The comparative study on adult surface ultrastructure of Harmonia axyridis Pallas ( Coleoptera : Coccinellidae )

Scanning electron microscopy was used to line scan 7 positions (pronotum, scutellum, elytra, abdominal sternites, head, maxillary palpus and labial palpus) of 11 Harmonia axyridis Pallas with different color patterns to make up for the lack of general morphological observation, and at the same time it was also used to further study the relationship of ultrastructure and predatory behavior of Harmonia axyridis Pallas to establish the important research foundation. The results show that the hair on the body of the elytra of the different H. axyridis directly stretches out from the surface of the hole and only few hairs on the body of the elytra stretched out from the concave surface of the elytra. Moreover, the hollow on the elytra edge increased significantly, though no two body hairs were produced on one hollow, and the granular material was borne on the first baseline web splices. It was observed that the hair densities of pronotum, scutellum, elytra, and abdominal sternites from the 11 types of H. axyridis Pallas showed some diversity. Likewise, the number of sensilla from the head, abdominal sternites, maxillary palpus, and labial palpus showed some differences. These findings highlight the fact that ultrastructure supplement was provided to the morphological aspect of H. axyridis.


INTRODUCTION
Harmonia axyridis Pallas, also called Ptychanatis axyridis or Leis axyridis, belongs to the Coleoptera Coccinellidae Synonychini Harmonia, which is also called Leis.This is important natural enemy of various pests, especially of arboreal aphids and scales.We had controlled Matsucoccus matsumurae (Kuwana), which damaged seriously Pinus spp.and caused major economic losses with H. axyridis Pallas in China and had achieved the desired effect (Liu, 1963;Pang et al., 2004).This insect has received increasing attention and it is studied thoroughly by geneticists because of its changing color patterns (Dobzhansky, 1924(Dobzhansky, , 1933;;Tan, 1946;Komai et al.. 1950;Geng and Tan, 1980).The argument regarding the difference between H. axyridis Pallas and Harmonia yedoensis (Takizawa) is manifested mainly on the ridge.Having a horizontal ridge before the end of the elytra is an important feature that distinguishes the H. axyridis Pallas from other members of the genera.In China, most H. axyridis Pallas have a horizontal ridge on the elytra proximal department.It had been pointed out that those without the ridge are H. yedoensis whereas those with the ridge are H. axyridis Pallas (Pang, 1984).The presence or absence of the ridge on the elytra end of H. axyridis Pallas from different regions is thought to be a geographical variation (Liu, 1963).However, other geneticists think that the presence of the ridge is controlled by Mendelian inheritance, which states that the one with the ridge is dominant whereas the other without the ridge is recessive, and that they are all H. axyridis Pallas (Liu, 1963;Geng and Tan, 1980).Mader (1932) classified H. axyridis Pallas into 93 varieties, whereas Korshefsky divided them into 105 varieties in the category list that he edited (Korshefsky, 1932).In China, H. axyridis Pallas beetles with black background are divided into 2 categories with 62 patterns whereas those with yellow background are divided into 11 categories with 33 patterns (Tan andLi, 1932-1933).
The varieties amounted to 95 patterns in all (Tan andLi 1932-1933).Research on H. axyridis Pallas has currently become very popular.The research done by Chinese scholars focused on finding and naming the color patterns of H. axyridis Pallas.Luo (1960) reported that the number of colors and patterns on the elytra of H. axyridis Pallas found in Shenyang and Gongzhuling was more than 50.The 1393 H. axyridis Pallas collected in Huairou and Yanqing Counties in Beijing were identified and classified into 50 varieties (Wu, 1987).Yuan et al. (1994) not only reported 176 varieties found in Changbai Mountain, but they also named each variant of H. axyridis Pallas with yellow background by spot serial numbers and mark nomenclature.
The high resolution and 3D image of the scanning electron microscopy (SEM) clearly shows the subtle structures on the body, which are difficult to observe and describe using general morphological means.The SEM was used to observe the fine surface structures of H. axyridis Pallas.This insect is easy to capture and the artificial feeding is simple, thus observing basic biological phenomena like mating, oviposition, hatching and larvae growth, molting, pupation and others is easy.Not only has smooth testing provided certain theoretical and practical evidence for classification research and use of the different color patterns of H. axyridis Pallas, but it can also be used to complement shape classification research.In addition, as a pleomorphic insect, H. axyridis Pallas is a very important experimental subject in genetics, molecular systematics, and molecular biology research, and has profound significance in basic theoretical exploration.

