Silencing of V-ATPase-E gene causes midgut apoptosis of Diaphorina citri and affects its acquisition of Huanglongbing pathogen

The Asian citrus psyllid, Diaphorina citri Kuwayama, is among the most important pests of citrus. It is the main vector of the Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas), which causes severe losses in citrus crops. Control of D. citri is therefore of paramount importance to reduce the spread of HLB. In this regard, using RNA interference (RNAi) to silence target genes is a useful strategy to control psyllids. In this study, using RNAi, we examined the biological functions of the V-ATPase subunit E (V-ATP-E) gene of D. citri, including its effect on acquisition of CLas. The amino acid sequence of V-ATP-E from D. citri had high homology with proteins from other insects. V-ATP-E was expressed at all D. citri life stages analyzed, and the expression level in mature adults was higher than that of teneral adults. Silencing of V-ATP-E resulted in a significant increase in mortality, reduced body weight, and induced cell apoptosis of the D. citri midgut. The reduced expression of V-ATP-E was indicated to inhibit CLas passing through the midgut and into the hemolymph, leading to a majority of CLas being confined to the midgut. In addition, double-stranded RNA of D. citri V-ATP-E was safe to non-target parasitic wasps. These results suggest that V-ATP-E is an effective RNAi target that can be used in D. citri control to block CLas infection.     

RNA interference of carboxyesterases causes nymph mortality in the Asian citrus psyllid,Diaphorina citri

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important pest of citrus. In addition, D. citri is the vector of Huanglongbing, a destructive disease in citrus, also known as citrus greening disease caused by Candidatus Liberibacter asiaticus. Huanglongbing causes huge losses for citrus industries. Insecticide application for D. citri is the major strategy to prevent disease spread. The heavy use of insecticides causes development of insecticide resistance. We used RNA interference (RNAi) to silence genes implicated in pesticide resistance in order to increase the susceptibility. The activity of dsRNA to reduce the expression of carboxyesterases including esterases FE4 (EstFE4) and acetylcholinesterases (AChe) in D. citri was investigated. The dsRNA was applied topically to the fourth and fifth instars of nymphs. We targeted several EstFE4 and AChe genes using dsRNA against a consensus sequence for each of them. Five concentrations (25, 50, 75, 100, 125 ng/μl) from both dsRNAs were used. The treatments with the dsRNA caused concentration dependent nymph mortality. The highest gene expression levels of both AChe and EstFE4 were found in the fourth and fifth nymphal instars. Gene expression analysis showed that AChe genes were downregulated in emerged adults from dsRNA‐AChe‐treated nymphs compared to controls. However, EstFE4 genes were not affected. In the same manner, treatment with dsRNA‐EstFE4 reduced expression level of EstFE4 genes in emerged adults from treated nymphs, but did not affect the expression of AChe genes. In the era of environmentally friendly control strategies, RNAi is a new promising venue to reduce pesticide applications.     

Effect of host deprivation on the foraging behavior of the Asian citrus psyllid parasitoid Tamarixia radiata: observations from the laboratory and the field

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a significant citrus pest and the parasitoid Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) has been released in various citrus‐producing areas in classical biological control programs targeting D. citri. We investigated the effect of host deprivation on the foraging behavior and patch utilization by T. radiata. In the laboratory, females deprived of hosts for 3 days tended to leave patches of 12 nymphs without parasitizing hosts during the ca. 30 min they spent in the patch before leaving. Moreover, half of these females failed to host feed, and those that did host feed, on average, needed more than 15 min to complete feeding. Conversely, non‐host‐deprived females parasitized on average three nymphs before leaving patches without host feeding during the ca. 39 min they spent in the patch. These laboratory observations were compared to mass‐reared female T. radiata that were released onto colonies of D. citri nymphs infesting citrus in the field. Release vials were provisioned with honey and these females had no opportunities to host feed over the 1‐ to 2‐day containment period prior to release. When introduced onto D. citri colonies, 68% T. radiata females abandoned D. citri patches prior to probing hosts, in part, because Argentine ants, Linepithema humile (Mayr) (Hymenoptera: Formicidae), tending colonies disturbed searching parasitoids. These results from laboratory and field studies are discussed in the context of classical biological control, with the aim of understanding how to manipulate host availability and ant activity so establishment rates and impact of T. radiata can be improved.     

