Ultrastructure of Microplitis croceipes (Cresson) (Braconidae : Hymenoptera) teratocytes

The developing embryo of the braconid, Microplitis croceipes (Braconidae : Hymenoptera), is encased in an extraembryonic serosal membrane. Hatching of the parasitoid within the larva of its habitual host, Hehothis virescens (Noctuidae : Lepidoptera), is initiated about 40 hr after oviposition when held at 25 ± 2°C. At this time, the monolayered serosal membrane begins to dissociate into individual cells (teratocytes). After dissociation, teratocytes become dispersed in the hemolymph of the host. The average number of teratocytes released from each parasitoid embryo is 914 ± 43. Teratocytes average 14.1 ± 2.4 μm in diameter when first released, and reach a maximum average diameter of 68.1 ± 4.6 μm 6 days after liberation. Newly released teratocytes have ovoid nuclei, simple mitochondria and a limited number of profiles of the endoplasmic reticulum, all of which indicate relative metabolic inactivity. The ramified nuclei, extensive endoplasmic reticulum, polymorphic mitochondria and accumulation of glycogen granules and lipid droplets observed in older teratocytes provide circumstantial evidence that protein synthesis is occurring. Within hours after dissociation, microvilli begin to cover the surface of the teratocytes. Anatomical deformation (blebs) that occurred on some older (8-day-old) teratocytes probably resulted from enlargement or expansion of microvilli.     

A study of uptake of radiolabeled host proteins and protein synthesis during development of eggs of the endoparasitoid,Microplitis croceipes (Cresson) (Braconidae)

The uptake of radiolabeled haemolymph and fat body proteins from fourth instar larvae of Heliothis zea (Boddie) by eggs of Microplitis croceipes (Cresson) was examined by SDS-polyacrylamide gel electrophoresis and by autoradiography. None of the ¹²⁵I-labeled haemolymph proteins was detected in eggs exposed to the proteins in vivo. Although several of the proteins were observed in eggs incubated with the labeled proteins in vitro, none of these proteins was degraded or resynthesized into new structural proteins during development of the embryo. Similarly, no significant uptake of labeled fat body proteins by the eggs could be detected in vitro. On the other hand, protein synthesis measured by incorporation of [³⁵S]methionine occurred throughout egg development. Proteins were synthesized at least 1 hr after the egg was deposited into the host. The protein patterns of eggs on one-dimensional SDS gels were complex and ranged in size from less than 18,500 to more than 330,000 mol. wt. The protein band patterns of the newly synthesized proteins showed some qualitative differences at 1–8, 16–32 and 40 hr after egg deposition. We conclude that eggs do not absorb or utilize the host apoproteins (or degradation products) but instead synthesize proteins de novo from free amino acids in the host haemolymph.     

Effects of female wasp accessory secretions, host fat body, and host hemolymph on protein synthesis and egg viability in Microplitis croceipes (Braconidae)

Effects of female wasp reproductive gland secretions, host fat body and hemolymph, and mechanical constriction of the parasitoid egg on protein synthesis were studied in eggs of Microplitis croceipes (Braconidae) dissected from the wasp ovary. Protein synthesis was measured by 35S-methionine incorporation in eggs held in tissue culture medium for 16 h after treatment. Synthesis was stimulated in oocytes obtained from three regions of the ovary (egg tube, reservoir, and calyx) by fat body and venom gland but not by calyx fluid. A combination of fat body, venom gland, and calyx fluid did not enhance the level of synthesis relative to that of fat body or venom gland alone. Host hemolymph inhibited protein synthesis when incubated directly with the dissected eggs but not when the eggs were collected from an artificial oviposition substrate (AOS) containing hemolymph. The inhibitory effect of the hemolymph is thought to be due to the occurrence of melanization. Mechanical constriction did not alter the rate of synthesis, confirming an earlier report that synthesis in newly deposited eggs in ongoing and is not dependent on mechanical activation during the act of oviposition. Mechanisms responsible for sustaining protein synthesis in eggs for 16 h in vitro after their exposure to host hemolymph in the AOSs or fat body and venom gland are not known. Only a small percentage (less than 2%) of dissected ovarial reservoir oocytes that were mechanically constricted and exposed to the venom gland, calyx fluid, and host fat body hatched in vitro. In contrast, an earlier study demonstrated that 38% of eggs oviposited by female wasps into AOSs developed and hatched.     