Insects
The materials used in the experiment were collected from the MaoEr Mountain experiment forest of the Northeast Forestry University.Eleven different color patterns of H. axyridis Pallas were tested according to the classification criteria of Yuan et al. (1994) as shown in Table 1.
One male H. axyridis Pallas was chosen from each color pattern.It was first washed with ddH2O, then 0.4 kg/L NaOH solution, and then 0.2 kg/L NaCl solution.The head, pronotum, scutellum, elytra, abdominal sternites, maxillary palpus, and labial palpus were removed using tweezers under the SMZ-140.These parts were ultrasonically cleaned in 70% ethanol for 10 min, then dehydrated in glutaraldehyde (2.5%) (v/v), and fixed for 12 h.After fixation, the specimens were immersed thrice in phosphoric acid buffer (pH = 7.4) for 20 min.After 3 to 4 times of cleaning with ddH2O, the specimens were dehydrated using 30, 50, 70, and 90% ethanol for 10 min at each concentration.They were finally dewatered for 20 min with 100% ethanol.After dehydration, the specimens were then cleaned for 20 min in acetone and were left to air dry (Chi et al., 2009).The samples to be scanned were attached to the SEM stage with conductive adhesive, sprayed with gold, and then observed using SEM (QUANTA FEI-200) at an accelerating voltage of 10 kV.The number of hairs of each specimen was observed and the differences between the color patterns of the H. axyridis Pallas were compared according to the area statistics.

Pronotum
There were many "dots" on the surface of the pronotum of H. axyridis Pallas.The results on morphological classification showed that the size and density of the dots are characteristics for morphological classification.There are slender hairy structures with about 60 to 100 μm in the lower part of the leading edge between the pronotum and head capsule.Considering the behavior of ladybirds, this kind of structure can avoid friction between the head capsule and pronotum and acts as a buffer agent when there is prey (Figure 1).There were 2 or 3 rows of structures similar to the styloconic sensillum on the edge of the pronotum (Figure 2).The number of "dot" (body hair) of the different color patterns of H. axyridis Pallas were statistically analyzed in the 300 μm × 300 μm area selected from the pronotum center (front, middle, trailing edge) to the pronotum edge (upper, lower) (Table 2).The density of the "dot" in the pronotum edge was more than the density in the pronotum center, and the density of the "dot" in pronotum of H. axyridis Pallas on a yellow background was higher than in H. axyridis Pallas with black background.Among the samples, the "dot" density in the pronotum of yellow background H. axyridis Pallas without spot was maximal, whereas the "dot" density in the pronotum of yellow background H. axyridis Pallas with 18 spots was minimal.

Scutellum
Scutellum is the inverted triangle with the bottom edge slightly longer than the side.The scutellum is located below the pronotum and between the two elytra.There is only one kind of body hair on the scutellum, those that stretched out of the holes directly.The body hair in the center of the scutellum is uniformly distributed, whereas the distribution is denser on the sides that connect the elytra (Figures 3 and 4).As shown in Table 3, under SEM, the number of microhairs (dots) on the small scutellum of the band of variant type was highest, the number of microhairs (dots) on the scutellum of the species with 4 spots on a black background was the least ( 22), and the number of other types of micro-hairs (dot) was between 25 to 33 n.The ratio of the bottom length and the side length was highest in the scutellum of the sample with 16 spots on a yellow background and the ratio was least for the sample with 2 spots on a yellow background.

Elytra
In the preparation of the elytra of the scanning specimens, the necessary parts to be observed were divided into six: upper left, upper right, left, right, lower left, and lower right (Figure 5).The elytra structure was divided into two kinds.One is the micro-hair with a length of 8 μm, which was developed in the depression structure and located in the center (Figures 6 and 7).The other kind is the salient spot.The top of the salient spot mushroomed.There were obvious boundaries on the surrounding epidermal tissue.This was a kind of epidermal salient structure that may be related with the secretion of pheromones of the epidermal tissue (Figure 8).The depressed structure on the edge of the elytra was bigger (Figure 9).The number of "dots" in a 300 μm × 300 μm area on the six parts of the elytra (upper left, upper right, middle left, middle right, lower left, and lower right) is shown in Table 4.The density of the left "dot" (body hair) on the elytra was larger than the right.The density of the "dot" on the upper right of the elytra was the highest, whereas the density of the "dot" on the right was the least.The density of the "dots" on the elytra of H. axyridis Pallas with black background was higher than that of the species with the yellow background.Also, the number of the "dot" on the elytra of the eye spot with black background was the largest.The number of "dots" on the elytra of name repetition of variant species was