Suitability of native North American Rutaceae to serve as host plants for the Asian citrus psyllid (Hemiptera: Liviidae)

The Asian citrus psyllid, Diaphorina citri Kuwayama, a vector of citrus huanglongbing, is now present in all citrus‐producing states in the USA and Mexico. In addition to citrus, the insect can reproduce on several other plant species in the Rutaceae family; orange jasmine (Murraya spp.) and curry leaf (Bergera koenigii) are among its preferred hosts. There are several indigenous Rutaceae species in North America, and some are popular ornamentals. A study was therefore initiated to determine the suitability of some of these plants for feeding and development of the psyllid in choice and no‐choice experiments. D. citri was found to reproduce successfully on Choisya ternata, C. arizonica and Helietta parvifolia in no‐choice tests, but preferentially selected orange jasmine and curry leaf for feeding and reproduction, in choice tests. On Amyris madrensis, A. texana and Zanthoxylum fagara, adult psyllids laid eggs which hatched, but no successful nymphal development was recorded beyond the first instars. No oviposition was recorded on Esenbeckia berlandieri, Ptelea trifoliata and Casimiroa tetrameria, although adult psyllids were able to survive on these species for several days. Results showed that C. ternata, C. arizonica and H. parvifolia can serve host plants of D. citri and this constitutes the first report of these plants serving as host for D. citri. The findings of the present study suggest that native rutaceous host plants can serve as host plants and thus affect D. citri population dynamics and the epidemiology of Huanglongbing, the deadly citrus greening disease whose pathogen is vectored by D. citri. Thus, area‐wide management of this pest also should target these riparian habitats where these host plants are present with D. citri biological control agents for sustainable management of this pest.     

Guava leaf volatiles and dimethyl disulphide inhibit response of Diaphorina citri Kuwayama to host plant volatiles

The Asian citrus psyllid, Diaphorina citri Kuwayama, vectors the causal pathogen of huanglongbing (HLB), which is likely the most important disease affecting worldwide citrus production. Interplanting citrus with guava, Psidium guajava L., was reported to reduce D. citri populations and incidence of HLB. We describe a series of investigations on the response of D. citri to citrus volatiles with and without guava leaf volatiles and to synthetic dimethyl disulphide (DMDS), in laboratory olfactometers and in the field. Volatiles from guava leaves significantly inhibited attraction of D. citri to normally attractive host‐plant (citrus) volatiles. A similar level of inhibition was recorded when synthetic DMDS was co‐released with volatiles from citrus leaves. In addition, the volatile mixture emanating from a combination of intact citrus and intact guava leaves induced a knock‐down effect on adult D. citri. Compounds similar to DMDS including dipropyl disulphide, ethyl‐1‐propyl disulphide, and diethyl disulphide did not affect the behavioural response of D. citri to attractive citrus host plant volatiles. Head‐space volatile analyses were conducted to compare sulphur volatile profiles of citrus and guava, used in our behavioural assays, with a gas chromatography‐pulsed flame photometric detector. DMDS, produced by wounded guava in our olfactometer assays, was not produced by similarly wounded citrus. The airborne concentration of DMDS that induced the behavioural effect in the 4‐choice olfactometer was 107 pg/ml. In a small plot field experiment, populations of D. citri were significantly reduced by deployment of synthetic DMDS from polyethylene vials compared with untreated control plots. Our results verify that guava leaf volatiles inhibit the response of D. citri to citrus host plant volatiles and suggest that the induced compound, DMDS, may be partially responsible for this effect. Also, we show that field deployment of DMDS reduces densities of D. citri and thus may have potential as a novel control strategy.     