Embryonic development of an endoparasitoid,Microplitis croceipes (Hymenoptera: Braconidae) in cell line-conditioned media

Summary Embryos of the parasitoidMicropolitis croceipes develop from pregerm band stage to first larval instar in cell culture medium conditioned by a cell line (IPLB-LdFB) derived from fat body from an atypical hostLymantria dispar. However, the percentage of eggs that develop normally to the first larval instar stage is significantly less than for those maintained in IPL-52B medium conditioned with host fat body tissue. Therefore, we examined the capacity of five insect cell lines to promote growth and development of pregerm band eggs in five media, IPL-52B, TC-199, TC-100, Grace’s, and ExCell 400. The developmental response ofM. croceipes was dependent both on the cell line and the cell culture medium used. TC-100, TC-199, and Grace’s media promoted development to the germ band stage without the need for conditioning with host tissue. IPL-52B supported development to the germ hand stage when a defined lipid concentrate was added. In IPL-52B medium, the IPLB-LdFB cell line promoted a significantly higher number of eggs developing to germ band relative to the other cell lines; however, none of the cell line-conditioned IPL-52B medium significantly stimulated egg hatch relative to the control medium. None of the cell line-conditioned Grace’s media had a significant effect on eggs attaining germ band stage compared with the Grace’s control medium. However, Grace’s medium conditioned with the IAL-TND1 and IPLB-LdFB cell lines promoted development beyond germ band, resulting in a significantly higher percentage of hatching eggs than the Grace’s control medium. Although the BCIRL-HZ-AMI cell line, which is derived from the parasitoid’s typical host, did not induce hatch in either IPL-52B medium or Grace’s medium, it promoted hatch in TC-199 and Excell 400 media. Fat body taken from the same species that the cell lines were derived from was a better predictor of a cell line’s embryotrophic activity in Grace’s medium rather than in IPL-52B medium. Thus, the composition of the medium and the species and tissue type of the cell line source must be evaluated interactively to determine optimal conditions for promoting development ofM. croceipes in vitro.     

Electroantennogram responses of a parasitic wasp, Microplitis croceipes, to host-related volatile and anthropogenic compounds

The parasitic wasp Microplitis croceipes (Cresson) (Hymenoptera: Braconidae) showed its own characteristic electroantennogram (EAG) response profiles to 13 host‐related (cis‐3‐hexenol, α‐pinene (R)‐(+)‐limonene (S)‐(–)‐limonene, trans‐β‐ocimene (±)‐linalool, (–)‐trans‐caryophyllene, α‐humulene, nerolidol, trans‐nerolidol, cis‐nerolidol, methyl jasmonate and indole) and four anthropogenic (2‐diisopropylaminoethanol, 2,2′‐thiodiethanol, 2‐methyl‐5‐nitroaniline and cyclohexanone) volatile compounds. These profiles were similar between males and females except for 2‐diisopropylaminoethanol, which elicited significantly larger EAG responses in males. Among the compounds tested, cis‐3‐hexenol, linalool and cyclohexanone elicited the largest EAG responses. EAG responses were not influenced by the age of wasps between 1 and 13 days after emergence. EAG responses were dose‐dependent, and highly EAG‐active compounds elicited significant EAG responses with less than 10 μg of the compounds at source. Quantification of compounds released from an odour cartridge indicates that release rate is highly dependent on the chemical nature of stimuli, showing up to 10 000‐fold differences in the amount released between different compounds when the same amount was loaded in the odour cartridge. Wasps having undergone a behavioural training regime to be attracted to either cyclohexanone or methyl jasmonate did not show any differences in EAG responses from those of untrained wasps.     