Abdominal sternites
The abdominal sternites of H. axyridis Pallas is composed of six sections.Sections 5 and 6 are the most important abdominal sternitess for classification and are often used as a reference feature to differentiate between males and females.The arc on the edge of abdominal sternites 5 and the central semicircle on the edge of abdominal sternites 6 were concave for males.The arcs on the edge of abdominal sternites 5 and on the middle of abdominal sternites 6 were salient for females.Under SEM, there were dense micro-hairs on the surface of the abdominal sternites perforation (Figures 10 and 12).The microhairs on the edges of the abdominal sternites were slightly longer, and when the internal microhairs were short, they were not obvious.In the intermediate department of salience on the first abdominal sternites, some small concave nests replaced the microhairs (Figure 11).The granular material was also observed using SEM.It could be secretion based on its shape and arrangement (Figure 13).It was found that there were no granular secretions on the surface of the microstructure of the abdominal sternites of female H. axyridis Pallas and the micro-hairs were thick types (Figure 14); there were granular structures on the surfaces of the abdominal sternites of the male H. axyridis Pallas (Figure 15).

Head
The head of the H. axyridis Pallas often retracted below the pronotum.Therefore, the front-end head can only be seen from the back of the insects.There is a pair of compound eyes, a pair of antennae, and a pair of maxillary and labial palpi on the head.The sensor type Li et al.

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Table 5.The density of dot on the head.

Variant
The on the head is one fold, predominantly sensilla chaetica (Figure 16).These were mainly distributed in the head front-end and behind the compound eyes (Figure 17), although a few were found on the side of the compound eyes (Figure 18).In addition, micro-hairs were distributed between the compound eyes (Figure 19).The sensilla of the front-end head and the side of the compound eye of the different H. axyridis Pallas are shown in Table 5.The body hair on the front-end head was more than that around the compound eyes.The body hair on the frontend head was the most for the variant with 2 spots on a black background and the least for the variant with 2 spots on a yellow background.The number of body hairs around compound eye was 5 to 8, whereas it was 12 to 42 on the front-end head.

Maxillary palpus
The maxillary palpi were divided into four sections in an adult H. axyridis Pallas.Ax-shaped, the paratelum is flourishing and flat (Figure 20).There is also a longitudinal groove on top.The width is also different from front to back, with both ends slightly wider while the central part is slightly narrower.There are spine-like protuberances on the groove (Figure 21).Similar to the antenna, the dorsal and ventral sides of the maxillary palpus are not symmetrical.There are also a larger number of ventral sensilla.Three sensors were observed in this study: sensilla chaetica (ch), sensilla trichodea (tr), and styloconic sensillum (st) (Yan et al., 1987).In addition, some special structures were discovered in the paratelum (Figure 25).
(3) Styloconic sensilla (st): located on the paratelum top and the groove and was about 1 to 2 μm in length (Figure 24).
The statistical analysis was done on the maxillary palpus sensilla chaetica (ch), sensilla trichodea (tr), and the special structure (Table 6).The number of sensilla in the band of the variant types with 2 spots on a black background, and 16 spots on a yellow background were larger, whereas the number of sensilla in name repetition of variant species, bimonthly spot on a black background, 19 spots on a yellow background, eyespot on a black background was fewer.The difference between the numbers of sensilla chaetica was about 20, whereas the difference in sensilla trichodea was much lower.The number of unknown structures for the 2 spots on a black background, 2 spots on a yellow background, and eye spot on a black background was more (8 to 9), whereas for the bands in the variant type with 16 spots on a yellow background number is less (3 to 4).The styloconic sensillum was not included in the results because of its large number and distribution.
(1) Sensilla chaetica: about 110 to 120 μm in length.There were 3 to 4 sensilla chaetica in the second section located on the abdominal side with an approximately linear arrangement.There was only one in the third section, also located in abdomen side (Figures 26 and 29).(2) Sensilla trichodea: divided into two kinds; sensilla trichodea 1 (tr1), about 10 μm in length, mainly located in the second section of the labial palpus and outside the dorsal (Figure 27), and sensilla trichodea 2 (tr2), about 2 μm in length, distributed in the paratelum of the labial palpus (third section).They are both the dorsal and ventral (Figure 28).
(3) Styloconic sensillum: distributed on the surface and top of the third section of the labial palpus, especially on the top.It was reported that it was a kind of taste sensor (Figure 29).
The statistical analysis of the labial palpus, sensilla trichodea 1 and sensilla trichodea 2 are shown in Table 7.There was no significant difference between the structure of the labial palpi and the sensor types among the 11 different color patterns of H. axyridis Pallas.However, there was a slight significant difference between the number of sensilla chaetica and sensilla trichodea.The sensor of the variant with 2 spots on a black background was the most whereas the sensor of the name repetition of variant species distributed was the least.The styloconic sensillum was not included in the 1 2 4 3                     results because of its large number.