Behavioral Response of <Emphasis Type="Italic">Tamarixia radiata</Emphasis> (Waterston) (Hymenoptera: Eulophidae) to Volatiles Emanating from <Emphasis Type="Italic">Diaphorina citri</Emphasis> Kuwayama (Hemiptera: Psyllidae) and Citrus

Tamarixia radiata Waterston (Hymenoptera: Eulophidae) is an effective idiobiont ectoparasitoid of the psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae), vector of the huanglongbing (HLB). We examined the olfactory responses of T. radiata to volatiles emanating from D. citri or plant volatiles using a custom designed T-maze olfactometer and open arena bioassays. We also examined the behavioral response of male and female T. radiata to conspecifics of the opposite sex to determine whether olfactory signals mediate mate location. T. radiata adults exhibited a sexually dimorphic response to volatiles emanating from D. citri and citrus. Female T. radiata responded positively to the odors emanating from D. citri nymphs in both olfactometer and open arena bioassays. However, female wasps showed no response to odors emanating from D. citri adults, D. citri honey dew secretions, intact citrus or orange jasmine leaves. Odors emanating from D. citri damaged citrus were not attractive to T. radiata females but stimulated attraction of wasps to D. citri on damaged plants. T. radiata females were not attracted to D. citri immatures when they were presented as visual cues. Male T. radiata did not show attraction to D. citri nymphs or other putative odors that were attractive to female T. radiata. In olfactometer bioassays, more male T. radiata responded to the odor of female conspecifics than blank controls in the absence of associated citrus host plant volatiles. Odors emanating from female T. radiata were not attractive to male T. radiata. Male or female T. radiata were not attracted to the odors emanating from same sex conspecifics. Both male and female T. radiata adults showed positive phototactic behavior. Collectively, our results provide behavioral evidence that: 1) female T. radiata use volatiles emanating from D. citri nymphs to locate its host and: 2) female T. radiata release a volatile pheromone that attracts male conspecifics.     

Delivery of dsRNA through topical feeding for RNA interference in the citrus sap piercing‐sucking hemipteran,Diaphorina citri

RNA interference (RNAi) is a powerful means to study functional genomics in insects. The delivery of dsRNA is a challenging step in the development of RNAi assay. Here, we describe a new delivery method to increase the effectiveness of RNAi in the Asian citrus psyllid Diaphorina citri. Bromophenol blue droplets were topically applied to fifth instar nymphs and adults on the ventral side of the thorax between the three pairs of legs. In addition to video recordings that showed sucking of the bromophenol blue by the stylets, dissected guts turned blue indicating that the uptake was through feeding. Thus, we called the method topical feeding. We targeted the abnormal wing disc gene (awd), also called nucleoside diphosphate kinase (NDPK), as a reporter gene to prove the uptake of dsRNA via this method of delivery. Our results showed that dsRNA‐awd caused reduction of awd expression and nymph mortality. Survival and lifespan of adults emerged from treated nymphs and treated adults were affected. Silencing awd caused wing malformation in the adults emerged from treated nymphs. Topical feeding as a delivery of dsRNA is highly efficient for both nymphs and adults. The described method could be used to increase the efficiency of RNAi in D. citri and other sap piercing‐sucking hemipterans.     