Computational modeling and experimental validation of odor detection behaviors of classically conditioned parasitic wasp,Microplitis croceipes

A prototype chemical sensor named Wasp hound® that utilizes five classically conditioned parasitoid wasps, Microplitis croceipes (Cresson) (Hymenoptera: Braconidae), to detect volatile odors was successfully implemented in a previous study. To improve the odor‐detecting ability of Wasp Hound®, searching behaviors of an individual wasp in a confined area are studied and modeled through stochastic differential equations in this paper. The wasps are conditioned to 20 mg of coffee when associated with food and subsequently, tested to 5, 10, 20, and 40 mg of coffee. A stochastic model is developed and validated based on three positive behavioral responses (walking, rotation around odor source, and self‐rotation) from conditioned wasps at four different test dosages. The model is capable to reproducing the behaviors of conditioned wasps, and can be used to improve the ability of Wasp Hound® to assess changes in odor concentration. The model simulation results show the behaviors of conditioned wasps are significantly different when tested at different coffee dosages. We conjecture that the searching behaviors of conditioned wasps are based on the temporal and spatial neuron activity of olfactory receptor neurons and glomeruli, which are strongly correlated to the training dosages. The overall results demonstrate the utility of mathematical models for interpreting experimental observations, gaining novel insights into the dynamic behavior of classically conditioned wasps, as well as broadening the practical uses of Wasp Hound. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:596–606, 2015     

Effect of host diet and preflight experience on the flight responses of Microplitis croceipes (Cresson)

Abstract. Female Microplitis croceipes without previous exposure to host diet, feeding damage or faeces, and with no oviposition experience (i.e. inexperienced) exhibited flight tunnel responses towards extracts of faeces from several diet–host species combinations, excluding artificial diet. Inexperienced females exhibited the following preference order towards faeces from Heliothis zea larvae fed selected diets: cotton > wild geranium > cowpea > artificial diet, suggesting and adaptation to 'preferred' host plants.
Females having a preflight experience with a plant-host complex (PHC) prior to testing exhibited a higher percentage of oriented flights in the wind tunnel than did inexperienced females. Oviposition Per se had little effect on the flight response of females. The increased flight response after an experience is apparently due to a combination of sensitization and associative learning. In a choice situation, a single experience was insufficient to establish a preference for the PHC experienced; however, repeated experience resulted in preferences, and the degree of preference tended to increase with the number of experiences.     

Functional morphology of antennal chemoreceptors of the parasitoid Microplitis croceipes (Hymenoptera: Braconidae)

The specialist parasitoid, Microplitis croceipes (Cresson) (Hymenoptera: Braconidae), uses chemical cues from plants damaged by herbivore-feeding and also plant by-products in host location and acceptance. These chemicals are detected by the wasp's antennae. We conducted scanning and transmission electron microscopy studies of male and female M. croceipes antennae and detected no distinct morphological differences in the chemoreceptors between the sexes. Male antennae are approximately twice as long as female antennae. We found five morphological sensillar types in both sexes: sensilla (s) trichodea were the most abundant and distributed over the whole antenna; s. chaetica were present in low numbers only on the scape and pedicel; and s. basiconica, s. coeloconica and elongated s. placodea were found only on the flagellum. Ultrastructural investigations revealed pore systems on s. basiconica and s. placodea. In s. placodea, sensory neurons run parallel to the longitudinal axis of the sensilla with terminal pores. We recorded responses from single olfactory receptor neurons in s. placodea to two plant-emitted volatiles, cis-3-hexenol and ocimene, and two anthropogenic compounds, cyclohexanone and 2-diisopropylaminoethanol. Male receptor neurons were more sensitive than those of females with significantly higher spike frequency being registered from neurons in males.     

The ultrastructure of the anal vesicle of the parasitoid,Microplitis croceipes (Hymenoptera:Braconidae)

The fine structural characteristics of epithelial cells of the anal vesicle in the hymenopteran parasitoid, Microplitis croceipes (Cresson), are similar to those of transport cells. Apical and basal infoldings, an abundance of mitochondria, ribosomes, rough endoplasmic reticulum, Golgi complexes and pinocytotic vesicles all indicate a transport function for these epithelial cells. The medial portions of both Malpighian tubules located within the anal vesicle also were examined and on the basis of morphology appear to be active. These observations support earlier physiological data which indicate that the anal vesicle functions in absorption of nutrients and excretion.     

