Morphological characteristics and distribution of antennal sensilla of Spodoptera litura (Lepidoptera: Noctuidae) using scanning electron microscopy

doi:10.21203/rs.3.rs-5285978/v1

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Morphological characteristics and distribution of antennal sensilla of Spodoptera litura (Lepidoptera: Noctuidae) using scanning electron microscopy

https://doi.org/10.21203/rs.3.rs-5285978/v1

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Spodoptera litura Fabricius (Lepidoptera: Noctuidae) is a major agricultural pest, primarily in Asia and Oceania. Chemical odor-based trapping is a major method used to control S. litura, and thus understanding the antennal sensilla of S. litura is critical for improving the efficacy of the attractants used in the pest control. In the present study, the S. litura antennal sensillum types were examined by low-voltage field emission scanning electron microscopy, and morphological descriptions were provided. A total of eight types and two subtypes of the antennal sensilla were identified, namely Böhm's bristles, sensilla trichoidea, sensilla basiconica, sensilla chaetica, sensilla coeloconica, sensilla styloconica, sensilla squamiformia (I and II), and sensilla auricillica (I and II). Among them, sensilla squamiformia II, and sensilla auricillica II are reported for the first time in S. litura. This study provides morphological information to aid in future electrophysiological tests on the antennal sensilla of S. litura.

Spodoptera litura

antennal sensilla

scanning electron microscopy

sensilla morphology

Spodoptera litura Fabricius (Lepidoptera: Noctuidae) is a significant agricultural pest in Asia and Oceania (Bragard et al. 2019). As an omnivorous insect, it can feed on hundreds of plant species from 40 families, including economically important crops such as beans, crucifers, cucurbits, and others in the families Gramineae, Malvaceae, and Solanaceae (Du et al. 2022; Sharma et al. 2022). S. litura occurs throughout China, especially in the middle and lower reaches of the Yangtze River and South China, where it impacts primarily tobacco and cruciferous vegetables (Zhou 2009; Yang et al. 2011). Historically, chemical pesticides have been the predominant method for controlling this moth (Yushima et al. 1974; Singh and Sachan 1993; Guerrero et al. 2014). However, varying levels of pesticide resistance have developed in this insect, leading to excessive spraying and environmental pollution (Ahmad et al. 2007; Bragard et al. 2019). In recent years, China has begun implementing pheromone trapping to monitor and control this pest (Yang et al. 2011; Sang et al. 2013). Pheromone trapping has proven to be environmentally friendly and has led to reduced reliance on chemical pesticides. Therefore, developing more effective odor-based trapping attractants has become an important focus to improve management of S. litura.

Antennal sensilla are the biological sensory organs that allow insects to detect and perceive external environmental information such as air flow, air temperature, and pheromones (Galizia and Rössler 2010; Schmidt and Benton 2020; Li et al. 2024). Identifying the types of antennal sensilla is an important prerequisite to clarify the function of antennal sensors, and also to improve the efficacy of pheromone or other odor-based attractants. Nevertheless, relatively few studies have focused on the antennal sensilla of S. litura. Liu et al. (2009) conducted the first study of antennal sensilla of S. litura and recorded five types: sensilla trichoidea, sensilla basiconica, sensilla chaetica, sensilla coeloconica, and sensilla auricillica. Thereafter, Aruna et al. (2019) also identified five types of antennal sensilla on S. litura (sensilla trichoidea, sensilla chaetica, sensilla coeloconica, sensilla auricillica, and sensilla styloconica) with sensilla styloconica being newly recorded in this insect. However, the number of sensilla types reported for S. litura in the above two studies (6 types), is relatively low when compared to many other Noctuidae. For example, as many as 15 sensilla types have been reported for Spodoptera frugiperda (Lepidoptera: Noctuidae) (J. E. Smith) (Li et al. 2024). Therefore, it is necessary to further clarify the sensillum types of S. litura and describe their characteristics.

The main objective of this study was to conduct a comprehensive examination and classification of the antennal sensilla of S. litura, using low-voltage field emission scanning electron microscopy. For each sensilla type, we described its morphological characteristics and distribution, as well as present one or more high-resolution photos. Providing such information will facilitate future research to improve odor attractants for this major agricultural pest.

Sampling insects

Newly emerged 1- to 2-day-old S. litura adults, both male and female, were used for scanning electron microscopy (SEM) observations. The adults were reared in the Insect Laboratory of Yunnan University. The rearing conditions were set at a temperature of 27 ± 1°C, relative humidity of 70 ± 10%, and a light/dark cycle of L12 h: D12 h. Larvae were fed an artificial diet following the formula described by Li et al. (1998). Fully mature larvae were put individually in a circular plastic box (30 mm diameter at bottom, 40 mm diameter at top, 30 mm in height) containing sand which they entered to pupate. Newly emerged adults were transferred to plastic boxes of the same size. Adult male and female moths were provided with a diet of 10% honey water to meet their nutritional needs until they were used for the antennal sensillum studies.