Seasonal flight activity by the Asian citrus psyllid in east central Florida

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is an important invasive citrus pest in the USA because it vectors a bacterium responsible for huanglongbing, a devastating disease of citrus. Information was lacking on seasonal aspects of flight activity by D. citri, which could have ramifications on psyllid management as well as our understanding of epidemiology of the disease. Of interest from a pest management standpoint would be whether D. citri regularly disperses to or away from citrus on a predictable schedule. In research presented here, seasonal flight activity by D. citri was investigated using yellow sticky traps deployed in citrus trees and in fallow areas adjacent to citrus. Results indicated that flight activity by both male and female D. citri away from citrus can occur at any time of the year with consistent dispersal activity during the spring. The research further indicated citrus is continually subject to infestation by immigrating adults and that there is no time during the year that a citrus grower could be assured immigration would not occur. Growers should be aware that adult dispersal occurs regularly during spring and they should time management tactics accordingly. Adult flight activity 2 m from a citrus tree was more pronounced at 1 m above ground than at 2 or 3 m high. At distances of 8–60 m from trees, numbers of adults on traps were similar among the three heights. Males and females were similar with respect to seasonal flight activity. Numbers of adults captured on traps distant from citrus were not correlated with wind speed, sunlight, or air temperature, but there was some evidence that relative humidity influenced flight activity. Although the D. citri life cycle is dependent on flush, data from these studies did not confirm that psyllid dispersal from citrus consistently increases as citrus flush abundance decreases.     

Effect of plant barriers and citrus leaf age on dispersal of Diaphorina citri (Hemiptera: Liviidae)

Studies designed to measure dispersal capacity of Diaphorina citri Kuwayama (Hemiptera: Liviidae) are needed to provide the epidemiological knowledge necessary to improve management of citrus huanglongbing. In this study, a mark–release–recapture technique was used to investigate whether 1) host or non‐host plants of D. citri can act as barriers for dispersing insects and 2) presence or absence of young citrus leaves influence movement of D. citri towards citrus plants. The experimental field consisted of four circular and adjacent areas containing citrus trees, Citrus sinensis (L.) Osbeck cv. ‘Hamlin’, planted in concentric circles at 18, 24 and 30 m from the release centre. Insect activity was monitored by recapturing at each distance using yellow stick traps. Dense plantings of tall non‐host plants of D. citri such as corn had no effect on insect dispersal towards citrus plants when compared to a shorter cover crop such as grass. In contrast, suitable host plants acted as traps decreasing movement of D. citri. Diaphorina citri dispersed at greater speeds in the absence of young leaves reaching 140 m within 6 hours after release, whereas in the presence of young leaves, individuals reached at most 60 m at 1 day after release. Results suggest that D. citri control measures may be more efficient during periods of highest vegetative activity when insects are less active. Moreover, the use of suitable host plants for D. citri as trap plants may be a potential tactic to prevent movement of insects into the crop.     

Trail Chemicals of the Convergens Ladybird Beetle, <Emphasis Type="Italic">Hippodamia convergens</Emphasis>, Reduce Feeding and Oviposition by <Emphasis Type="Italic">Diaphorina citri</Emphasis> (Hemiptera: Psyllidae) on Citrus Plants

We investigated feeding and oviposition behavior of the Asian citrus psyllid, Diaphorina citri, when exposed to the foraging trails of the convergens ladybird beetle, Hippodamia convergens. Diaphorina citri females feeding on citrus leaves directly exposed to the ladybird adults or treated with trail extract excreted significantly less honeydew droplets than controls. The trail chemicals of the ladybird beetle also decreased oviposition by D. citri females on citrus. In a no-choice experiment, D. citri females preferred to oviposit on control flush and plants than those with ladybird trail-extract treatments. In two-choice experiments, 68.0% of D. citri released into cages exhibited strong selection preference for settling and eventual oviposition on control plants than plants treated with ladybird trail extract. Diaphorina citri eggs were found on all new leaf flush of control plants, whereas only 29.5% of flush on treatment plants were selected for oviposition. The trail chemical deposited by the convergens ladybird beetle elicits repellency of D. citri feeding and oviposition. Therefore, the trail chemicals my contain components that could be useful for behavior-based management of D. citri and HLB disease by reducing psyllid feeding and oviposition.     