Nutrient absorption by the anal vesicle of the braconid wasp,Microplitis croceipes

The function of the anal vesicle of Microplitis croceipes in nutrient absorption was investigated. When larvae were incubated in low-concentrations of several ¹⁴C-labeled nutrient solutions, ¹⁴C-trehalose, -glucose and -amino acids accumulated in the body of control and head-ligated larvae but failed to accumulate in larvae with the anal vesicle ligated. When larvae were incubated in a more concentrated solution of trehalose, ¹⁴C-trehalose also accumulated in the body of control and head-ligated larvae and accumulation of trehalose was reduced in the body of anal-vesicle-ligated larvae. The results indicate that the anal vesicle functions in the absorption of trehalose, glucose and amino acids. Trehalose, when present in high concentrations, was also absorbed cutaneously to some extent. The lipid, triolein, appeared to be absorbed cutaneously and absorption was unaffected by vesicle ligation. The present study also indicates that disaccharides may be absorbed as disaccharides and rapidly converted to insoluble products in the parasitoid larvae.     

The role of learning in adult food location by the larval parasitoid,Microplitis croceipes (Hymenoptera: Braconidae)

Wind tunnel experiments were conducted to determine roles of odor learning in food foraging of the larval parasitoid,Microplitis croceipes (Hymenoptera: Braconidae). Females that had neither fed on sucrose water nor experienced any odor and females that had experienced an odor without feeding failed to respond to any odors in a wind tunnel. Most of the females that had fed without an odor also did not respond to odors. However, most of the females that had experienced an odor during feeding on sucrose water flew to the odor. These results indicate that when females experience an odor during feeding, they learn to associate the odor with food and subsequently respond to the odor. As age of females increased, their response to an experienced odor increased, peaked 2 to 5 days after emergence, and then decreased. With an increasing number of odor experiences while feeding, accuracy of females choosing the experienced odor increased. Females that experienced an odor while feeding three to five times chose the experienced odor 90% of the time. When females experienced an odor while feeding five times, the memory of food associated odor lasted at least 2 days. When they experienced food with two odors successively, they could memorize both odors, and multiple experiences did not cause memory interference. Even when females had learned a food-associated odor, their response to the learned odor ceased after several visits on patches containing the odor but no food. Such negative experience may cause switching of food searching to new odors by females.     

Impact of recombinant baculovirus field applications on a nontarget heliothine parasitoid, Microplitis croceipes (Hymenoptera: Braconidae)

The kill times of two viruses infectious to the heliothine pest complex indigenous to Texas cotton have been significantly reduced by expressing a scorpion toxin gene. Autographa californica nucleopolyhedrovirus (NPV) and Helicoverpa zea NPV express the toxin only in permissive lepidopteran hosts. The toxin, however, could indirectly harm members of upper trophic levels that feed upon and parasitize infected larvae producing the toxin. In this study, the effects of recombinant and wild-type viruses on Microplitis croceipes (Cresson) were studied in cotton using Heliothis virescens (F.) (Lepidoptera: Noctuidae) as hosts. Two recombinant viruses, their two wild-type progenitor viruses, and untreated cotton served as the five treatments of study. Larvae were previously parasitized 2 and 4 d before being confined for 72 h to cotton terminals treated with field rates of virus or left untreated. The sexes of adult M. croceipes that emerged from the recovered H. virescens larvae were determined and their head capsule widths were measured. Polymerase chain reaction (PCR) searched their extracts for virus DNA. There were no differences in percentage emergence and sex ratios of parasitoids among recombinant, wild-type, and control treatments. Significantly more wasps emerged from the 4-d cohort, but these wasps were significantly smaller than wasps from the 2-d cohort regardless of treatment. Finally, PCR found only 15-25% of the recovered H. virescens larvae and none of the emergent M. croceipes had detectable levels of viral DNA. Recombinant and wild-type viruses had a similar, minimal impact on emergent wasps, and the probability of virus dispersal via parasitoids is low in the system tested.     

Expression and characterization of a novel teratocyte protein of the braconid,Microplitis croceipes (cresson)

Microplitis croceipes wasps overcome host immunity by inducing changes in host physiology using factors derived from the embryo and/or larva. Teratocytes of some parasitic wasps circulate in the host hemolymph after egg hatch and synthesize proteins (TSPs), some of which are secreted to alter host physiology in support of endoparasitoid development. TSPs appear to alter host physiology, at least in part, by inhibiting synthesis of certain proteins. M. croceipes teratocytes synthesize a 13.9 kDa protein (TSP14), which inhibits synthesis of host proteins that are linked to larval growth and development. A cDNA encoding TSP14 was generated by RT-PCR from teratocyte RNA, and cloned into yeast expression vectors to produce sufficient recombinant protein for functional analyses. RecTSP14 was produced using the yeast expression system at a concentration of more than 300 micrograms/L. The recTSP14 inhibited in vitro translation of larval Heliothis virescens RNA, with the activity sensitive to boiling, protein concentration, incubation time, and storage temperatures. Although recTSP14 inhibited translation of some cellular RNAs in vitro, the in vivo incorporation of [35S]-methionine into proteins of selected insect and mammalian cell lines was not inhibited. These findings suggest that recTSP14 entry is cell type-specific and required to inhibit synthesis of target protein(s).     

