Microscopic morphology and distribution of the antennal sensilla in the double‐spined bark beetle, Ips duplicatus (Coleoptera: Curculionidae)

The double‐spined spruce bark beetle, Ips duplicatus, has become an infamous secondary pest of Norway spruce, causing extensive ecological and economic destruction in many Central European countries. Antennae are the primary olfactory organs that play a fundamental role in insect‐host chemical communication; therefore, understanding morphology is crucial before conducting electrophysiological investigations. Here, we present our analysis of sensilla types on the antennal surface of I. duplicatus for the first time, using high‐resolution‐scanning electron microscopy. We studied the external morphological characteristics of antennae and the types, numbers, and distribution of the antennal sensilla in males and females. Our results revealed the presence of five different types of morphologically distinct sensilla: sensilla chaetica, sensilla basiconica, sensilla trichodea, sensilla coeloconica, and Böhm's sensilla. We observed two subtypes of sensilla chaetica (SChI and SChII), four subtypes of sensilla basiconica (SBI, SBII, SBIII, and SBIV), three subtypes of sensilla trichodea (STrII, STrIII, and STrIV) and two subtypes of sensilla coeloconica (SCoI and SCoII), respectively in I. duplicatus males and females. Minor differences in length and numbers between the sexes for some sensilla types were found. Distribution maps for different sensillar types were constructed, and specific areas for the respective sensilla were found. Possible functions of observed sensilla types are discussed. The present study provides a basis for future electrophysiological studies to understand how I. duplicatus detects ecologically important olfactory cues.

• A total of 6 main types and 11 antennal sensilla subtypes were observed in male and female Ips duplicatus.
• Minor sex-specific differences were seen in the length and numbers in several sensilla types.

| INTRODUCTION
Most bark beetles (Coleoptera: Curculionidae, Scolytinae) are natural decomposers of dead and dying trees in forests, and several species are considered economically significant conifer pests that also attack living trees.The double-spined bark beetle, Ips duplicatus (Sahlberg, 1836), originally native to Fennoscandia, Siberia, and East Asia, has spread recently to Central Europe and is expanding southward (Wermelinger et al., 2020).The primary host tree of I. duplicatus is Norway spruce (Picea abies (L.) Karst.), the most cultivated conifer in Europe (Grodzki, 2012;Holuša et al., 2010).In endemic phases, I. duplicatus colonizes the uppermost stem and the crown of the weakened or dying spruce or spruce trees, often attacked by European spruce bark beetle (Ips typographus [Linnaeus, 1758;Schlyter & Olle, 1993]).However, in outbreaks, I. duplicatus can also infest living trees (Kašák & Foit, 2015;Knížek et al., 2019).The current I. duplicatus population increases in Central Europe, and its southwest expansion has worsened the already problematic situation in spruce forests (Wermelinger et al., 2020).I. duplicatus shares similar biology as I. typographus; however, due to its specific host preferences and different bionomy, its management is different and less effective than that for I. typographus (Holuša et al., 2010).
Antennal morphology and the distribution of different sensillar types have been published for several bark beetle species of the genus Ips, including I. typographus, I. sexdentatus, I. pini, I. subelongatus, I. confusus, and other Ips species (Faucheux, 1989(Faucheux, , 1994;;Hallberg, 1982;Payne et al., 1973;Shi, Zhang, Liu, Xu, et al., 2021;Shi, Zhang, Liu, Zhang, et al., 2021).However, we do not have any literature reporting morphological data about the sensilla types and distribution of I. duplicatus.Information about antennal morphology and distribution of the sensillar types is essential for further physiological studies related to the olfaction of I. duplicatus that govern the specific behavior and host preferences.We provide external morphology of different sensillar types on I. duplicatus antennae and maps of different sensilla on the antennal surface.We compare and discuss our morphological findings with the available literature for other Ips species.

| Insects
Logs of Norway spruce (Picea abies) infested by I. duplicatus were collected in the Kostelec nad Černými lesy (49 59 0 39 00 , 14 51 0 33 00 , Czech Republic) and maintained in insect rearing chambers at the Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague until beetles developed.Then the logs were debarked, and adult beetles were collected and stored in plastic boxes with small breathing holes in a refrigerator at 4 C until used for experiments.
Five individuals of each sex were selected for observations using scanning electron microscopy (SEM).