Sample preparation and SEM observation

SEM observations were conducted at the Modern Analytical Testing Center of Yunnan University. After being killed, the antennae were dissected from male and female S. litura adults under a stereomicroscope (Nikon SMZ1500, Nikon Instrument, Japan). The treatment of the antennae was slightly modified from the steps given in Guo et al. (2022). Briefly, the dissected antennae were soaked in a 75% ethanol solution and subjected to ultrasonic cleaning for 30 s to remove surface contaminants. Afterwards, the antennae were rinsed with n-hexane for cleaning, with the process repeated three times for 15 min each. Subsequently, a series of dehydration steps were carried out using 80%, 90%, and 100% ethanol, with each step lasting 15 min. After the samples were dried, they were affixed to sample holders using conductive adhesive, and a thin layer of gold was sputter-coated on the samples using a BAL-TEC SCD005 under conditions of 30 mA for 300s. The prepared samples were then observed and photographed using an advanced low-voltage field emission scanning electron microscope (LVSEM) (Nova NanoSEM 450, FEI, USA) operating at voltages between 3–10 kV and magnifications ranging from 250 − 60,000x.

Terminology, measurements and statistical analysis

The identification and classification of the antennal sensilla were based on the external morphology and surface features according to Schneider (1964) and Zacharuk (1980). Antennal measurements were taken on 20 intact male and 20 intact female antennae, including total length, basal diameter, and the number of subsegments. The length and basal diameter of each sensillum type were measured using Image J 1.38 software (National Institutes of Health) and reported as the mean ± SE (standard error). For each type of sensillum found, we attempted to measure 30 replicates for both males and females. However, for some sensillum types that were found beneath scales (e.g., sensilla squamiformia Ⅱ), it was not possible to reach this goal.

Data analysis was conducted using SPSS 26.0 software (IBM). Differences between males and females for various sensilla measurements were statistically analyzed using Student's t-test, with an alpha level of P < 0.05. Similarly, size differences among the lateral sensilla chaetica (LSC), central sensilla chaetica (CSC), and dorsal sensilla chaetica (DSC) of both males and females were statistically analyzed using ANOVA and Tukey’s mean separation test, with an alpha level of P < 0.05. Microscopic and electron microscopic images were processed using Adobe Photoshop CC 2017 software (Adobe Systems).

The basic morphology of the antennae

The antennae of Spodoptera litura were filamentous and composed of three segments: a scape, a pedicel, and a flagellum (Fig. 1). The scape segment was connected to the head; followed by the pedicel and then the flagellum (Figs. 1; 2a, b). Both the scape and the pedicel had muscle structure, indicating that they were able to rotate. These first two segments were totally covered by scales and had two sensilla types: Böhm's bristles (BB) and sensilla squamiformia II (SSQ II) (Figs. 4a, b; 9c, d). Böhm's bristles were located at the basal portion of the scape; while sensilla squamiformia II were scattered on the surface of the pedicels. These sensilla types are both regarded as mechanical sensilla, and likely play a role in regulating the spatial orientation of the antennae (Schneider 1964). The average length and basal diameters of the scape and pedicel were significantly longer and larger in males compared to females (Table 1).

Table 1

Mean (SE) length and basal diameter of the antennal segments of male (N = 20) and female (N = 20) *Spodoptera litura.*
Antennal segments	Mean lengths (µm)		Mean basal diameters (µm)
Antennal segments	Female	Male	Female	Male
Scape	350.50 ± 7.58 b	393.35 ± 10.85 a	260.30 ± 7.75 a	278.25 ± 5.93 a
Pedicel	171.05 ± 4.88 b	187.50 ± 4.46 a	197.95 ± 3.31 b	222.00 ± 6.40 a
Flagellum	8.89 ± 1.70 b	9.86 ± 0.66 a	NA	NA
Total	9.38 ± 0.17 b	10.45 ± 0.07 a	NA	NA
For length and diameter measurements separately, values comparing sexes within a row followed by different letters were significantly different (paired t-test, p < 0.05).

The flagellum was composed of several subsegments (Fig. 1), gradually becoming thinner towards the tip. The flagellum consisted of 83.0 ± 0.5 subsegments (n = 20) on average in females and 83.2 ± 0.6 subsegments (n = 20) in males, with no significant difference between the sexes (t = 0.249, df = 38, p = 0.805). However, there was a significant difference in flagellum length between sexes (t = 5.51, df = 38, p < 0.001), being longer on average in males than females (Table 1). At the distal end of the flagellum, there were conical structures covered with petal-like folds, and the tip was adorned with 2 or 3 small cones, which were sensilla styloconica (SS) (Fig. 2c).