DISCUSSION
The magnifier, dissecting microscope, optical microscope, and other tools combined with the general classification of morphological and anatomical structures, both domestic and foreign, were used to study H. axyridis Pallas.Tan (1946) thought that the coleopteran colors of H. axyridis Pallas are black, khaki, red or orange.After the vivo H. axyridis were made into dried specimens, the color of the elytra turned to yellow from red or orange after a month.This is the same as the phenomenon observed when saving the specimens in the testing Li et al. 5789  process.The color of the elytra turned yellow because of the oxidation in the air or the chemical changes in their body.Either way, it showed that the pigment contained in the yellow, orange, or red elytra is of the same kind of material.Therefore, according to the classification, the elytra are sufficient for classification into two-color types: the black and yellow types.This is one of the bases that we followed in our trial for selecting specimens.At present, there is no documented research on the surface structures of H. axyridis Pallas.We observed that the surface fine structures, the labial and maxillary palpi of H. axyridis Pallas under SEM were consistent with the previous description.There were two hairs in one depression on the edge of the elytra in two ladybirds used in the comparative study of the surface fine structure between H. vigintioctomaculata and H. vigintioctopunctat by Zhang et al. (2002).This result was not found in the scanned observation of H. axyridis Pallas.Regarding the insertion of body hairs in the depression of the elytra, Holloway (1985Holloway ( , 1997) ) had done similar studies with the Anthribidae and Lucanidae families, as well as other insects.They thought that the depth of the grooves and the insertion of the body hairs could be a basis for classification (Holloway, 1985(Holloway, , 1997)).Sensilla trichodea have different functional types; for instance, they are usually responsible for pheromone reception in the antennae of Lepidoptera (Hallberg et al., 1994).Sensilla trichodea are observed in the labial palpi and maxillary palpus of H. axyridis, as for the aggregative habit and research prey of H. axyridis, we assumed that Sensilla trichodea are responsible for their aggregative and predatory behavior, they may be pheromone receptors.
Functionally, sensilla chaetica have been ascribed to mechano-and chemore-ception in the coccinellids (Broeckling and Salom, 2003).The sensilla chaetica of Psylliodes chrysocephala is suggested to be in contact with chemosensilla that responds to chemicals presented in plant surface waxes when it comes in contacts with a leaf having antennae (Isidoro et al., 1998).The sensilla chaetica herein described resembles those of other insects and are abundant in the labial palpi and maxillary palpus of H. axyridis.It is premature to assign any particular function to these sensilla.
There was no obvious difference between the depth of the grooves on the elytra and the position of the body hair in all 11 different variants of H. axyridis Pallas.Therefore, it could not be used as a basis for classification.Whether the depth of the grooves on the elytra and the insertion of the body hair can be used as the basis of classification remains unclear.In addition, under SEM granular material was observed on the male web splices baseline.These materials are likely secreted substances based on their shape and arrangement.Some material similar to granular secretions with uneven sizes and varying shapes were also found.The composition and the role of these secretions are not known hence, further studies are needed.

Figure 1 .
Figure 1.The front edge of pronotum.

Figure 2 .
Figure 2. The back edge of pronotum.

Figure 18 .
Figure 18.The hair around the eyes.

Figure 19 .
Figure 19.The hair interspace the eyes.

Figure 23 .
Figure 23.The groove on the apical segment.

Table 2 .
The density of "dot" on the surface of the pronotum.

Table 3 .
Comparison of the length of each side and number of scutellum on eleven phenotypes.

Table 4 .
The density of dot on the surface of elytra.

Table 6 .
The statistic of the sensilla and structure on the maxillary palpi.

Table 7 .
The statistics of sensilla on the labial palpus.