| SEM analyses
Beetles were cleaned using an air blower to remove dirt from their surfaces.The antennae of each individual beetle were dissected under a stereomicroscope (Nikon, Japan).At first, antennae were primarily fixed for 24 h in 2.5% glutaraldehyde in 0.5 M cacodylate buffer (pH -7.2), followed by post-fixation in 2% OsO4 in the same buffer for 4 h.
Then, the antennae were washed twice with distilled water for 10 min.Fixed antennae were dehydrated by passing through a series of ethanol with increasing ethanol concentrations in water (35%, 50%, 70%, 96%, and 100%, with 10 min of incubation at each step).Antennae were further dried using a critical point dryer (Bal-Tec CPD 030).
Preparations were then sputter-coated with a gold layer (20 nm thickness) in an ion sputter coater (Bal-Tec SCD 050) and observed under a JEOL JSM-IT200 scanning electron microscope and JEOL IT800 high-resolution scanning electron microscope (high-resolution SEM) at 3, 5, 10, and 15 kV with a working distance of 3-5 mm.The antennal morphology and sensilla types, numbers, and distribution were studied on five antennae from adults of both sexes.
Sensilla categorization was based on external morphological criteria like size, shape, presence or absence of pores, and other features such as the attachment of the sensilla with the cuticle (flexible or inflexible socket) (Nowi nska & Brożek, 2017).

| Statistical analyses
Image J v.1.53q(Schneider et al., 2012) was used to measure and quantify each sensilla type.The software allows you to set a defined scale and measure different parameters of selected region such as length, width, diameter, and area, using the specific tools.The length of the sensilla was measured from the sensilla tip to the base of the sensilla, and basal width was determined at the bottom of the sensilla (n = 10 per sensilla type per specimen in each sex).The length, basal width, and total numbers of sensilla of each respective category were compared between the sexes by Bonferroni multi-comparison test using GraphPad Prism v.9.0 trial version for Windows, GraphPad Software (www.graphpad.com).

| General antennal morphology
The antennae of Ips duplicatus are elbowed with seven flexibly connected segments: the scape (on the proximal side), five funicular segments (from proximal to distal: pedicel (F1), F2, F3, F4, F5), and the antennal club (the most distal side) (Figure 1a).The segments between the scape and the antennal club are smaller, flexible, and collectively considered as funicle.The funicle has five bowl-shaped linked segments, with their depth increasing and diameter decreasing distally from F5 to F (Figure 1a and Supplementary Table 1).The segment F1 connected to the scape is also known as the pedicel and is the largest of all funicular segments.The pedicel of the left antenna is slightly bent towards the left side, while the pedicel of the right antenna is curved to the right side.The mean length of the whole antenna is approximately 755.99 ± 1.69 μm.The scape is the longest part of the antenna and is, on average, 354.23 ± 2.05 μm long, followed by the antennal club wide and oval shaped with an average length of 220.66 ± 2.43 μm in the direction of the antennal axis with an average width of 180.70 ± 0.53 μm.The club is slightly bulging (convex) on both sides.The surface of the scape, funicle, and dorsal surface of the club have a scalelike layer on the outer surface.These scales are also visible on the proximal area of the ventral side of the club.The sensilla on the surface of the scape, funicular segments, and dorsal side of the antennal club are sparse, and only a few types are present (Supplementary Figure 1).The majority of sensilla are located on the ventral side antennal club, specifically in its most distal three-fourth area (Figure 1b).The sensilla are systematically organized into three sensory areas, referred to here as A, B, and C bands here (distal sensory band C, middle sensory band B, and proximal sensory band A) (Figure 1b).

| Sensilla types and distribution
Two types of sensilla chaetica (SChI and SChII), four types of sensilla basiconica (SBI, SBII, SBIII, and SBIV), three subtypes of sensilla trichodea (STrII, STrIII, and STrIV), two types of fluted cone-shaped sensilla coeloconica (SCoI and SCoII), and Böhm's sensilla (BS) were identified on I. duplicatus antennae in both sexes (Table 1).Table 1 summarizes the respective features (length, basal width, socket characterization, presence of pores in the sensillar cuticular wall or at the tip, tip shape, etc.) and numbers of each sensilla type present on the ventral area of the antennal club.On the dorsal surface of the club, only sensilla chaetica type II, sensilla trichodea type III, and Böhm's sensilla were seen (Supplementary Figure 1).

