and Figure 9B,C).In both species, the immunohistochemical studies showed a diffuse, weak intensity of TTX labeling in the cytoplasm of endotheliocytes and proboscis musculature; also, intense TTX labeling was revealed in the secretory granules of gp4 (Figures 2, 8C and 9D).

Proboscis
The proboscises in K. alborostrata and K. manchenkoi were composed of the endothelium, three muscle layers, and the glandular epithelium (Figure 3B).In the epithelia of both K. alborostrata and K. manchenkoi, five types of glandular cells (gp1-gp6) were identified (Table 3, Figures 8A,B and 9B,C).In both species, the immunohistochemical studies showed a diffuse, weak intensity of TTX labeling in the cytoplasm of endotheliocytes and proboscis musculature; also, intense TTX labeling was revealed in the secretory granules of gp4 (Figures 2, 8C and 9D).

Discussion
The microscopic data, as well as a chromatographic analysis, showed an uneven TTXs distribution in the bodies of both K. alborostrata and K. manchenkoi (Figure 2).Despite the close relationship of the nemertean species under study, the pattern of 5,6,11-trideox-yTTX distribution within the K. manchenkoi body, according to the HPLC-MS/MS analysis, was different from that in K. alborostrata, which was consistent with the differences in the localization of TTXs-positive cells.Thus, the high concentration of 5,6,11-trideoxyTTX in the precerebral and mouth regions of K. alborostrata could likely be a result of its accumulation in gc1 (Figure 4D).However, as followed from the microscopic examination, the total frequency of the occurrence of g6, g8, and g11 in K. alborostrata was lower than that of gc1 and gc4 in the precerebral and mouth regions, which could lead to a decrease in the 5,6,11-trideoxyTTX concentration in the anterior, middle, and posterior body regions (Table 1, Figure 2).On the contrary, gc1 was not TTXs-positive in K. manchenkoi, and the increase in the toxin concentration from the precerebral to posterior body regions could result from the greater abundance of TTXs-bearing glandular cells of the intestine, g8 and g10 (Figures 2 and 7G).The proboscises of both studied nemertean species had similar TTXs-bearing glandular structures, gp4, whose abundances were equal, as well as similar toxin proportions (Figures 2, 7C and 8D; Tables 1 and 2).
The localization of TTXs within the body in TTXs-bearing animals may indicate their physiological role [33].Thus, TTXs were detected in the glandular cells of the proboscis