Insecticide odour interference with food-searching behaviour of Microplitis croceipes (Hymenoptera: Braconidae) in a laboratory arena

Response to odours plays an important role in resource location by natural enemies, particularly by parasitoid wasps. While a considerable research effort has been dedicated to studying the effects of insecticide intoxication on natural enemy search behaviour, it is yet unknown if the odours themselves interfere with distant chemoreception. We investigated this issue using the food-searching behaviour of Microplitis croceipes (Cresson)(Hymenoptera: Braconidae) in laboratory arenas as a model system. Odours of imidacloprid (Genesis®), spinosad (Entrust®), esfenvalerate (Asana®), methamidophos (Monitor®), and vanilla were tested for their ability to interfere with wasp response to the odour of honey. The wasps did not contact the chemicals. Honey odour was generally effective in triggering food-searching behaviour in both honey-fed (and thus conditioned to associate honey odour with food) and unfed, naïve wasps. Mixing honey with imidacloprid and spinosad did not affect wasp responses. The remaining compounds (esfenvalerate, methamidophos, and vanilla) significantly reduced the proportion of positively responding fed wasps, but only methamidophos had such an effect on the unfed wasps. Negative methamidophos effects became completely reversed when wasps were forced to feed on honey in the presence of methamidophos odour. Our results suggest that odours and provision of food may potentially be used to keep beneficial natural enemies away from insecticide-treated areas.     

The function of the anal vesicle in respiration and excretion in the braconid wasp,Microplitis croceipes

An investigation into the function of the anal vesicle of the larval braconid wasp, Microplitis croceipes demonstrated that the anal vesicle has no significant rôle in respiration. The experimental evidence also suggested that these larvae are falculatively anerobic. The anal vesicle does appear to function in the excretion of ammonia, HCO₃^? and possibly the excretion of arginine and alanine. Although the anal vesicle does function in the absorption of salts and is permeable to water it does not function as an effective osmoregulator.     

Polymorphic microsatellite loci in the parasitic wasp Cotesia glomerata (Hymenoptera: Braconidae)

Five polymorphic microsatellite loci were isolated in a gregarious endoparasitoid, Cotesia glomerata, using an enrichment protocol. We detected two to 15 alleles per locus in a sample of 50 female wasps collected from a field population near Zürich, Switzerland. The observed heterozygosities ranged from 0.21 to 0.92. These markers are currently being used in our studies of sex determination in this species.     

Cloning and expression profile of ionotropic receptors in the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidae)

Ionotropic receptors (IRs) mainly detect the acids and amines having great importance in many insect species, representing an ancient olfactory receptor family in insects. In the present work, we performed RNAseq of Microplitis mediator antennae and identified seventeen IRs. Full-length MmedIRs were cloned and sequenced. Phylogenetic analysis of the Hymenoptera IRs revealed that ten MmedIR genes encoded “antennal IRs” and seven encoded “divergent IRs”. Among the IR25a orthologous groups, two genes, MmedIR25a.1 and MmedIR25a.2, were found in M. mediator. Gene structure analysis of MmedIR25a revealed a tandem duplication of IR25a in M. mediator. The tissue distribution and development specific expression of the MmedIR genes suggested that these genes showed a broad expression profile. Quantitative gene expression analysis showed that most of the genes are highly enriched in adult antennae, indicating the candidate chemosensory function of this family in parasitic wasps. Using immunocytochemistry, we confirmed that one co-receptor, MmedIR8a, was expressed in the olfactory sensory neurons. Our data will supply fundamental information for functional analysis of the IRs in parasitoid wasp chemoreception.     