| Sensilla chaetica
Sensilla chaetica (SCh) are long aporous sensilla with toothed projections and flexible (deep and wide) sockets (Figure 2).In I. duplicatus, sensilla chaetica were generally projected at an angle greater than 45 from the antennal club surface.They comprise 12% of the total observed sensilla.Based on their length and branching pattern, sensilla chaetica were categorized into two subtypes (Figure 2a, b).Shorter and slender sensilla chaetica type I (SChI) with a length of 21.2-46.5 μm in males and 21.9-38.9μm in females, respectively, had toothed projection oriented in only one plane (saw-toothed with bilateral branching) and visible longitudinal grooves on the wall surface (Figure 2c, f-h).
T A B L E 1 General morphological characteristics based on external appearance and distribution of different sensilla types in Ips duplicatus.SChII on the scape and funicular segments were remarkably longer than those observed on the antennal club surface.SPs were often observed close to both types of sensilla chaetica (Figure 2d).

| Sensilla basiconica
Sensilla basiconica (SB) were the most frequent sensilla type (66% of the total sensilla) observed within the sensory epithelium of the club (Figure 3).All SB have inflexible (fused) sockets.Sensilla basiconica were categorized into four subtypes based on their length, basal width, porosity, tip shape, and wall structure (Figure 3a).The most abundant sensilla basiconica type were sensilla basiconica type I (SBI) (Figure 3b).These sensilla types were straight, multiporous with a pointed tip.The pores of SBI formed longitudinal slit-like depressions on the wall surface (Figure 3c).The pore density was 40 pores/um 2 .The SBI length was around 9.3-11.7 μm long in males and 8.6-13.2μm long in females.Sensilla basiconica (SBII) were also multiporous with pointed tips but comparatively shorter and thicker than SBI (Figure 3d and Table 2).SBII were around 5.8-10.2μm long in males and 6.3-10.9μm in females.SBII have a lower pore density of 20 pores/um 2 compared to SBI.The pores collectively resembled pit-like depressions on the sensillar wall surface (Figure 3d).Sensilla basiconica type III (SBIII) were uniporous with peg-like appearance (length and basal width range: 5.8-10.9μm and 1.1-2 μm, respectively), a slightly tapered tip, and a smooth wall with slight depressions (Figure 3e, f).The range of length of SBIII was around 3.6-6.8μm in males and 3.3-7.1 μm in females, respectively.Sensilla basiconica type IV (SBIV) were the shortest basiconica type with an inflexible (fused) socket, smooth wall, and an uniporous round tip (Figure 3g) with the length ranging from 3 to 5 μm in males and 3.4-6 μm in females.

| Sensilla trichodea
This category (STr) is rather non-homogeneous in its parameters and covers around 19% of the total sensilla.Considering their length as a classification parameter, ranging from 12.24 to 30.62 μm, they fit between the sensilla basiconica and sensilla chaetica (Table 2).We classified these sensilla types into three categories (Figure 4a).The longest sensilla trichodea type III (STrIII) (length range: 21.5-44.8μm in males and 18.9-47.6μm in females) were distinctly curved sensilla with a flexible socket, smooth wall, and a single terminal pore (Figure 4b,g,h).Sensilla trichodea type II (STrII) were shorter and slender than STrIII (length range: 15.7-34.5 μm in males and 13.7-36.4μm in females, respectively), elongated and tapering towards the tip (Figure 4e), with an inflexible (fused) socket, and a multiporous wall (Figure 4f).The pore density calculated was 30 pores/um 2 for STrII.
The shortest sensilla trichodea type IV (STrIV) with length range of 11.1-15.8μm in males and 9.5-16.2μm in females, respectively, had a sharp pointed tip, porous wall surface, and fused sockets (Figure 4c,   d).STrIV were easily distinguishable from STrII since they bulged in the middle and tapered towards the tip.The pore density was 15 pores/um 2 .We did not observe sensilla trichodea type I, which was reported in previous studies in Ips species (Supplementary Table 2).