Discussion
The microscopic data, as well as a chromatographic analysis, showed an uneven TTXs distribution in the bodies of both K. alborostrata and K. manchenkoi (Figure 2).Despite the close relationship of the nemertean species under study, the pattern of 5,6,11-trideoxyTTX distribution within the K. manchenkoi body, according to the HPLC-MS/MS analysis, was different from that in K. alborostrata, which was consistent with the differences in the localization of TTXs-positive cells.Thus, the high concentration of 5,6,11-trideoxyTTX in the precerebral and mouth regions of K. alborostrata could likely be a result of its accumulation in gc1 (Figure 4D).However, as followed from the microscopic examination, the total frequency of the occurrence of g6, g8, and g11 in K. alborostrata was lower than that of gc1 and gc4 in the precerebral and mouth regions, which could lead to a decrease in the 5,6,11-trideoxyTTX concentration in the anterior, middle, and posterior body regions (Table 1, Figure 2).On the contrary, gc1 was not TTXs-positive in K. manchenkoi, and the increase in the toxin concentration from the precerebral to posterior body regions could result from the greater abundance of TTXs-bearing glandular cells of the intestine, g8 and g10 (Figures 2 and 7G).The proboscises of both studied nemertean species had similar TTXs-bearing glandular structures, gp4, whose abundances were equal, as well as similar toxin proportions (Figures 2, 7C and 8D; Tables 1 and 2).
The localization of TTXs within the body in TTXs-bearing animals may indicate their physiological role [33].Thus, TTXs were detected in the glandular cells of the proboscis and integument of some nemerteans, which gave reason to hypothesize a contribution of TTXs to prey immobilization during hunting and defense against predators [16][17][18]26].
The assumption was also confirmed by in vivo studies that demonstrated the secretion of TTXs-containing mucus from the integument [14,34].The present study revealed the localization of the toxins in the glandular cells of the proboscis and integument epithelium of K. alborostrata and K. manchenkoi as well.However, the discovery of TTXs in the glandular cells of the intestine of both species was unexpected, and, therefore, no data concerning the secretion of the toxin in the digestive system and its role had been obtained in previous studies.
In the proboscis, TTXs-positive cells are located in the so-called "epithelial ridge" (Figure 9D) which is formed on the proboscis' ventral side [35].When the proboscis is everted, the glandular epithelium appears on its external surface, and the cells of the epithelial ridge become distributed all over the extensive area of the proboscis.As was reported in a previous study, while hunting, nemertean comes in contact with its prey with the ventral side of its proboscis [36].Therefore, a contribution of TTXs-bearing glandular cells to prey immobilization can be assumed for some of nemertean species.The first presumptive mechanism of TTXs utilization during hunting is the introduction of the toxin into the prey's body using pseudocnidae, the structures with an internal hollow thread-like tubule (core) [37][38][39][40][41] that supposedly penetrate the prey's integument [29,37,41].Another possible mechanism of TTXs delivery into the prey's body is the use of a venomous mixture of enzymes and proteinaceous pore-forming toxins produced by the proboscis that can promote TTXs distribution over tissues [42].Thus, a comprehensive investigation should be carried out to elucidate both hypothesized TTXs delivery mechanisms.
5,6,11-trideoxyTTX was the only TTX analogue with quantified concentration in both species.Earlier, it was demonstrated that the toxic effect of 5,6,11-trideoxyTTX is minimal [43], and its contribution to nemertean's toxicity is ambiguous.A recent behavioral study has reported that 5,6,11-trideoxyTTX elicits chemotaxis of pufferfish Takifugu alboplumbeus and attracts them [44].The role of 5,6,11-trideoxyTTX in the communication of nemerteans has not been studied yet.Moreover, it should be noted that the TTXs profile of predators is not a constant and depends on the profile of TTXs sources, which may vary between different periods and localities [45].An assumption can be made that TTXs-positive cells described here may also be involved in accumulation of toxic TTX analogues other than 5,6,11-trideoxyTTX that was dominant in the species studied here.Thus, the invariability of TTXs-accumulating structures was demonstrated for C. simula individuals from different localities [17,26].
The K. alborostrata and K. manchenkoi specimens examined in the present study appeared to possess common TTXs-positive gc4 in the integument that may be responsible for the secretion of the toxic mucus on the surface all over the body of both nemertean species, thus, contributing to repelling predators [14,15,34].A microscopic examination of K. alborostrata also revealed gc1 whose bodies were TTXs-positive, in contrast to their ducts, which were not immunostained.Therefore, this cell type could not be involved in TTXs secretion.The function of TTXs storage could be assumed for gc1.However, further research is needed to verify this assumption.
The presence of TTXs in the glandular cells of the intestine was demonstrated in the present study for the first time.One TTXs-positive glandular cell type was revealed in the digestive system of K. alborostrata, and two cell types were revealed in that of K. manchenkoi.The distribution of TTXs-positive glandular cells within the digestive system also varied between these species.Thus, in K. alborostrata, the TTXs-positive glandular cells were spread evenly within the intestine epithelium and were absent from both the foregut and the buccal cavity.In K. manchenkoi, TTXs-positive glandular cells were located within the digestive system, and their number/abundance increased from the buccal cavity to the posterior region of the intestine (Figure 2).Earlier, the only known TTXs-bearing cell type in the nemertean's digestive system was enterocytes, which were assumed to uptake toxins from food [17,18,26].The presumed way of TTXs uptake by the glandular cells of the digestive system is the toxin's migration from enterocytes to glandular cells through the epithelium cells of the digestive system.The role of the toxins in the glandular cells of the nemertean's digestive system, especially in the posterior intestine, seems ambiguous.Several studies have demonstrated that TTXs may help protect TTXs-bearing animals against parasitic infections, including intestinal ones [46,47].However, the contribution of TTXs secretion into the intestine to the reduction in infections or other processes in nemerteans should be considered comprehensively.

