Adipokinetic hormone signaling mediates the enhanced fecundity of Diaphorina citri infected by 'Candidatus Liberibacter asiaticus'.

Diaphorina citri serves as the primary vector for 'Candidatus Liberibacter asiaticus (CLas),' the bacterium associated with the severe Asian form of huanglongbing. CLas-positive D. citri are more fecund than their CLas-negative counterparts and require extra energy expenditure. Therefore, understanding the molecular mechanisms linking metabolism and reproduction is of particular importance. In this study, we found adipokinetic hormone (DcAKH) and its receptor (DcAKHR) were essential for increasing lipid metabolism and fecundity in response to CLas infection in D. citri. Knockdown of DcAKH and DcAKHR not only resulted in the accumulation of triacylglycerol and a decline of glycogen, but also significantly decreased fecundity and CLas titer in ovaries. Combined in vivo and in vitro experiments showed that miR-34 suppresses DcAKHR expression by binding to its 3' untranslated region, whilst overexpression of miR-34 resulted in a decline of DcAKHR expression and CLas titer in ovaries and caused defects that mimicked DcAKHR knockdown phenotypes. Additionally, knockdown of DcAKH and DcAKHR significantly reduced juvenile hormone (JH) titer and JH signaling pathway genes in fat bodies and ovaries, including the JH receptor, methoprene-tolerant (DcMet), and the transcription factor, Krüppel homolog 1 (DcKr-h1), that acts downstream of it, as well as the egg development related genes vitellogenin 1-like (DcVg-1-like), vitellogenin A1-like (DcVg-A1-like) and the vitellogenin receptor (DcVgR). As a result, CLas hijacks AKH/AKHR-miR-34-JH signaling to improve D. citri lipid metabolism and fecundity, while simultaneously increasing the replication of CLas, suggesting a mutualistic interaction between CLas and D. citri ovaries.

Natural Infection of Murraya paniculata and Murraya sumatrana with CLas in Java.

The phloem-limited bacterium, 'Candidatus Liberibacter asiaticus' (CLas), is the putative causal pathogen of the severe Asiatic form of huanglongbing (citrus greening) and is most commonly transmitted by the Asiatic citrus psyllid (ACP), Diaphorina citri. CLas severely affects many Citrus species and hybrids and has been recorded in the Citrus relative, orange jasmine, Murraya paniculata (L.) Jack (syn. M. exotica L.). In this study, 13 accessions of three Murraya species (M. paniculata, M. sumatrana Roxb. and M. lucida (G.Forst.) Mabb,) and the Papuan form of a putative hybrid (M. omphalocarpa Hayata) were identified morphologically and molecularly based on sequence identity of the matK-5'trnK region of the chloroplast genome, and infection on these plants under field conditions was determined by PCR and qPCR on 2-4 occasions over 14 months. CLas was repeatedly detected in leaflet midribs by PCR and qPCR on four and three accessions of M. paniculata and M. sumatrana, respectively. It was not detected in leaflet midribs of single accessions of M. lucida and M. omphalocarpa. The species identification of the CLas-positive accessions was further confirmed using all the molecular taxonomic markers consisting of the six fragments of the maternally inherited chloroplast genome and part of the nuclear-encoded ITS region. The results indicated that natural infection of M. paniculata and M. sumatrana with CLas can occur in Java. This is the first demonstration of the natural infection of M. sumatrana with CLas. Further studies are required to determine if infections persist in the absence of D. citri.

Infection with 'Candidatus Liberibacter asiaticus' improves the fecundity of Diaphorina citri aiding its proliferation: A win-win strategy.