Scales were the most prominent structures on the antennal surface, covering the entire surface of the scape and the pedicel, as well as the dorsal side of the flagellum, occupying almost 50% of the antennal surface area (Figs. 2a, 2b, 2d). Most of the sensilla found on S. litura were primarily distributed on the ventral side of the flagellum, with a few sensilla located under the scales (Fig. 3a). Each subsegment had two rows of scales arranged in an overlapping tile pattern, where the scales of the previous subsegment covered the base of the next subsegment (Fig. 2d). The scales looked somewhat like shields protecting the antenna, with parallel vertical ridges on their surfaces (Fig. 2d). The average length of scales was significantly different between the sexes, being 71.01 ± 0.91 µm in females and 80.99 ± 1.15 µm in males (t = 6.95, df = 58, p < 0.001).

Types of antennal sensilla

Eight types of antennal sensilla and two subtypes were identified in the present study, namely Böhm’s bristles (BB), sensilla trichoidea (ST), sensilla basiconica (SB), sensilla chaetica (SC), sensilla coeloconica (SCO), sensilla styloconica (SS), sensilla squamiformia I and II (SSQ I and SSQ II), and sensilla auricillica I and II (SAU I and SAU II).

Böhm's bristles

Böhm's bristles (BB) were only found on the surface of the scape and pedicel, distributed in clusters at the base of the two segments, near the intersegmental region (Fig. 4a). These sensilla looked like thorns in appearance, arising from a socket-like depression, gradually tapering from the base to the tip, and ending in a sharp point. They had a smooth surface without surface pores, suggesting their role was mostly mechanical (Fig. 4b, Table 2). Average BB basal diameter was not significantly different between the sexes (t = 1.22, df = 58, p = 0.227); while the mean BB length was significantly (t = 5.54, df = 58, p < 0.001) longer in males than that in females (Table 3).

Table 2

Morphological features and location of the antennal sensilla observed on *Spodoptera litura* adult moths
Sensilla types	Shape	Wall surface	Pores	Locations*
Böhm's bristles, BB	Thorn	Smooth	Aporous	S, P
Sensilla trichoidea ST	Slender hair	Helical ridges	Multiporous	Flv
Sensilla basiconica, SB	Small cone	Longitudinal ridges	Multiporous	Flv
Sensilla chaetica, SCh	Upright spine	Striped	Multiporous	Fld, Flv, Fll, Flt
Sensilla coeloconica, SCO	Chrysanthemum-like	Longitudinal ridges	Multiporous	Flv, Flt
Sensilla styloconica, SS	Cylindrical	Intricate ridges	Multiporous	Flv, Flt
Sensilla squamiformia I, SSQ I	Narrow willow leaf	Longitudinal grooves	Aporous	Fld
Sensilla squamiformia II, SSQ II	Spindle	Vertical stripes	Aporous	S, P
Sensilla auricillica I, SAU I	Rabbit ear-like	Longitudinal ridges	Multiporous	Flv
Sensilla auricillica II, SAU II	Enlarged ears-like	Smooth	Aporous	Flv
* S = scape, P = pedicel, Fld = dorsal side of flagellomeres, Flv = ventral side of flagellomeres, Fll = lateral side of flagellomeres, Flt = terminal flagellomere.