Incidence of “Candidatus Liberibacter asiaticus” in a Florida population of Asian citrus psyllid

The incidence of a bacterium “Candidatus Liberibacter asiaticus” was assessed in a Florida population of Asian citrus psyllid, Diaphorina citri. The bacterium is the presumed causal agent of Asiatic huanglongbing, a serious citrus disease. Adult D. citri were periodically collected between May 2010 and September 2012 in a block of diseased trees located in east‐central Florida. The psyllids were individually subjected to molecular analyses (quantitative polymerase chain reaction assays using HLBaspr primers) to determine whether the bacterium was present and, if so, the population level of the pathogen based on qPCR cycle threshold (CT) values. Significantly greater percentages of females tested positive for the pathogen than males, but there were no significant differences between females and males with respect to population levels of the pathogen within the psyllids. No significant differences were found among the three D. citri colour morphs with respect to percentages of adults testing positive for the pathogen. Among 47 sample dates, a mean of 17.5% of adults per sample date tested positive (CT < 36) for the pathogen with a mean CT value of 31.1. The incidence of the pathogen was generally higher during late fall or early winter and often lower during mid‐ to late summer. There was a significant negative correlation between percentages of D. citri testing positive and air temperature. Increases in the incidence of the pathogen may not necessarily correspond to increases in transmission, as a number of factors both internal and external to D. citri can influence transmission. Transmission rates may be highest during periods when D. citri infestation levels are large, a high percentage of adults carry a high population of the pathogen in their salivary glands, and citrus flush is abundant.     

Chemical and behavioral analysis of the cuticular hydrocarbons from Asian citrus psyllid,Diaphorina citri

Huanglongbing (HLB) is the most destructive disease of citrus worldwide. The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the vector of the phloem‐inhabiting bacterium, Candidatus Liberibacter asiaticus, which is presumed to cause HLB in Florida citrus. Laboratory and field studies were conducted to examine the behavioral responses of male and female D. citri to their cuticular extracts. In olfactometer assays, more male D. citri were attracted to one, five, or 10 female cuticular extract equivalent units than blank controls. The results were confirmed in field studies in which clear or yellow traps baited with 10 female cuticular extract equivalent units attracted proportionately more males than clear traps baited with male cuticular extract or unbaited traps. Analyses of cuticular constituents of male and female D. citri revealed differences between the sexes in chemical composition of their cuticular extracts. Laboratory bioassays with synthetic chemicals identified from cuticular extracts indicated that dodecanoic acid attracted more males than clean air. Traps baited with dodecanoic acid did not increase total catch of D. citri as compared with blank traps at the dosages tested; however, the sex ratio of psyllid catch was male biased on traps baited with the highest lure loading dosage tested (10.0 mg).     

Characterization of the voltage‐gated sodium channel of the Asian citrus psyllid,Diaphorina citri

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important insect pest of citrus. It is the vector of ‘Candidatus’ Liberibacter asiaticus, a phloem‐limited bacterium that infects citrus, resulting in the disease Huanglongbing (HLB). Disease management relies heavily on suppression of D. citri populations with insecticides, including pyrethroids. In recent annual surveys to monitor insecticide resistance, reduced susceptibility to fenpropathrin was identified in several field populations of D. citri. The primary target of pyrethroids is the voltage‐gated sodium channel (VGSC). The VGSC is prone to target‐site insensitivity because of mutations that either reduce pyrethroid binding and/or alter gating kinetics. These mutations, known as knockdown resistance or kdr, have been reported in a wide diversity of arthropod species. Alternative splicing, in combination with kdr mutations, has been also associated with reduced pyrethroid efficacy. Here we report the molecular characterization of the VGSC in D. citri along with a survey of alternative splicing across developmental stages of this species. Previous studies demonstrated that D. citri has an exquisite enzymatic arsenal to detoxify insecticides resulting in reduced efficacy. The results from the current investigation demonstrate that target‐site insensitivity is also a potential basis for insecticide resistance to pyrethroids in D. citri. The VGSC sequence and its molecular characterization should facilitate early elucidation of the underlying cause of an established case of resistance to pyrethroids. This is the first characterization of a VGSC from a hemipteran to this level of detail, with the majority of the previous studies on dipterans and lepidopterans.     