| Sensilla coeloconica
Sensilla coeloconica (SCo) were fluted cone-shaped structures with deep longitudinal grooves on their wall and inflexible (fused) sockets (Figure 5) covering about 2% of the total number of sensilla.Two types of SCo were classified based on the differences in their tip shape.SCo type I had a pointed tip with a length range from 5.9-8.1 μm in males and 5.5-8.3μm in females (Figure 5a, b), whereas SCo type II had a round and bulgy tip with a length range of 5.8-7.4μm in males and 5-7.9 μm in females, respectively (Figure 5c, d).

| Böhm's sensilla
The sensilla type, called Böhm's sensilla (BS), were present on the base of the scape and pedicel (Figure 6a).They were short (length range: 3.9-6.9μm in males and 8.4-15.5 μm in females, respectively), straight or slightly curved hairs with their base in a flexible (deep and wide) socket and a smooth wall surface, typically angled 45 to the cuticle of the antennal surface (Figure 6b).

| Surface pores
The SPs were present homogeneously on the ventral side of the antennal surface, the funicular segments, and the scape (Figure 6a, d).
Some were also observed on the dorsal surface (Supplementary Figure 1).The pore diameter was 0.5 μm.We observed around 28 of these pores in males whereas approximately 18 in females (Supplementary Table 1).The exact number could not be estimated since the pores were difficult to observe since they were often hidden between different sensilla on the antennal surface.The pores were sometimes associated with sensilla with flexible sockets but otherwise   diffusely scattered among sensilla on the antennal club surface (Figure 6c).

| Distribution, dimensions, and numbers among sexes
The structural features and distribution pattern of sensilla on the antennal surface was approximately same in males and females.After statistical analyses using Bonferroni's multicomparison test, we noted minor variations concerning the length of a few sensilla types and numbers among the sexes (Table 2).The total length of the antenna and other antennal segments showed no significant differences between the sexes.The mean length of SChII was significantly different when comparing females and males (p-value of <.0001); however, the average length differences in other sensilla types were non-significant (Figure 7a and Table 2).The mean basal width of SChII was significantly higher in females than in males ( p-value <.0058).
Similarly, the mean basal width of BS was considerably higher in females than in males, with a p-value of .0058(Figure 7b and Table 2).
The average number of SBI was significantly different in males than in females ( p < .0163),whereas the mean number of STrIV was significantly higher in females than in males (p < .0026)(Figure 7c and Table 2).
We mapped the distribution pattern of different sensilla types present on the antennal surface in I. duplicatus (Figure 8).The two types of sensilla chaetica, SChI and SChII, often can be seen as forming the boundary within the sensory bands around the sensilla basiconica and sensilla trichodea, whereas SChII is primarily seen on the funicular segments and scape and sometimes on Band A (Figure 8a).
BS was exclusively present at the base of the scape and pedicel with only 1% of the total sensilla (Figure 8a).The sensilla basiconica types
Based on our study, it is not clear whether these differences reflect specificities related to the technique used in previous studies or whether they indicate a specific adaptation for I. duplicatus.
There have been several studies of Ips spp.sensilla so far, with different classification and nomenclature (Faucheux, 1989(Faucheux, , 1994;;Hallberg, 1982;Payne et al., 1973;Shi, Zhang, Liu, Zhang, et al., 2021).Supplementary Table 2 summarizes the classification and nomenclature of sensilla in the present study and previously studied Ips species facilitating a clear understanding and avoiding any future confusion.We kept our classification consistent with previous reports and followed a new nomenclature only for those sensilla types which were not reported previously and do not fit within the existing classification.
As also observed for other Ips species (Faucheux, 1989(Faucheux, , 1994;;Shi, Zhang, Liu, Xu, et al., 2021;Shi, Zhang, Liu, Zhang, et al., 2021) and bark beetles in general, sexual dimorphism in I. duplicatus was not too prominent, and if present, it generally refers to only minor differences in abundance and length of some sensillar types.This finding indicates that in I. duplicatus the sensilla probably have similar functions in both sexes.Interestingly, we observed significantly longer sensilla chaetica with multilateral branching (SChII) in females, the wider multiporous sensilla basiconica (SBI), and their higher number in males.In addition, small sex-specific differences were determined for multiporous sensilla trichodea STrIV, which were present in slightly higher numbers in females.Further experiments are needed to determine whether these differences reflect sex-specific differences related to mating or host selection.Morphologically different sensillar categories are supposed to have specific physiological functions (Hallberg, 1982;Hansson & Stensmyr, 2011;Keil, 1999;Schneider, 1964).Below, we discuss the possible physiological roles of the different morphological types observed in I. duplicatus.