The evolution of insect vector-pathogen relationships has long been of interest in the field of molecular ecology. One system of special relevance, due to its economic impacts, is that between Diaphorina citri and 'Candidatus Liberibacter asiaticus' (CLas), the cause of the severe Asian form of huanglongbing. CLas-positive D. citri are more fecund than their CLas-negative counterparts, boosting opportunities for pathogens to acquire new vector hosts. The molecular mechanism behind this life-history shift remains unclear. Here, we found that CLas promoted ovarian development and increased the expression of the vitellogenin receptor (DcVgR) in ovaries. DcVgR RNAi significantly decreased fecundity and CLas titer in ovaries, extended the preoviposition period, shortened the oviposition period and blocked ovarian development. Given their importance in gene regulation, we explored the role of miRNAs in shaping these phenotypes and their molecular triggers. Our results showed that one miRNA, miR-275, suppressed DcVgR expression by binding to its 3' UTR. Overexpression of miR-275 knocked down DcVgR expression and CLas titer in ovaries, causing reproductive defects that mimicked DcVgR knockdown phenotypes. We focused, further, on roles of the Juvenile Hormone (JH) pathway in shaping the observed fecundity phenotype, given its known impacts on ovarian development. After CLas infection, this pathway was upregulated, thereby increasing DcVgR expression. From these combined results, we conclude that CLas hijacks the JH signalling pathway and miR-275, thereby targeting DcVgR to increase D. citri fecundity. These changes simultaneously increase CLas replication, suggesting a pathogen-vector host mutualism, or a seemingly helpful, but cryptically costly life-history manipulation.

Lower reproductive rates of Asian citrus psyllid (Hemiptera: Psyllidae) on 'Tahiti' acid lime than on 'Valencia' sweet orange

ABSTRACT The 'Tahiti' acid lime and orange trees are hosts of 'Candidatus Liberibacter asiaticus' (CLas), the pathogen associated with the severe Asian form of huanglongbing (HLB), the most devasting citrus disease. They are also hosts of the vector of CLas, the Asian citrus psyllid (ACP) Diaphorina citri Kuwayama. Relatively small numbers of lime trees occur in gardens and small orchards near large commercial 'Valencia' sweet orange orchards in Brazil. Applications of insecticides to suppress populations of ACP on the lime trees are usually nil or less frequent than in the orange orchards. Abundance of the psyllid on lime trees may therefore increase the risk of CLas spreading to the orchards. Because the abundance of the psyllid is influenced by the suitability of the trees as hosts, we compared reproductive potential of the insect on the two hosts in a controlled environment chamber (CEC) and in a greenhouse (GH). Daily temperature and relative humidity averaged 22ºC and 60% inside the CEC and 24°C and 70% inside the GH. Two pairs of adult male and female psyllids were caged for 3 days on new shoots and the fecundity and durations of development and survival of eggs and nymphs evaluated. Overall, acid 'Tahiti' was 3.5 times less suitable to ACP than 'Valencia'. Fecundity and survival of nymphs were 27% and 59% lower, and the life cycle 34% longer on 'Tahiti' than on 'Valencia'. Potential impacts of the results on CLas spread and HLB control are discussed.

Susceptibility of Diaphorina citri to Irradiation with UV-A and UV-B and the Applicability of the Bunsen-Roscoe Reciprocity Law.

Populations of Diaphorina citri decline with elevation and, in a study in Bhutan, were rarely found above 1200 m ASL. The impact of ultraviolet (UV) radiation, particularly UV-B, on immature stages of the psyllid was proposed as limiting factor. As no studies have been undertaken on the influences of UV radiation on the development of D. citri, we examined the effects of UV-A and UV-B on different stadia of the psyllid. In addition, compliance with the Bunsen-Roscoe reciprocity law was examined. Irradiation with UV-A marginally reduced egg hatch and the survival times of emerging nymphs. Early instar nymphs were little affected by this waveband, but the survival of adults was reduced at the higher doses used. With UV-B, egg hatch and the survival times of early and late instar nymphs declined in proportion to UV-B dose. A dose of 57.6 kJ m-2 d-1 reduced the survival time of only adult females. Female fecundity was reduced at high UV-A and UV-B doses but increased at low doses. The Bunsen-Roscoe law held true for eggs and early instar nymphs for different durations and irradiances of UV-B. Eggs and nymphs had ED50 values for UV-B lower than the daily fluxes of this wavelength experienced worldwide. Thus, UV-B could be a factor causing the psyllid to be scarce at high elevations.