Table 3

Mean length and basal diameter of the antennal sensilla present on adult female and male *Spodoptera litura* moths
Sensilla types	Mean lengths (µm)		Mean basal diameters (µm)
Sensilla types	Female	Male	Female	Male
Böhm's bristles, BB	16.77 ± 0.36 b	22.72 ± 0.62 a	2.18 ± 0.05 a	2.28 ± 0.53 a
Sensilla trichoidea ST	67.17 ± 1.12 b	81.60 ± 3.00 a	2.98 ± 0.08 b	3.76 ± 0.11 a
Sensilla basiconica, SB	12.46 ± 0.51 a	13.42 ± 0.54 a	2.38 ± 0.47 b	2.60 ± 0.08 a
Sensilla Chaetica, Central, CSC	74.34 ± 1.02 a3	75.65 ± 1.63 a2	4.54 ± 0.08 b2	5.17 ± 0.17 a2
Sensilla Chaetica, Dorsal, DSC	79.62 ± 1.44 a2	79.86 ± 1.09 a2	4.22 ± 0.99 b2	4.78 ± 0.11 a2
Sensilla Chaetica, Lateral, LSC	87.05 ± 1.29 b1	102.49 ± 2.38 a1	5.36 ± 0.12 b1	6.77 ± 0.14 a1
Sensilla coeloconica, SCO	NA*	NA	9.84 ± 0.19 b	10.68 ± 0.97 a
Sensilla styloconica, SS	21.83 ± 0.35 a	22.55 ± 0.40 a	6.20 ± 1.15 a	6.28 ± 0.19 a
Sensilla squamiformia I, SSQ I	49.37 ± 0.51 b	53.32 ± 0.68 a	2.07 ± 0.05 a	2.21 ± 0.05 a
Sensilla squamiformia II, SSQ II	66.27 ± 2.25 a	70.81 ± 1.96 a	2.01 ± 0.07 a	2.13 ± 0.06 a
Sensilla auricillica I, SAU I	14.13 ± 0.74 a	14.47 ± 0.75 a	2.60 ± 0.11 a	2.86 ± 0.12 a
Sensilla auricillica II, SAU II	12.18 ± 0.17 a	12.37 ± 0.29 a	NA*	NA
Values comparing sexes within a row for either length or basal diameter and followed by different letters were significantly different (paired t-test, p < 0.05). Mean values within a column for CSC, DSC and LSC followed by the same letter were not significantly different (ANOVA and Tukey mean separation test, p < 0.05). * NA = Not available. The mean lengths of SCO and basal diameters of SAU II were not measured.

Numerous microtrichia were observed around BB (Fig. 4b). The microtrichia did not have structures or a socket-like depression at their base, were angled with the surface at approximately 90 degrees, and were shorter and much slender compared to BB (Fig. 4b).

Sensilla trichoidea

Sensilla trichoidea (ST) were the most abundant sensilla type found on the antennae of S. litura, accounting for approximately 70% of all sensilla found. ST were primarily located on the ventral side of the flagellum (Figs. 3a, 5a). ST gradually tapered from base to tip, ending with a rounded and blunt apex (Fig. 5b). The ST angled away from the antenna surface and had helical ridges along their length (Fig. 5a, b, Table 2). Average ST length (t = 5.75, df = 58, p < 0.001) and basal diameter (t = 5.50, df = 58, p < 0.001) were significantly larger in males than females (Table 3).

Sensilla basiconica

Sensilla basiconica (SB) were the second most abundant sensilla type, being located on the ventral side of the flagellum, with 5–12 SB per subsegment. The SB were widest at their base, short, and with a slender and blunt tip (Fig. 6a, b). The SB surface was characterized by longitudinal ridges with numerous pores between ridges (Fig. 6b, Table 2). SB are often regarded as chemical sensilla (Chapman, 1982; Nation, 2001; Schneider, 1964). Average SB length did not differ significantly between the sexes (t = 1.30, df = 58, p = 0.201); however, average SB basal diameter was greater in males than females (t = 2.40, df = 58, p = 0.021) (Table 3).

Sensilla chaetica

Sensilla chaetica (SCh) resembled an upright spine, with its base arising from a socket-like depression, then tapering slightly and ending with a blunt, rounded, multiporous tip (Fig. 7a, b) (Table 2). SCh were distributed on all subsegments of the flagellum, with slightly longer SCh being located near the flagellum tip. There were 6 SCh per subsegment near the middle of flagellum in both females and males (n = 15 females and 15 males). Pores were clearly visible at the SCh tip (Fig. 7c, d). Based on their location around each subsegment, SCh are often classified as lateral sensilla chaetica (LSC), central sensilla chaetica (CSC), and dorsal sensilla chaetica (DSC) (Fig. 3a, b; Table 3) (Gargi et al. 2022; Li et al. 2024). Average length (F = 65.596, df = 2, p < 0.001) and basal diameter (F = 54.818, df = 2, p < 0.001) differed significantly among LSC, CSC and DSC in males. For example, in males, average LSC length was significantly longer than either CSC or DSC, with no significant difference found between CSC and DSC) (Table 3). Similarly, average LSC basal diameter was significantly thicker than either CSC or DSC, with no significant difference found between CSC and DSC. In females, average LSC length was significantly longer than either DSC or CSC, and DSC were longer than CSC (Table 3; F = 25.697, df = 2, p < 0.001). Likewise, in females, average LSC basal diameter was significantly thicker than both CSC and DSC, with no significant difference found between CSC and DSC (Table 3; F = 35.266, df = 2, p < 0.001).