| Sensilla chaetica
This sensillar type have a flexible socket and long, either bilaterally or multilaterally branched hair that project outwards from the antennal surface well above other sensillar types.Two types of sensilla chaetica with the same morphology were also observed in other Ips species (Faucheux, 1994;Shi, Zhang, Liu, Xu, et al., 2021;Shi, Zhang, Liu, Zhang, et al., 2021), though there are differences when it comes to nomenclature (Supplementary Table 2).The morphological description and location of this sensilla type in I. duplicatus match with the previous studies in I. typographus and I. confusus (Borden & Wood, 1966;Faucheux, 1989;Hallberg, 1982;Shi, Zhang, Liu, Zhang, et al., 2021).
The cross-sections of sensilla chaetica in I. typographus (Hallberg, 1982); shows a basal cuticular ring of articulating membrane, circular solid hair shafts filled with an electron dense material, and a sensory process that terminates as a tubular body in the basal part of the hair.These parameters suggest that sensilla chaetica serves the mechanoreception function (Borg & Norris, 1971;Hallberg, 1982;Moeck, 1968).Though we have not performed the cross-section of sensilla chaetica in I. duplicatus, the external morphological similarity between sensilla chaetica in I. typographus and respect to its surroundings and detect wind movement (Payne et al., 1973).It may also serve as "displacement detectors" as reported in some beetle species (Borg & Norris, 1971;Henderson & Wadhams, 1981;Moeck, 1968;Wadhams et al., 1982).Alternatively, sensilla chaetica might be involved in fly speed detection during flying as suggested by the electrophysiological recordings from bilaterally branched sensilla chaetica in Scolytus scolytus showing that these types of sensilla respond to airflow (Wadhams and Angst unpublished data mentioned in Sivalinghem, 2012).Alternatively, sensilla chaetica might possibly serve as auditory organs (Borden & Wood, 1966) and can be involved in bark beetle acoustic communication that mediates beetle interaction during mating, various social or defensive interactions, and dispersion under the bark (Barr, 1969;Borden & Wood, 1966;Dobai et al., 2018;Hofstetter et al., 2019;Rudinsky, 1979;Rudinsky et al., 1976;Wilkinson et al., 1967).Until now, no tympanal organs in bark beetles have been found (Borden & Wood, 1966).As long sensilla on the antenna can easily vibrate (Yack & Hoy, 2003), sensilla chaetica might be involved in the perception of sound and/or substrate vibrations (Rudinsky et al., 1976;Schmitz, 1972;Sivalinghem, 2012;Swaby & Rudinsky, 1976).Unilateral and bilateral branching may represent specific adaptations with respect to the perception of specific sound/ vibration parameters.We noted that in I. duplicatus, multilaterally branched sensilla chaetica (SChII) are significantly longer and thicker in females than in males.This may reflect the greater need by ovipositing females to orient under the bark to ensure uniform dispersion.Similar sex-specific differences were reported in I. typographus, I. sexdentatus (Faucheux, 1989), and Tryptodendron lineatum (Moeck, 1968).These differences may reflect the sex-specific differences in premating behavior and/or during oviposition (Hofstetter et al., 2019;Rudinsky, 1979).As compared to other bark beetle species (Bedoya, Brockerhoff, et al., 2019;Bedoya, Nelson, et al., 2019), the genus Ips is characterized by a relatively smaller number of sensilla chaetica (Faucheux, 1989;Shi, Zhang, Liu, Xu, et al., 2021;Shi, Zhang, Liu, Zhang, et al., 2021), which can reflect the relative importance in sound communication in different taxonomic categories (Bedoya et al., 2020;Hofstetter et al., 2019).