Effective molecular detection of Diaphorina citri Kuwayama (Hemiptera: Liviidae) in bulk insect samples from sticky traps

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is the principal vector of citrus greening (huanglongbing) disease. Invasion of new areas by the vector increases the risk of further spread of the disease and has economic impacts on the global citrus industry. Effective implementation of vector surveys is essential to contain disease outbreaks. This is especially true in countries such as Japan, where most of the major citrus‐producing areas are free from citrus greening. Recently, vector surveys have been routinely conducted to maintain ‘disease‐free’ and ‘disease‐ and vector‐free’ areas in Japan, and improvement of methods that can detect D. citri in native insect populations is imperative. Here, we developed a method of using conventional and real‐time PCR to detect D. citri among bulk insects captured in sticky traps without the need for preliminary differentiation steps based on morphology. DNA fragments of D. citri were specifically detected by both conventional and real‐time PCR in a mixture of a 10^?3 dilution (ca. 0.008–0.009 ng/μl) of D. citriDNA and 100 ng/μl of bulk insect DNA, indicating that small body parts such as pieces of leg or parts of wings of D. citri were detectable in the bulk insect samples. No misleading amplification of fragments from the other psyllid species and citrus pests we used occurred under our PCR conditions. Our results suggest that the technique is applicable to extensive surveys of D. citri in early warning programmes.     

Selection of Bacillus thuringiensis strains in citrus and their pathogenicity to Diaphorina citri (Hemiptera: Liviidae) nymphs

Bacillus thuringiensis (Bt) toxins are effective in controlling insect pests either through the spraying of products or when expressed in transgenic crops. The discovery of endophytic Bt strains opened new perspectives for studies aimed at the control of sap‐sucking insects, such as the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae), a vector of “Candidatus Liberibacter spp.,” associated with citrus huanglongbing (HLB). In this study, translocation of endophytic Bt strains in citrus seedlings inoculated with Bt suspension delivered by soil‐drench, and their systemic pathogenicity to D. citri nymphs were investigated. The pathogenicity of three wild‐type Bt strains against D. citri third‐instar nymphs was demonstrated. Among the 10 recombinant strains tested (each of them harboring a single cry or cyt gene), 3 can be highlighted, causing 42%–77% and 66%–90% nymphal mortality at 2 and 5 d after inoculation, respectively. The isolation of Bt cells from young citrus shoots and dead nymphs, and PCR performed with specific primers, confirmed the involvement of the Bt strains in the psyllid mortality. This is the first report showing the translocation of Bt strains from citrus seedling roots to shoots and their potential to control D. citri nymphs that fed on these soil‐drench inoculated seedlings. The Bt strains that caused the highest mortality rates have the potential to be used as bioinsecticides to control D. citri and the identified genes can be used for the production of transgenic Bt citrus.     

Sex‐specific responses of Asian citrus psyllid to volatiles of conspecific and host‐plant origin

Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is the primary vector of Candidatus Liberibacter spp. bacteria that cause citrus greening, a disease of worldwide importance. Olfactometry was employed to test responses of D. citri to odours from intact citrus plants (Mexican lime, Citrus aurantifolia, sour orange, Citrus aurantium, Marsh grapefruit, Citrus paradisi and Valencia orange, Citrus sinensis), citrus plants previously infested with D. citri, and odours of conspecifics including nymphs, adult insects of same and opposite sex, and their products (honeydew), both alone and in combination. In contrast to other studies, psyllids of both sexes were attracted to volatiles of undamaged Mexican lime leaves, whereas undamaged grapefruit attracted only females, and leaves of Valencia and sour orange did not attract either sex. All four plant species attracted female psyllids when previously infested, but only Mexican lime and sour orange‐attracted males. Thus, Citrus species appear to vary in the production of both constituitive and induced volatiles that attract adult psyllids. Volatiles emitted by nymphs did not attract either sex, but psyllid honeydew was attractive to males, likely due to female pheromone residues. Males oriented to the odour of females, whereas the reverse was not true, and neither males nor females oriented to same‐sex volatiles. The addition of conspecific cues (adults, nymphs or honeydew) did not increase female attraction to previously infested leaves, but male response was increased by the presence of adults and honeydew, regardless of plant species. Thus, female psyllids appear to orient more strongly to volatiles of plant origin, whereas males respond more strongly to cues emanating from females and conspecific excretions. These results suggest that female psyllids drive the initial colonization of host plants, whereas males orient to females and infested plants. Identification of the specific volatiles involved may permit their use in monitoring and management of this pest.     