Sensilla coeloconica

Sensilla coeloconica (SCO) were the most complex sensilla found in S. litura, consisting of a central cone (CP), surrounded by multiple spines (SP) (Fig. 8a). The central cone (CP), was conical in shape with longitudinal ridges on its surface and a pore at the tip, and was surrounded by approximately 12–15 spines (Fig. 8a, Table 2). The spines (SP) had longitudinal ridges and were significantly longer than the central cone, bending inward from their base and forming a dome-like structure that enveloped the central cone, similar to a chrysanthemum flower (Fig. 8a). Two to eight SCO were present on the ventral side of each flagellum subsegment and at the terminal end (Table 2). The size of SCO varied significantly between the sexes, with the basal diameter being significantly larger in males than females (t = 3.90, df = 58, p < 0.001) (Table 2).

Sensilla styloconica

Sensilla styloconica (SS) were present at the distal end of each subsegment, usually one per subsegment along the ventral side (Figs. 3a; 8b). The SS were cylindrical in shape, with 1–3 blunt protrusions at the tip that were angled at about 45 degrees to the surface (Fig. 8b, c, d; Table 2). SS with three blunt protrusions were found only in the terminal subsegments, while SS with 1–2 blunt protrusions appeared in the other subsegments. Several small pores were irregularly distributed on the surface of SS (Fig. 8d). There were no significant differences between sexes in average SS length (t = 1.35, df = 58, p = 0.181) or basal diameter (t = 0.33, df = 58, p = 0.745) (Table 3).

Sensilla squamiformia I

Sensilla squamiformia I (SSQ I) were found in all subsegments of the flagellum, located in socket-like depressions that appeared to be connected with the antenna through the membrane within the depression. SSQ I were widest near their center, gradually narrowing towards the base and tip (Fig. 9a, b). The SSQ I surface had a serrated longitudinal ridge structure (Fig. 9a). No pores were detected on SSQ I (Fig. 9a, Table 2). There were 3–5 SSQ I on each subsegment of the flagellum, primarily on the dorsal side (Fig. 9b). The base of each SSQ I was covered by scales, with just the upper part appearing above the scale surface. The average length of SSQ I was significantly longer in males than females (t = 4.68, df = 58, p < 0.001), while average basal diameters did not differ significantly between the sexes (t = 1.92, df = 58, p = 0.06).

Sensilla squamiformia II

Sensilla squamiformia II (SSQ II) were another subtype of sensilla detected on S. litura (Fig. 9c). SSQ II were similar in morphology to SSQ I, but different in their distribution and size. SSQ II were abundant and distributed over the entire scape but found only sparsely on the lateral sides of the pedicel; whereas, SSQ I were present only on the subsegments of the flagellum (Fig. 9d) (Table 2). In addition, SSQ II had longitudinal ridges similar to SSQ I, but the SSQ II were much slender with a slightly curved tip (Fig. 9a, c; Table 2). Moreover, the average length of SSQ II was significantly longer than SSQ I in both males (t = 9.907, df = 47, p < 0.001) and females (t = 8.935, df = 47, p < 0.001) (Table 3). However, there were no significant differences between the sexes regarding SSQ II average length (t = 1.52, df = 36, p = 0.137) or average basal diameter (t = 1.27, df = 36, p = 0.211).

Sensilla auricillica I

Sensilla auricillica I (SAU I) rose from socket-like depressions and had the shape of a rabbit’s ear (Fig. 10a). There were pores on the SAU I surface. Average SAU I length (t = 1.66, df = 58, p = 0.103) and basal diameter (t = 0.33, df = 58, p = 0.743) did not differ significantly between the sexes (Table 3). There was a large depression in these sensilla that gradually narrowed toward the tip (Fig. 10a). There were longitudinal ridges along the length of these sensilla (Fig. 10a). The pores on SAU I were scattered sparsely on the ventral side of the flagellum (Fig. 10a, Table 2).

Sensilla auricillica II

Sensilla auricillica II (SAU II) showed obvious differences in morphology and distribution from SAU I (Fig. 10; Table 2). The SAU II had an overall tongue-like shape, with its tip being only slightly narrower than its central region, and having 3 small protrusions at the tip (Fig. 10b). SAU II had some longitudinal ridges on their surface but they were not as prominent as on SAU I (Fig. 10b). SAU II were present in all subsegments of the flagellum, near the junction of the ventral and lateral surfaces, and usually with 1–4 SAU II being found in each subsegment. Average SAU II length did not differ significantly between the sexes (t = 0.57, df = 58, p = 0.569) (Table 3).

Antennal sensilla are important sensory organs for insects, allowing them to receive and perceive external information, locate host plants, avoid predators, and seek mates (Schneider 1964; Chapman 1982; Nation 2001). The needs of insects to perceive these various types of external information promoted the evolution of the complex variety of insect antennal sensilla types (Schneider 1964; Chapman 1982; Li et al. 2024). Identifying the types of the antennal sensilla on a given insect facilitates our understanding of the mechanisms by which an insect can sense environmental stimuli, as well as possibly contributing to the development of innovative odor attractants, which can be used in insect pest control (Malo et al. 2004; Ansebo et al. 2005).