| Sensilla trichodea
This morphological category is inconsistent among all sensillar types found in I. duplicatus.The only common characteristic of I. duplicatus is that they are longer than the sensilla basiconica type and shorter than the sensilla chaetica type.Sensilla trichodea in I. duplicatus form two distinct categories.The first category includes two subtypes of multiporous sensilla with inflexible sockets that differ by wall structure (STrII are smooth-walled while STrIV are pitted).The second category represents the long terminal pore sensillum with a flexible socket (STrIII).STrII corresponds to trichodea type II in some Ips species (Borden & Wood, 1966;Faucheux, 1989Faucheux, , 1994;;Payne et al., 1973) (Supplementary Table 2).
The cross-sections of the multiporous sensilla performed in I. typographus (Hallberg, 1982) show single-walled sensilla with numerous pores.The multiporous trichoid sensilla were described in majority of investigated Ips species so far (Borden & Wood, 1966;Faucheux, 1989Faucheux, , 1994;;Payne et al., 1973).The multiporous sensilla are expected to have an olfactory function (Andersson et al., 2009;Borden & Wood, 1966;Hallberg, 1982;Payne et al., 1973).The wall pores allow volatile molecules to penetrate the sensillar lumen to activate the olfactory receptor neurons.The olfactory function of multiporous sensilla has been confirmed by many electrophysiological investigations performed in I. typographus (Andersson et al., 2009;Kandasamy et al., 2019;Schiebe et al., 2019).Sensilla trichodea type IV (STrIV) observed in I. duplicatus does not match with any trichodea sensilla types reported previously.Because of its distinct structure and characteristic pore features on the wall surface, the probability of misclassification can be excluded.Further studies are needed to determine the physiology of these two olfactory trichoid sensilla.
In our study on I. duplicatus, we did not see sensilla trichodea type I, which was observed previously at the base of the scape and pedicel of many Ips species (Payne et al., 1973;Shi, Zhang, Liu, Zhang, et al., 2021) and in T. lineatum (Moeck, 1968).Sensilla trichodea type I are Böhm's sensilla with proprioceptive function, though no histological information nor the characteristics of the socket are available.

| Sensilla basiconica
Our study provided evidence for four categories of sensilla basiconica in I. duplicatus.This category is also not morphologically  et al., 2009;Biswas et al., 2023;Borden & Wood, 1966;Dickens et al., 1978).Sensilla basiconica type III and IV (SBIII and SBIV) are uniporous peg-shaped hairs with slight depressions on the wall surface present predominantly in the distal club region.Only SBIII was reported previously in I. typographus (Shi, Zhang, Liu, Zhang, et al., 2021).The exact function of these pegs is not known; however, their uniporous nature indicates the contact chemoreception.

| Sensilla coeloconica
The sensilla coeloconica (SCo) has a distinct shape with longitudinal grooves on the wall surface and have previously been reported in many Ips species (Chen et al., 2010;Dickens et al., 1978;L opez et al., 2018;Whitehead, 1981).In most of the studied species so far, only one type of sensilla coeloconica has been described.Our study is the first to report two different types of sensilla coeloconica.
The cross-section performed on SCo in I. typographus (Hallberg, 1982) shows a "double-walled sensilla" with finger-like radial channels connecting the sensillar surface with neuronal sensory processes within the hair lumen.The "double-walled sensilla" are innervated by 2-6 sensory cells with unbranched sensory processes terminating in the apical part of the hair.Below the hair, one of the sensory processes exhibits a lamellar pattern like that of pore less sensilla in which thermoreception has been demonstrated (Altner, 1977).
On the other hand, the "double-walled sensilla" of I. typographus have a similar structure as certain chemoreceptive sensilla (Altner, 1977;Altner et al., 1977).Thus, in different insect species, SCo may have various functions, such as hygroreception, thermo-hygroreception and olfactory reception.SCo in moths and flies are known to have an olfactory role and olfactory receptor neurons are tuned to compounds like acids, aliphatic aldehydes, amines, and ketones (De Bruyne & Baker, 2008;Pophof et al., 2005;Yao et al., 2005).
We observed the mirror arrangement of SCo on both antennal clubs, with SCo more frequently distributed on the lateral regions of the antennal clubs, indicating a potentially highly specific function.We found no previous literature reporting this kind of arrangement.Further investigation can explain this specific arrangement and modalities of SCo.

