Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), and huanglongbing disease do not exist in the Stapleton Station area of the Northern Territory of Australia

Abstract  A series of specimens of the Asian citrus psyllid, Diaphorina citri , collected from the Northern Territory (NT) in 1915 was recently rediscovered in the Natural History Museum, London. Surveys were conducted in 2002 on suitable hosts in the locality of the 1915 collections to see if the infestation had persisted. These failed to detect either D. citri or the bacterium that it transmits and that causes huanglongbing disease in citrus. It is presumed that D. citri was eradicated fortuitously by the removal of all citrus plants above latitude 19°S during an eradication program for citrus canker in the NT from 1916 until 1922.     

Repellent effect of guava leaf volatiles on settlement of adults of citrus psylla, Diaphorina citri Kuwayama, on citrus

Abstract  The Asiatic citrus psyllid ( Diaphorina citri Kuwayama [Hemiptera: Sternorrhyncha: Psyllidae]) is a vector of huanglongbing (citrus greening), a devastating disease of citrus caused by phloem-limited bacteria. Growing guava ( Psidium guajava ) as an intercrop appears to be a successful means of reducing psyllid numbers within citrus orchards; however, the mechanism by which such a reduction is achieved is unknown. To determine the repellent effect of guava leaf and factors attributed to this activity, responses of adult psyllids to guava leaf and its odor were evaluated in cage tests and Y-tube olfactometer test. The results showed that guava leaf possessed a repellent effect against the adult citrus psyllids. Fewer psyllids were found on citrus leaves in the presence of guava foliage than in its absence. Young and old guava leaf showed equal repellent activity. By covering the guava shoots with net cloth, it was revealed that the repellent effect of guava leaf against adult psyllids on citrus was attributed to the volatile compounds, rather than physical factors. The olfactometer response of adult psyllids to guava leaf odor was dosage-dependent. Between guava odor and control, only 35.00%, 25.00% and 16.25% of the psyllids moved toward guava odor when presented with 5.0, 10.0 and 15.0 g of guava shoots, respectively. The olfactometer experiments also showed that both male and female psyllids responded similarly to the guava leaf odor.     

Nine polymorphic microsatellite loci from the psyllid Cacopsylla pruni (Scopoli), the vector of European stone fruit yellows

Cacopsylla pruni is the vector of European stone fruit yellows, a quarantine disease of Prunus trees. Nine polymorphic microsatellite markers were developed from enriched DNA libraries. Allelic variability was assessed in a collection of 149 females obtained from five localities covering a large geographical area in France. The number of detected alleles ranged from 8 to 37. Within the localities, observed and expected heterozygosities averaged across loci ranged from 0.39 to 0.55, and from 0.68 to 0.81, respectively. A heterozygote deficiency was detected for almost all loci, possibly due to a high null allele frequency. Other possible causes of the homozygote excess (mode of reproduction, inbreeding, assortative mating or Wahlund effect) are discussed. These variable microsatellite loci can provide tools to assess overall genetic variation in this important vector species. They will be used to search for population structure and migration patterns of C. pruni.     

Morphological description of the mouthparts of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae)

Scanning (SEM) and transmission (TEM) electron microscopy were used to elucidate the morphology of the rostrum, as well as the mandibular and maxillary stylets of the psyllid Diaphorina citri, vector of phloem-inhabiting bacteria associated with citrus huanglongbing (HLB) disease. D. citri has a cone-shaped rostrum that extends behind the pair of prothoracic coxae. The stylet bundle comprises a pair of mandibular (Md) and maxillary (Mx) stylets with a mean length of 513.3 μm; when retracted, their proximal portions form a loop and are stored in the crumena (Cr). Serial cross-sections of the rostrum revealed that the mandibles are always projected in front of the maxillary stylets. The two maxillary stylets form the food and salivary canals, with diameters of 0.9 μm and 0.4 μm respectively. These two canals merge at the end of the stylets forming a common duct with a length of 4.3 μm and a mean diameter of 0.9 μm. The acrostyle, a distinct anatomical structure present in the common duct of aphid maxillary stylets, was not observed by TEM in the ultrathin cross-sections of the common duct (CD) of D. citri. This study provides new information on D. citri mouthparts that may help to understand the feeding behaviour of this important vector of HLB-associated bacteria.     