Proteomic maps of subcellular protein fractions of the Asian citrus psyllid Diaphorina citri,the vector of citrus huanglongbing

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is an insect vector that transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas) associated with the destructive citrus disease, citrus huanglongbing (HLB). Currently, D. citri is the major target in HLB management, although insecticidal control and disruption of the D. citri–CLas interactions both face numerous challenges. The present study reports the subcellular proteomic profiles of D. citri, encompassing the three main subcellular protein fractions: cytosol, mitochondria and microsomes. After optimization, subcellular proteins of both high and low abundance are obtained by two‐dimensional gel electrophoresis (2‐DE). A total of 1170 spots are detected in the 2‐DE gels of the three subcellular fractions. One hundred and sixty‐four differentially expressed proteins are successfully identified using liquid chromatography‐dual mass spectroscopy. An efficient protocol for subcellular protein fractionation from D. citri is established and a clear protein separation is achieved with the chosen protein fractionation protocol. The identified cytosolic proteins are mainly metabolic enzymes, whereas a large portion of the identified proteins in the mitochondrial and microsomal fractions are involved in ATP biosynthesis and protein metabolism, respectively. Protein–protein interaction networks are predicted for some identified proteins known to be implicated in pathogen–vector interactions, such as actin, tubulin and ATP synthase, as well as insecticide resistance, such as the cytochrome P450 superfamily. The findings should provide useful information to help identify the mechanism responsible for the CLas–D. citri interactions and eventually contribute to D. citri control.     

The process of population increase and patterns of resource utilization of two spider mites,Oligonychus ununguis (Jacobi) andPanonychus citri (McGregor), under experimental conditions (Acari: Tetranychidae)

Summary Pattern of population growth and characteristics of habitat utilization and of migration by two species of spider mites were studied under experimental conditions. The population growth ofOligonychus ununguis (Jacobi) on a chestnut occurred only on a single mite-release leaf over a long period, and few individuals moved away. Most of the 2nd progeny generation females of this species emigrated from the mite-release leaf as well as the sapling by means of ballooning threads. During this growth period, population density on the mite-release leaf levelled off, whereas that on the sapling increased. In contrast, the foundress ofPanonychus citri (McGregor) on citrus actively moved over several neighbouring leaves, and until the 2nd progeny generation females emerged, individuals were distributed over all the sapling leaves by means of walking. Emigration from the sapling was not observed until the 2nd progeny females emerged, and after that the mites emigrated by means of ballooning threads. The population density ofP. citri on the sapling levelled off and was rather decreased on the mite-release leaf at the time of mite emigration. Comparing the changing pattern of the relative degree of aggregation (m ^*/m) measured in two different units between these two species, the pattern ofm ^*/m in 1 cm² on the mite release leaf inO. ununguis resembled that of the unit of leaves on the sapling inP. citri. This result as well as behavioural observations indicate that migration ofO. ununguis is the movement from leaf to leaf and that ofP. citri from sapling to sapling. It is, therefore, concluded that the boundary of the microhabitat is a single leaf forO. ununguis but sapling or foliage forP. citri. This work was presented in Annual Meeting of Jap. Soc. Appl. Ent. Zool., 1981 in Okayama.