Conclusions
The present study provides the first comprehensive overview of TTXs distribution within the body and ТТХs-accumulating structures in the low-toxic nemerteans K. alborostrata and K. manchenkoi.As a result, toxins have been detected in the glandular cells of the proboscis and integument epithelium of these nemerteans, which is consistent with the general hypothesis concerning the TTXs contribution to hunting and self-defense.However, TTXs have been discovered in the glandular cells of the intestine of both species.Nevertheless, no data concerning the secretion of the toxin in the digestive system and its function were obtained in previous studies.The newly characterized TTXs-positive glandular cells may become an incentive to study the unique unknown physiological role of TTXs in nemerteans.

Sample Collection and Preparation
Kulikovia alborostrata (28 specimens) and K. manchenkoi (12 specimens) were collected from rhizoids of the biennial brown alga Saccharina sp. at a depth of 0.5-2.5 m in Spokoynaya Bay, Sea of Japan (42.7090 • N, 133.1809 • E), in August 2023 (Figure 10).The animals were kept in containers with aerated seawater at 17 • C for three days without feeding.Then, the proboscis was cut off from each specimen, and its body was divided into five fragments: the precerebral, mouth, and anterior, middle, and posterior regions (Figure 11).To prepare a homogenate of K. alborostrata, these body regions of 25 specimens were pooled together; for a homogenate of K. manchenkoi, the same body regions of 9 specimens were pooled together.Then, both homogenates were used for the extraction of TTX and its analogues.Three specimens of each species were used for both immunohistochemical and morphological studies of all fragments.glandular cells may become an incentive to study the unique unknown physiological role of TTXs in nemerteans.

Sample Collection and Preparation
Kulikovia alborostrata (28 specimens) and K. manchenkoi (12 specimens) were collected from rhizoids of the biennial brown alga Saccharina sp. at a depth of 0.5-2.5 m in Spokoynaya Bay, Sea of Japan (42.7090° N, 133.1809°E), in August 2023 (Figure 10).The animals were kept in containers with aerated seawater at 17 °C for three days without feeding.Then, the proboscis was cut off from each specimen, and its body was divided into five fragments: the precerebral, mouth, and anterior, middle, and posterior regions (Figure 11).To prepare a homogenate of K. alborostrata, these body regions of 25 specimens were pooled together; for a homogenate of K. manchenkoi, the same body regions of 9 specimens were pooled together.Then, both homogenates were used for the extraction of TTX and its analogues.Three specimens of each species were used for both immunohistochemical and morphological studies of all fragments.

Species Identification
The nemertean species were identified by sequencing the cytochrome c oxidase subunit I (COI) gene according to the protocol described by Chernyshev and Polyakova [48] (see Supplementary Materials).Amplification of polymerase chain reaction (PCR) was carried out using the Folmer's primers [49].The sequences of the COI gene were submitted to the DDBJ/ENA/GenBank databases under the accession numbers OR883925 (K.alborostrata) and OR883927 (K.manchenkoi).

Extraction and HPLC-MS/MS Analysis of TTX and Its Analogues
The nemertean extracts were prepared, and the analysis was carried out according to the protocol described by Vlasenko with coauthors [7].The concentrations of the toxins were calculated following the procedure of Chen with coauthors [50].The full protocol is provided in the Supplementary Materials.

Species Identification
The nemertean species were identified by sequencing the cytochrome c oxidase subunit I (COI) gene according to the protocol described by Chernyshev and Polyakova [48] (see Supplementary Materials).Amplification of polymerase chain reaction (PCR) was carried out using the Folmer's primers [49].The sequences of the COI gene were submitted to the DDBJ/ENA/GenBank databases under the accession numbers OR883925 (K.alborostrata) and OR883927 (K.manchenkoi).