Morphological and molecular characterization of a Hirsutella species infecting the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), in Florida

The Asian citrus psyllid, Diaphorina citri Kuwayama, is an invasive pest that vectors citrus greening disease, which recently was detected in Florida. Mycosed adult D. citri were collected at four sites in central Florida between September 2005 and February 2006. Observation of the cadavers using scanning electron microscopy revealed that the pathogen had branched synnemata supporting monophiladic conidiogenous cells. A high-fidelity polymerase chain reaction (PCR) assay was used to amplify the 18S rRNA, 28S rRNA and beta-tubulin genes of the pathogen for phylogenetic analysis. The morphological and genetic data indicated that the pathogen was a novel isolate related to Hirsutella citriformis Speare. PCR assays using isolate-specific primers designed from the unique putative intron region of the beta-tubulin sequence distinguished the psyllid pathogen from five related Hirsutella species. The pathogen was maintained in vivo by exposing healthy D. citri to the synnemata borne on field-collected cadavers. Infected psyllids had an abundance of septate hyphal bodies in their hemolymph and exhibited behavioral symptoms of disease. In vitro cultures of the pathogen were slow-growing and produced synnemata similar to those found on mycosed D. citri. In laboratory bioassays, high levels of mortality were observed in D. citri that were exposed to the conidia-bearing synnemata produced in vivo and in vitro.     

Suitability of Asian citrus psyllid, Diaphorina citri, as prey for ladybeetles

Given the apparent importance ofladybeetles as biological control agents of theinvasive Asian citrus psyllid, Diaphorinacitri Kuwayama (Homoptera: Psyllidae), aseries of experiments were undertaken to assessthe nutritional suitability of this pest as aprey item. Five species of Coccinellidae wereshown to develop successfully on a diet ofpsyllid nymphs and four species produced viableeggs. Eggs of the flour moth, Ephestiakuehniella Zeller (Lepidoptera: Pyralidae)were used as a reference diet. Larvae of Curinus coeruleus Mulsant, Cyclonedasanguinea L., Exochomus childreniMulsant, Harmonia axyridis Pallas, andOlla v-nigrum Mulsant all had survival ona diet of D. citri nymphs not differentfrom 100%, although developmental times wereextended and adult dry weights were reducedrelative to the Ephestia egg diet. Species were ranked (highest to lowest) forlarval performance on the D. citri dietrelative to the Ephestia egg diet as:E. childreni, 0.85;O. v-nigrum,0.82; C. coeruleus, 0.80;H.axyridis, 0.71;C. sanguinea, 0.48. Most females of C. sanguinea ceasedoviposition on the second day followingtransferal to the D. citri diet, but thefecundity and fertility of females of the otherspecies were not different from those feedingon Ephestia eggs. Generation times onthe Ephestia egg diet at 24°C(time to egg hatch + larval developmental time+ pupation time + adult prereproductive period)were (mean ± SEM): C. coeruleus, 56.7± 1.32 d;C. sanguinea, 28.0 ±0.88 d;E. childreni, 60.8 ± 1.96 d;H. axyridis, 32.2 ± 1.47 d;O.v-nigrum, 25.8 ± 1.12 d. Adult females ofC. coeruleus, H. axyridis and O.v-nigrum consumed the most psyllids in oneh, C. sanguinea was intermediate, andE. childreni consumed the least.     

Effects of the fungus Isaria fumosorosea (Hypocreales: Cordycipitaceae) on reduced feeding and mortality of the Asian citrus psyllid,Diaphorina citri (Hemiptera: Psyllidae)

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is an important citrus pest primarily because it transmits bacteria putatively responsible for huanglongbing, a serious disease of citrus. We present a study on the effects of blastospore and conidial formulations of Isaria fumosorosea Wize on feeding rates and mortality of adult psyllids in laboratory bioassays. Information on quantities of honeydew droplets was used to make inferences on feeding rates. Psyllids treated with the blastospore formulation of I. fumosorosea produced fewer honeydew droplets compared to the conidial treatment and control beginning within the first 24 h after treatment. The highest daily mean number of droplets thereafter never exceeded 2.4 drops compared to 4 and 8 for the conidial treatment and control, respectively. The mean number (±SEM) of honeydew droplets produced per psyllid per day over 7 days was significantly higher in the control (5.5±0.5) compared to the blastospore treatment; however, there were no significant differences between the treatments. Psyllids treated with the conidial formulation of the pathogen showed no significant reduction in feeding activity until 4 days after treatment. One and 2 day's post-exposure, mortality of psyllids in the blastospore treatment ranged from 8 to 25% compared to 0% in the conidial and control treatments. By 7 days post-exposure, psyllid mortality reached 100% under both fungal treatments compared to none in the controls. This study documented that adult psyllids infected by I. fumosorosea (PFR 97) produce less honeydew than healthy psyllids and suggests that they may feed less, which could potentially reduce the spread of huanglongbing.     