Phylogeny of the parasitic wasp subfamily Euphorinae (Braconidae) and evolution of its host preferences

The braconid subfamily Euphorinae is a large, cosmopolitan group of endoparasitoid wasps. The majority of species attack adult hosts, a strategy that is rare among parasitic wasps, but there are also many species that attack nymphs and larval stages. Euphorine hosts may belong to a variety of insect orders (Coleoptera, Hemiptera, Hymenoptera, Neuroptera, Psocoptera, Orthoptera and Lepidoptera) although most euphorine tribes are confined to Coleoptera. Here we investigate the phylogenetic relationships of the Euphorinae based on molecular data (3 kb of nucleotide data from four markers: 18S, 28S, CAD and COI) and propose a higher‐level classification based upon the resulting phylogeny. We also infer the evolution of host associations and discuss the diversification of the Euphorinae. Results from both Bayesian inference and maximum‐likelihood analysis show that the subfamily, as previously circumscribed, is paraphyletic. We propose that the subfamily be expanded to include the tribes Meteorini and Planitorini (Mannokeraia + Planitorus), so that it corresponds to a clade that is strongly supported as monophyletic in our analyses. Based on our results, a revised higher classification of the Euphorinae is proposed, in which 52 extant genera and 14 tribes are recognized. We reinstate the genus Microctonus belonging to the tribe Perilitini, and synonymize Ussuraridelus with Holdawayella, Sinuatophorus with Eucosmophorus. Furthermore, we propose the following tribal rearrangements: Spathicopis and Stenothremma are transferred to Perilitini; Tuberidelus, Eucosmophorus and Plynops to Cosmophorini; Ecclitura to Dinocampini; Chrysopophthorus, Holdawayella and Wesmaelia to Helorimorphini; Proclithroporus and Heia to Townesilitini. The monotypic tribe Cryptoxilonini is synonymized with Cosmophorini. The genera Pygostolus and Litostolus are placed in a separate tribe, Pygostolini, previously recognized as a subtribe among the Centistini. Parsimony‐based ancestral state reconstructions suggest that the ancestor of Euphorinae was a parasitoid of lepidopteran larvae, and that a host shift to larval Coleoptera occurred only in one clade of the Meteorini, some members of which secondarily shifted back to larval lepidopteran hosts. In the remainder of the subfamily, there was an initial shift from larval to adult coleopterans, followed by subsequent shifts to adults or larvae of Hemiptera, Hymenoptera, Neuroptera, Orthoptera and Psocoptera.     

中红侧沟茧蜂气味结合蛋白MmedOBP18的原核表达及配体结合特性

【目的】研究中红侧沟茧蜂Microplitis mediator气味结合蛋白MmedOBP18在触角中的表达定位,解析MmedOBP18重组蛋白的配体结合特性。【方法】原核表达中红侧沟茧蜂气味结合蛋白MmedOBP18;采用荧光免疫组织化学技术研究MmedOBP18在中红侧沟茧蜂雌蜂触角中的表达定位;通过荧光竞争结合实验分析MmedOBP18重组蛋白与99种候选配体的结合特性。【结果】在原核表达系统中成功表达MmedOBP18重组蛋白。荧光免疫定位结果显示MmedOBP18主要表达在触角锥形感器Ⅰ内的淋巴液中。荧光竞争结合实验结果表明,MmedOBP18重组蛋白能够与16种候选配体结合,与低挥发性植物挥发物2-十三酮、十二醛、十四酸和十一酸的结合能力最强,其解离常数K_i值分别为5.21, 6.42, 6.49和6.58μmol/L;而且MmedOBP18重组蛋白与非挥发性植物次生物质棕榈酸、棉酚、槲皮素及亚麻酸也有较强的结合能力,K_i值分别为3.86, 5.07, 5.08和6.51μmol/L;此外,MmedOBP18重组蛋白与鳞翅目昆虫性信息素组分顺-9-十四碳烯醛及顺-11-十六碳醛表现出较强的结合能力,K_i值分别为9.09和11.67μmol/L,提示该蛋白在寄主定位过程中可发挥重要的作用。【结论】中红侧沟茧蜂气味结合蛋白MmedOBP18能够结合长链低挥发和非挥发性化合物,推测其在嗅觉和味觉识别中发挥双重功能,主要参与对寄主和寄主生境的化学信息的近距离识别。