Extraction and HPLC-MS/MS Analysis of TTX and Its Analogues
The nemertean extracts were prepared, and the analysis was carried out according to the protocol described by Vlasenko with coauthors [7].The concentrations of the toxins were calculated following the procedure of Chen with coauthors [50].The full protocol is provided in the Supplementary Materials.

Immunohistochemical Studies
For the fluorescence microscopy analysis, body fragments from the proboscis, precerebral, mouth, anterior, middle, and posterior body regions of the animals were treated according to the method described by Malykin and coauthors [26] with modifications.To confirm the specificity of the immunoreactions, the negative control was carried out according to Sato with coauthors [51].The cross-reactivity of polyclonal anti-TTX antibodies with several TTX analogues, including 5,6,11-trideoxyTTX, was demonstrated earlier [15,52].The full protocol is provided in the Supplementary Materials.

Morphological Studies
Morphological studies of nemertean body fragments from the proboscis, precerebral, mouth, anterior, middle, and posterior regions were carried out according to Pereverzeva and coauthors [16].The full protocol is provided in the Supplementary Materials.

Figure 1 .
Figure 1.Representative high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) chromatograms of a standard 5,6,11-trideoxyTTX solution and extracts of Kulikovia alborostrata and K. manchenkoi.The black and red curves represent two different mass transitions (described in each chromatogram).

Figure 1 .
Figure 1.Representative high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) chromatograms of a standard 5,6,11-trideoxyTTX solution and extracts of Kulikovia alborostrata and K. manchenkoi.The black and red curves represent two different mass transitions (described in each chromatogram).

Figure 6 .
Figure 6.Light (A,B,D) and immunofluorescence (C,E) micrographs of transverse sections through digestive tract of Kulikovia alborostrata.Red color indicates TTX-like immunoreactivity.(A) Panoramic view of buccal cavity epidermis.(B) Panoramic view of foregut.(C) Distal part of foregut epithelium with TTX-positive type 6 glandular cells (arrows).(D) Middle region of intestinal epithelium.Arrow points to phagosome.(E) Intestinal epithelium with TTX-positive glandular cells.Arrows point to phagosomes.Letter designations: g1-g11, type 1-11 glandular cells of digestive system.

Figure 6 .
Figure 6.Light (A,B,D) and immunofluorescence (C,E) micrographs of transverse sections through digestive tract of Kulikovia alborostrata.Red color indicates TTX-like immunoreactivity.(A) Panoramic view of buccal cavity epidermis.(B) Panoramic view of foregut.(C) Distal part of foregut epithelium with TTX-positive type 6 glandular cells (arrows).(D) Middle region of intestinal epithelium.Arrow points to phagosome.(E) Intestinal epithelium with TTX-positive glandular cells.Arrows point to phagosomes.Letter designations: g1-g11, type 1-11 glandular cells of digestive system.

Figure 10 .
Figure 10.Sampling locality (A) of Kulikovia alborostrata (B) and K. manchenkoi (C).The images of nemerteans were taken with a reflex camera in macro mode.

Figure 11 .
Figure 11.Diagram of division of Kulikovia alborostrata and K. manchenkoi for tetrodotoxin extraction

Figure 10 .
Figure 10.Sampling locality (A) of Kulikovia alborostrata (B) and K. manchenkoi (C).The images of nemerteans were taken with a reflex camera in macro mode.

Figure 10 .
Figure 10.Sampling locality (A) of Kulikovia alborostrata (B) and K. manchenkoi (C).The images of nemerteans were taken with a reflex camera in macro mode.

Figure 11 .
Figure 11.Diagram of division of Kulikovia alborostrata and K. manchenkoi for tetrodotoxin extraction and immunohistochemical and morphological studies.

Figure 11 .
Figure 11.Diagram of division of Kulikovia alborostrata and K. manchenkoi for tetrodotoxin extraction and immunohistochemical and morphological studies.

Table 3 .
Characteristics of glandular cells in the integument, digestive system, and proboscis of Kulikovia alborostrata and K. manchenkoi.