不同离体寄主及其成熟度对亚洲柑桔木虱存活的影响

【目的】亚洲柑桔木虱Diaphorina citri Kuwayama的虫口密度及活动与柑桔黄龙病的田间传播、流行有十分密切的关系,寻找合适的室内饲养条件能够便于观察和研究其生物学特性和传病机制。【方法】本文以九里香Murraya exotica(L.)Mant.、酸桔Citrus sunki Hort.ex Tanaka和马水桔Citrus reticulata Blanco.cv.Mashuiju 3种寄主植物不同成熟度离体梢为研究材料,(1)比较亚洲柑桔木虱卵分别在3种寄主植株和离体嫩梢上的孵化率;(2)比较3种寄主不同成熟度离体梢对各龄若虫存活率和蜕皮的影响;(3)比较3种寄主不同成熟度离体梢对成虫存活率的影响。【结果】(1)亚洲柑桔木虱在植株嫩梢上卵的孵化率高于离体嫩梢,九里香表现最明显;(2)低龄若虫在叶片未完全展开的离体嫩梢上存活率最高,而高龄若虫在完全展开的嫩梢上最高;(3)用离体成熟梢饲养柑桔木虱成虫存活率高于离体嫩梢,酸桔和马水桔离体梢饲养的存活率较九里香高。【结论】用寄主植物离体梢饲养的亚洲柑桔木虱卵孵化率和若虫、成虫存活率都较高而且较稳定,该方法可用于这种木虱的室内繁殖中。     

Isolation and characterization of an Isaria fumosorosea isolate infecting the Asian citrus psyllid in Florida

A fungal pathogen that killed adult Diaphorina citri Kuwayama (Asian citrus psyllid) (Hemiptera: Psyllidae) in Florida citrus groves during the fall of 2005 was identified and characterized. Investigation of this pathogen is important because D. citri vectors citrus greening disease (Huanglongbing), which was reported in Florida in 2005. The morphological and genetic data generated herein support identification of the fungus as Isaria fumosorosea Wize (Ifr) (=Paecilomyces fumosoroseus) (Hypocreales: Cordycipitaceae) from the Asian citrus psyllid (Ifr AsCP). Koch’s postulates were fulfilled after the fungus was isolated in vitro and transmitted to healthy psyllids, which then exhibited a diseased-phenotype similar to that observed in the field. Both in vitro growth characteristics and two Ifr AsCP-specific molecular markers discriminated the psyllid pathogen from another local Ifr isolate, Ifr 97 Apopka. These molecular markers will be useful to track the dynamics of this disease in D. citri populations. The potential for utilizing Ifr to complement existing psyllid pest management strategies is discussed.     

Development of simple sequence repeat markers for the Japanese population of the Asian citrus psyllid,Diaphorina citri (Hemiptera: Psyllidae)

We developed nine simple sequence repeat (SSR) markers for the Japanese population of Diaphorina citri Kuwayama, which is known as the vector for citrus greening disease or huanglongbing, for use in population genetic studies. Analysis of 50 individuals collected from Tokunoshima Island showed that allelic diversity ranged from 5 to 24 alleles per locus, and observed and expected heterozygosities ranged from 0.100 to 0.939 and from 0.222 to 0.926, respectively. These SSR markers can be used to understand the population genetics of D. citri, and the obtained information may be useful in the estimation of population genetic structure, gene flow and invasion routes of D. citri.     

Polymerase chain reaction primers for polymorphic microsatellite loci from the Korean sandfish,Arctoscopus japonicus

We developed 12 polymorphic microsatellite markers from Arctoscopus japonicus by screening an enriched genomic library using polymerase chain reaction (PCR) techniques. The average of alleles size was 16.2, and the average observed and expected heterozygosities were 0.59 and 0.78, respectively. The observed genotypic frequencies in five loci were significantly deviated from Hardy–Weinberg expectations. The high variability revealed in this study suggested that these microsatellite loci should provide useful markers for population genetics of A. japonicus.     