Prior to the present study, six types of the antennal sensilla had been recorded in Spodoptera litura, namely sensilla trichoidea, sensilla basiconica, sensilla chaetica, sensilla coeloconica, sensilla styloconica, and sensilla auricillica (Liu et al. 2009; Aruna et al. 2019). In the current study, we identified a total of eight antennal sensilla and two subtypes, of which sensilla squamiformia II, and sensilla auricillica II were recorded for the first time.

From an evolutionary perspective, the types of antennal sensilla present on a given insect species are often similar, but not necessarily the same, as those found on closely related species (Schneider, 1964). In the moth family Noctuidae, nine types of antennal sensilla have been recorded to date: Böhm's bristles, sensilla trichoidea, sensilla chaetica, sensilla coeloconica, sensilla styloconica, sensilla squamiformia, sensilla auricillica, sensilla basiconica, and uniporous peg sensilla. These types of antennal sensilla have been observed in Athetis lepigone (Noctuidae) (Hu et al. 2021), Copitarsia consueta (Noctuidae) (Castrejón-Gómez et al. 1999), Helicoverpa armigera (Noctuidae) (Rani et al. 2021a), H. assulta (Noctuidae) (Koh et al. 1995), Mythimna separata (Noctuidae) (Chang et al. 2015), S. frugiperda (Noctuidae) (Malo et al. 2004; Li et al. 2024) and S. littoralis (Seada 2015). The antennal sensilla recorded in the current study were shared with many of the above noctuids, except for sensilla squamiformia II and sensilla auricillica II. This similarity in the antennal sensilla types among most several noctuids indicates a common evolutionary origin and similar requirements for receiving and perceiving external information.

The sensilla and scales are the most obvious features on the antennal surface in S. litura. The antennal sensilla are primarily distributed on the ventral side of the flagellum, with the scales being the most widespread and abundant non-sensory structures present, being found on the scape, pedicel, and dorsal side of the flagellum. This distribution pattern of sensilla and scales in the antenna of S. litura is consistent with many other Noctuidae (Koh et al. 1995; Chang et al. 2015; Roh et al. 2016; Yang et al. 2017; Zhang et al. 2019; Hu et al. 2021; Rani et al. 2021a). Koh et al. (1995) proposed that the arrangement of scales helped prevent mechanical damage to the antenna and the antennal sensilla. Others have suggested that the scales could help capture and concentrate odors, thus enhancing the insect’s ability to perceive and accurately locate the odor (Van der Pers et al. 1980; Castrejón-Gómez et al. 1999).

In terms of function, the antennal sensilla of S. litura can be classified into three categories: mechanical sensilla, chemical sensilla, and temperature and humidity sensilla. Both Böhm's bristles and sensilla squamiformia are considered mechanical sensilla, responsible for detecting mechanical stimuli derived from the external environment (Schneider 1964; Kristensen 2003). Böhm's bristles are mainly found on the intersegmental membranes between the scape and pedicel, allowing for the orientation of the antenna to be adjusted (Koh et al. 1995; Castrejón-Gómez et al. 1999). Numerous microtrichia were also observed around Böhm’s bristles (Fig. 4b) and are generally considered to have no sensory function (Hu et al. 2021; Rani et al. 2021a).

Sensilla squamiformia, another mechanical sensilla, has been widely recorded in species of Noctuidae, such as A. lepigone (Hu et al. 2021), H. armigera (Rani et al. 2021a), M. separata (Chang et al. 2015), S. frugiperda (Malo et al. 2004; Li et al. 2024), and S. littoralis (Seada 2015). Because sensilla squamiformia are in sockets with no surface pores, some researchers suggested that these sensilla may sense air vibrations rather than perceive chemical molecules (Steinbrecht 1997; Bawin et al. 2017). Sensilla squamiformia I and sensilla squamiformia II were similar in shape but were significantly different in size in the present study. Considering that sensilla squamiformia I and II were located on different parts of the antenna (Table 2, Fig. 9b, d), we classified sensilla squamiformia II as a separate subtype of sensilla squamiformia.

The chemosensory antennal sensilla identified in S. litura include sensilla trichoidea, sensilla basiconica, sensilla chaetica, sensilla coeloconica, and sensilla auricillica. Pores on the surface of the antennal sensilla are the key feature that defines these sensilla as having a chemosensory function, given that they serve as channels for external chemicals to enter and interact with internal sensory receptors (Schneider 1964; Zacharuk 1980).