| Böhm's sensilla
BS are usually seen exclusively on the base of the scape and pedicel of the antenna in I. duplicatus, also reported as "böhm's bristles" in I. subelongatus (Shi, Zhang, Liu, Xu, et al., 2021) and as "böhm sensilla" in I. typographus (Shi, Zhang, Liu, Zhang, et al., 2021).In Curculio nucum, BS are present on the base of the scape and pedicel (Faucheux et al., 2019).Their location and distribution suggest a propioceptive role (Merivee et al., 1999).They possess a flexible deep socket and smooth wall surface.These are known to monitor the antennal positioning and movements during the flight (Dong et al., 2020).

| Surface pores
The SPs were about 0.5 um wide and were present on both sides of the club without any association with sensilla.These structures might be similar to the previously reported "glandular pores" in I. sexdentatus and I. typographus (Faucheux, 1989).However, the glandular pore diameters were not provided, and were associated with sensilla chaetica.Alternatively SP can represent "mechanosensory cuticle sensillum" reported on the antennal club of I. typographus (Hallberg, 1982)."Mechanosensory cuticle sensillum" terminates within a cavity of the cuticle with an approximate diameter of 2-2.5 um (Hallberg, 1982).Since I. typographus is significantly bigger in overall size than I. duplicatus, we can say that the dimensions of SP and "mechanosensory cuticle sensillum" are relatively similar.Alternatively, SP might be glands meant for secretion of the antennal epicuticular layer of the antennae and their sensilla (Bin et al., 1989;Dahms, 1984;Faucheux, 1994;Faucheux & Kundrata, 2015;Romani et al., 2019;Skilbeck & Anderson, 1994;Weiss et al., 2011).

| CONCLUSION
Ips duplicatus is a serious conifer pest that shares the same host and Our microscopic results revealed important information about the general morphology and the distribution of various functionally important sensilla in I. duplicatus.We found the sexual dimorphism in I. duplicatus is unrelated to the general antennal morphology and concerns only minor variations with the number and length of different sensilla types.This discrepancy in morphological properties can be associated with variation in the biophysical characteristics of different hair types, allowing them to be sensitive to different mechanical stimuli.We also provided comparative information on sensilla typology and its external characteristics in Ips species as an attempt to establish the general sensilla nomenclature for future studies in this genus.
Overall, the present study provides a map of I. duplicatus olfactory equipment and establishes a basis for future olfaction-based and electrophysiological investigations of this destructive forest pest.

(
220.66 ± 2.43 μm) and the funicular segments.The antennal club is F I G U R E 1 The general morphology of Ips duplicatus antenna (female).(a) Ventral side of the antennal club showing funicular segments (F1-F5), pedicel (F1) and scape; (b) Three sensory bands on the antennal club (A, B, and C).
The sensory bands A and B form two parallel wave-like stripes across antennal club separated by a strip of the plain cuticle.The sensory band B has a deeper curve in the middle compared to sensory bands A and C. A hint of the third distal sensillar band C almost merges with the middle sensory band B area on the antennal club.Oval-shaped pit-like structures scattered randomly among the sensilla, termed here as surface pores (SPs), were observed on both the ventral side and the dorsal part of the club and other antennal segments.
SChI are present on all antennal sections, including the dorsal and ventral side of the club.Sensilla chaetica type II (SChII) were longer and thicker, with a length range of 23.9-79.2μm in males and 29.2-221.3μm in females, respectively, with multilateral branching (Figure2d, i-k).The socket shape was different in both types of sensilla chaetica, with SChI having a deeper socket than SChII (Figure2h, k).

F
I G U R E 3 Ips duplicatus antenna highlighting sensilla basiconica and its subtypes, (a) Antennal sensory band C showing clusters of sensilla showing different types of sensilla basiconica (b) sensilla basiconica type I (SBI) with slit-like depressions on the wall surface and fused socket, (c) magnified SBI tip showing pores (indicated by arrows), (d) sensilla basiconica type II (SBII) with the porous wall surface, (e) peg-shaped sensilla basiconica type III (SBIII) with fused socket and a pointed tip, (f) closer look of SBIII tip with the pore-like structure on the tip and, (g) sensilla basiconica type IV (SBIV) with the pore-like structure on the blunt tip.T A B L E 2 The length range, mean length, basal width, numbers, and respective p-values (mean ± SE) of the different sensilla type in male and female Ips duplicatus along with their percent total distribution.