Highly polymorphic tetranucleotide microsatellite loci for the eastern Canary Island lizard,Gallotia atlantica

We describe 10 polymorphic tetranucleotide microsatellite loci from the eastern Canary Island lacertid lizard, Gallotia atlantica. Loci were isolated from a partial genomic library that had been enriched for AAAG repeat sequence. All loci were highly polymorphic (eight alleles or more) with observed heterozygosities from 0.75 to 1.00. At least four loci were successfully amplified and polymorphic in the Gran Canarian lacertid, Gallotia stehlini. These loci will be used to examine correlations between patterns of gene flow and recent volcanism on the island of Lanzarote.     

Isolation and characterization of nine polymorphic microsatellite loci from the desert locust,Schistocerca gregaria

Because of the scarcity of polymorphic genetic markers, only a few genetic studies on the population structure of the desert locust, Schistocerca gregaria, have been carried out. We isolated and characterized nine polymorphic dinucleotide microsatellite loci. These markers were evaluated using individuals from Niger and Senegal. Seven of these microsatellite markers are also applicable to the nongregarious subspecies Schistocerca gregaria flaviventris. Cross‐species applicability was limited to one of the loci in the sister species S. americana and in the locust Locusta migratoria.     

Isolation and characterization of 18 new polymorphic microsatellite loci for the swordfish, Xiphias gladius

Eighteen microsatellite loci (13 di- and 5 tri-repeats) were isolated from swordfish and characterized in two populations from the Atlantic Ocean and Mediterranean Sea. The number of alleles per locus ranged from two to 29 and the observed heterozygosity from 0.302 to 0.953. All but one locus conformed to Hardy-Weinberg expectations and there was no evidence for linkage disequilibrium between loci.     

Newly developed PCR primers for polymorphic microsatellite loci from the marine isopod Limnoria sp.

Nine microsatellite loci are isolated and characterized for the marine isopod Limnoria sp., a model invertebrate species for the study of connectivity of marine brooders with high potential of dispersal through rafting. Analysis of 35 individuals of one population revealed a mean number of alleles of 12.66 and mean expected heterozygosity of 0.83, indicating a high level of variability at most loci. These polymorphic microsatellite loci should be useful to the study of the geographical distribution of the genetic diversity of this species of Limnoria.     

Development of 10 polymorphic microsatellite loci isolated from the mountain beaver, Aplodontia rufa rufa (Rafinesque)

We developed 10 microsatellite markers for the mountain beaver, Aplodontia rufa rufa. In three populations of A. r. rufa, the number of alleles for these loci ranged from monomorphic to nine. Average observed heterozygosities in these populations ranged from 0.29 to 0.60. We also tested previously published markers from the endangered subspecies A. r. nigra in A. r. rufa populations.     

PERMANENT GENETIC RESOURCES: Development of polymorphic microsatellite loci for the endangered Topeka shiner, Notropis topeka

We have developed a set of eight polymorphic microsatellite markers for the endangered Topeka shiner, Notropis topeka. Allelic diversity at each of these loci was assessed in a single isolated population from eastern South Dakota, USA. The allelic diversity ranged from four to 15 alleles. These are the first microsatellite markers to be reported for this species. These markers are being used in a more thorough study of the population structure throughout the remaining range of this species.     

Variable microsatellite loci isolated from the Asian honeybee, Apis cerana (Hymenoptera; Apidae)

We developed 12 polymorphic microsatellite loci for the Asian honeybee, Apis cerana using the magnetic particle method. Eight of these 12 were highly polymorphic, having four to seven alleles with an expected heterozygosity of 0.38 to 0.78. The primers also produce polymorphic products in related honeybee species such as Apis nigrocincta. These loci can be used to study parameters associated with genetic structure, such as paternity frequency and worker reproduction.     

New polymorphic microsatellite loci for population studies in the European anchovy, Engraulis encrasicolus (L.)

Eleven microsatellite loci were developed in the European anchovy, Engraulis encrasicolus, and tested in samples from two geographically distant populations (Atlantic and Mediterranean Sea). Number of alleles ranged from eight to 28 and observed heterozygosity from 0.440 to 0.920. There was no evidence of linkage disequilibrium, although two loci are indeed linked. All loci were in Hardy-Weinberg equilibrium, except for one locus in the Atlantic and two loci in the Mediterranean sample. These three loci plus two more showed evidence for null alleles.