Sensilla trichoidea are one of the most common antennal sensilla types found in Lepidoptera (Malo et al. 2004; Seada 2015; Roh et al. 2016; Zhang et al. 2019; Rani et al. 2021b). Sensilla trichoidea can perceive sex pheromones and plant odors, indicating that these sensilla are essential for insects to locate mates and their host plants (Seada 2015; Roh et al. 2016; Yang et al. 2017; Zhang et al. 2019; Rani et al. 2021a). Pores on the surface of sensilla trichoidea have been recorded in several moths species such as Cydia pomonella (Tortricidae) (Roh et al. 2016), C. succedana (Tortricidae) (Roh et al. 2016), Diaphania angustalis (Crambidae) (Zhang et al. 2019), Dioryctria rubella (Pyralidae) (Xu et al. 2021), H. armigera (Rani et al. 2021a), M. separata (Chang et al. 2015), and S. littoralis (Seada 2015). In our study, numerous pores were observed on the surface of sensilla trichoidea (Fig. 5c), indicating that they likely serve as chemosensory organs.

Sensilla basiconica have been recorded in almost all Lepidoptera with filiform antennae, and their distribution pattern on the antennal surface is generally the same in all Noctuidae studied to date (Liu and Liu 1984; Chang et al. 2015; Rani et al. 2021a). The surface of sensilla basiconica is characterized by numerous small pores and grooves, suggesting that these sensilla have an olfactory function such as pheromone and plant odor detection (Liu and Liu 1984; Chang et al. 2015; Zhang et al. 2019; Rani et al. 2021a). Koh et al. (1995) reported that the sensilla basiconica of H. assulta are porous, fluted, and thin-walled, and have a role in odor detection.

Sensilla chaetica are very common in the antennae of the noctuid moths, being found in each subsegment of the flagellum (Malo et al. 2004; Seada 2015; Hu et al. 2021). These sensilla had the widest distribution on the antennae of S. litura compared to the others types found, being present on the dorsal, lateral and ventral sides of the flagellum. In some studies, sensilla chaetica are referred to as central chaetica, lateral chaetica, and dorsal chaetica according to their locations on the surface of the flagellum (Malo et al. 2004; Gargi et al. 2022; Li et al. 2024). This naming method was adopted in our study. The size, shape and distribution of sensilla chaetica are similar among all Noctuidae studied to date, suggesting they have a similar perception function among species (Chang et al. 2015; Yuan et al. 2017; Hu et al. 2021; Rani et al. 2021b). In several studies of Lepidoptera, sensilla chaetica have been the longest sensilla detected (Faucheux 1991; Chang et al. 2015; Rani et al. 2021a, 2021b; Li et al. 2024). Some authors have proposed that sensilla chaetica help protect other sensilla on the same antenna (Chang et al. 2015; Rani et al. 2021a), while others have suggested that they may have a taste function in addition to perceiving odors (Popescu et al. 2013; Seada 2015; Bawin et al. 2017; Li et al. 2024).

Sensilla coeloconica exhibit a distinctive morphology among the many types of antennal sensilla of Lepidoptera, characterized by a central cone surrounded numerous spines (Flower and Helson 1974; Bawin et al. 2017; Rani et al. 2021a). Sensilla coeloconica have been mostly found on the ventral surface of the flagellum, but their number and shape vary among moth species (Malo et al. 2004; Chang et al. 2015; Seada 2015; Rani et al. 2021b). For example, sensilla coeloconica of S. frugiperda have a distinct petal-like structure that encloses the sensilla in a cavity, and the upper part of the spines bends more inward to form a distinct dome-like structure (Li et al. 2024). Many authors have suggested that the surrounding spines serve to protect the central cone from mechanical damage (Yang et al. 2009; Rani et al. 2021a). In addition, Binyameen et al. (2012) suggested that sensilla coeloconica have an olfactory function in S. littorali. Moreover, in several recent studies, sensilla coeloconica were considered multifunctional, i.e., capable of perceiving temperature, humidity, water vapor, pheromones, and plant odors (Seada 2015; Yang et al. 2017; Zhang et al. 2019; Rani et al. 2021a, 2021b).

Sensilla auricillica have been widely reported in Lepidoptera, including Earias vittella (Nolidae) (Rani et al. 2021b), Athetis lepigone (Hu et al. 2021), and Cydia succedana (Tortricidae) (Roh et al. 2016), and S. frugiperda (Li et al. 2024). Two subtypes of sensilla auricillica were observed in our study of S. litura. These two subtypes have been also recorded in the tortricids C. pomonella and C. succedana (Roh et al. 2016). Sensilla auricillica are porous sensilla and considered involved in detection of host volatiles and pheromones (Faucheux 2006). Hu et al. (2021) also proposed that sensilla auricillica had an olfactory function in A. lepigone. Electrophysiological studies have demonstrated that sensilla auricillica of the tortricid C. pomonella can respond to phytochemicals and sex pheromones (Ansebo et al. 2005).