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I G U R E 4 Ips duplicatus antenna highlighting sensilla trichodea and its subtypes, (a) Group of sensilla trichodea types on the antennal club(indicated by arrows) including (b) sensilla trichodea type II (STrII) and sensilla trichodea type III (STrIII), (c and d) sensilla trichodea type IV (STrIV) with fused socket, bulged middle, porous wall surface and pointed tip, (e) sensilla trichodea type II (STrII) with fused socket, pointed tips and (f) multiporous wall surface, (g) sensilla trichodea type III (STrIII) with deep flexible socket, blunt tip and (h) terminal pore.F I G U R E 5 Ips duplicatus antenna highlighting sensilla coeloconica and its subtypes (a) sensilla coeloconica type I (SCoI) with longitudinal grooved wall surface and (b) sharp tip, (c) sensilla coeloconica type II (SCoII) with (d) round tip.F I G U R E 6 Ips duplicatus antenna highlighting (a) Böhm's sensilla (BS) on the scape (highlighted square) and surface pore (SP) indicated by an arrow, (b) detailed view of BS with a deep socket and round tip, (c) SPs on the antennal club surface (indicated by arrows) and, (d) magnified view of SPs.F I G U R E 7 Graph showing the comparison of length (a), width (b), and numbers (c) of different sensilla types in Ips duplicatus antenna in females and males.Bars represent means and SE.(Bonferroni's multiple comparisons tests; n = 5 per sex).
SBI and SBII, were uniformly distributed on the sensory band A and B. SBI were denser in the middle of the sensory bands A and B. Very few were observed on the distal band C. Shorter types of sensilla basiconica (SBIII and SBIV) were primarily observed in most distal club area C (Figure8b).Among all the sensilla trichodea types, STrIII is typically present within the proximal boundaries of sensillar bands and at the periphery of the club, and the distribution is quite distinct and uniform.STrII was more abundant in the antennal club's C and B sensory areas, whereas STrIV was spotted more within A and B sensory bands.STrIV were often seen around SBI and SBII forming a peripheral borderline on the sensory band A and B (Figure8c).Both types of sensilla coeloconica (SCoI and SCoII) were more abundant on the sensory bands B and C and sometimes observed on the sensory band A. These sensilla often occurred in pairs close to each other, and the distribution pattern was quite interesting.Both sensilla coeloconica I and II were more frequently distributed on the middle and to the right side of the left antenna and vice versa; rarely, 1 or 2 sensilla coeloconica were observed on the same side of the antenna (Figure9a, b).

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. duplicatus allows us to conclude that sensilla chaetica in I. duplicatus also have a mechanoreceptive role.Their locations on the scape may enable I. duplicatus to determine the positions of the antennae with F I G U R E 9 Map of sensilla coeloconica distribution on ventral side of Ips duplicatus.(a) Sensilla coeloconica type I (SCoI) and sensilla coeloconica type II (SCoII) on the left antenna; (b) sensilla coeloconica type I (SCoI) and sensilla coeloconica type II (SCoII) on the right antenna.
homogenous.They are shorter and wider in comparison with sensilla chaetica and trichodea and form two distinct groups.Sensilla basiconica type I (SBI) are the most numerous type occupying about three-fourths of the total area of the antennal club surface.They are highly dense in the sensory bands A and B and represent multiporous sensilla with slitlike depressions suggesting their possible role in olfactory detection.SBI in I. duplicatus has features similar to single-walled sensillum type I reported by Hallberg, 1982 in I. typographus.The sensilla basiconica type II (SBII) is not described in Hallberg, 1982 in I. typographus but is mentioned by Faucheux (1989) in I. sexdentatus and I. typographus.SBII has lower pore density when compared to SBI.These two types of SB are known to be sensitive to general odors like plant compounds and pheromones validated by electrophysiological studies (Andersson has similar biology as compared with I. typographus, which is a model bark beetle for studying olfaction.Numerous morphological and electrophysiological investigations have been conducted for I. typographus, but we have no information about the sensillar equipment including the typology and functions of different sensilla in I. duplicatus.The study addressed the research gap concerning the types of sensilla and their distribution and possible role in I. duplicatus.