Sensilla styloconica are commonly found on the filiform antennae of Lepidoptera (Koh et al. 1995; Castrejón-Gómez et al. 1999; Malo et al. 2004; Chang et al. 2015; Seada 2015; Aruna et al. 2019; Rani et al. 2021a; Li et al. 2024;). Sensilla styloconica have usually been found on the ventral distal portion of each subsegment of the flagellum. Overall, the number, morphological characteristics and distribution of sensilla styloconica are similar in the Noctuidae (Gómez et al. 1999; Malo et al. 2004; Castrejón- Chang et al. 2015; Seada 2015; Aruna et al. 2019; Rani et al. 2021a; Gargi et al. 2022; Li et al. 2024). The blunt protrusions at the tip of sensilla styloconica are a distinguishing feature of this sensilla type. The number of blunt protrusions in S. litura varied from one to three in our study, which was also consistent with the report by Aruna et al. (2019). A similar pattern has been reported in other Noctiudae (Koh et al. 1995; Castrejón-Gómez et al. 1999; Malo et al. 2004; Chang et al. 2015; Hu et al. 2021; Rani et al. 2021a; Li et al. 2024). Based on the number of the blunt protrusions, Rani et al. (2021a) and Zheng et al. (2014) classified these sensilla into two subtypes, but all other authors did not use the number of blunt protrusions as a morphological feature to assign subtype classification (Koh et al. 1995; Castrejón-Gómez et al. 1999; Malo et al. 2004; Galizia and Rössler 2010; Zheng et al. 2014; Chang et al. 2015; Seada 2015; Aruna et al. 2019; Hu et al. 2021; Rani et al. 2021a; Li et al. 2024). Although we noted variation in the number of blunt protrusions, we did not classify sensilla styloconica into subtypes.

Sensilla styloconica are often categorized as humidity and temperature sensing sensilla, given that no pores are generally observed on their surface (Roh et al. 2016; Yang et al. 2017; Rani et al. 2021b). However, pores have been recorded on sensilla styloconica in the noctuid M. separata, suggesting that these sensilla may perceive chemical stimuli (Chang et al. 2015). In our study of S. litura, we observed pores irregularly distributed on the surface of sensilla styloconica (Fig. 8d). Such observations suggest that there may be multiple types of sensilla styloconica, some of which can perceive only humidity and temperature, and others that can perceive chemical stimuli as well.

Our study demonstrated that Spodoptera litura possess eight types and two subtypes of the antennal sensilla: Böhm's bristles, sensilla trichoidea, sensilla basiconica, sensilla chaetica, sensilla coeloconica, sensilla styloconica, sensilla squamiformia (I and II), and sensilla auricillica (I and II). Sensilla squamiformia II and sensilla auricillica II were reported for the first time. Detailed low-voltage field emission scanning electron microscope images and descriptions were provided for each sensillum type. This study provided the morphological information to aid in future neurophysiological testing of the antennal sensilla of S. litura.

Author Contributions Conceptualization: Y-P. Li, J. Cao, H. Ye; Methodology: H-Y. Zhou, Q-L.Hu, M-M. Jiang, Y-Yang, Y-P. Li; Investigation: H-Y. Zhou, Q-L.Hu, M-M. Jiang; Writing—original draft preparation: Y-P. Li, H. Ye, R.A. Haack, H-Y. Zhou; Writing—review and editing, Y-P. Li, H. Ye, R. A. Haack; Funding acquisition: H. Ye; J. Cao; Supervision: J. Cao; Project administration: J. Cao; All authors have read and agreed to the published version of the manuscript. All authors have read and agreed to the published version of the manuscript.”

Funding This research was funded by the National Natural Science Foundation of China, Grant No. 32271563, 32471568; Science and Technology Planning Project in Key Areas of Yunnan Province, Grant No. 202001BB050002; Science and Technology Program of Yunnan Province, Grant No.202401AS070151.

Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests The authors declare no competing interests.

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Morphological characteristics and distribution of antennal sensilla of Spodoptera litura (Lepidoptera: Noctuidae) using scanning electron microscopy

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Abstract

Figures

Introduction

Materials and Methods

Sampling insects

Sample preparation and SEM observation

Terminology, measurements and statistical analysis

Results

The basic morphology of the antennae

Types of antennal sensilla

Böhm's bristles

Sensilla trichoidea

Sensilla basiconica

Sensilla chaetica

Sensilla coeloconica

Sensilla styloconica

Sensilla squamiformia I

Sensilla squamiformia II

Sensilla auricillica I

Sensilla auricillica II

Discussion

Conclusion

Declarations

References

Additional Declarations

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