Effect of Horticultural Mineral Oil on Huanglongbing Transmission by Diaphorina citri Kuwayama (Hemiptera: Psyllidae) Population in a Commercial Citrus Orchard in Sarawak, Malaysia, Northern Borneo.

Diaphorina citri Kuwayama transmits a destructive citrus disease caused by a fastidious bacterium 'Candidatus Liberibacter asiaticus' (CLas) designated as Huanglongbing (HLB) which posed a risk of detrimental threat to the Malaysian citrus industry. All D. citri life stages show a lumped habit on young flushes and its population fluctuations was closely related to accessibility of young flushes. The study aimed to investigate if the appearance of young flush shoots on citrus influences ACP population fluctuation and if horticultural mineral oil (HMO) could reduce spread of HLB transmission by ACP in a commercial healthy orchard. Field research was carried out from 1 April 2011 to 1 December 2014 in a 2-year-old 1 ha citrus farm that consisted of 200 PCR-certified disease-free grafted non-bearing honey tangerine (Citrus reticulata L.) in southwestern Sarawak, Malaysia. The experiment had two treatments namely control (unsprayed) and nC24 HMO with four replications arranged in a simple randomized block design. ACP eggs, nymphs, and adults per flush shoot was assessed and HLB incidence was monitored for visual inspection of the citrus trees for the current existence of usual signs of characteristic symptoms of HLB such as yellowing shoots, leaf mottling, and corky or enlarged veins on leaves. HLB-specific primer was employed in 16S rDNA polymerase chain reaction to detect the CLas gene in diseased trees. Increase in abundance of D. citri is mainly affected by the citrus flushing cycles and their life stages are completed on these flush shoots. Relative degree of aggregation index for D. citri adults increased during periods of cyclic production of new flush. HMO-treated plots produced a significantly lower percentage up to 11.43% of diseased trees against 42.20% in untreated control plots. HMO is effective against D. citri and recommended to be incorporated in the IPM program to prevent infection and reduce the spread of HLB.

Murraya paniculata and Swinglea glutinosa as Short-Term Transient Hosts of 'Candidatus Liberibacter asiaticus' and Implications for the Spread of Huanglongbing.

Murraya paniculata and Swinglea glutinosa are aurantioid hosts of the Asian citrus psyllid (ACP) Diaphorina citri, the principal vector of 'Candidatus Liberibacter asiaticus' (Las). Las is the pathogen associated with huanglongbing (HLB), the Asian form of which is the most devastating disease of Citrus species and cultivars (Rutaceae: Aurantioideae). M. paniculata is a common ornamental and S. glutinosa is grown as an ornamental, a citrus rootstock, and a hedgerow fence plant. Because of the uncertain status of these plants as reservoirs of Las, a series of cross-inoculation bioassays were carried out in different environments, using infected Valencia sweet orange (Citrus × aurantium) infected shoot tops as a source of inoculum and D. citri nymphs and adults reared on M. paniculata and S. glutinosa to inoculate pathogen-free Valencia orange plantlets. In contrast to sweet orange, Las was more unevenly distributed and reached much lower titers in M. paniculata and S. glutinosa. Infections in M. paniculata and S. glutinosa were also transient. Very few insects that successfully acquired Las from M. paniculata and S. glutinosa were able to transmit the pathogen to healthy citrus. Transmission rates were low from M. paniculata (1.0%) and S. glutinosa (2.0%) and occurred only in a controlled environment highly favorable to Las and ACP using 10-day-old adults that completed their life cycle on Las-positive plants. Our study showed that in HLB-endemic areas, M. paniculata and S. glutinosa can be deemed as epidemiologically dead-end hosts for Las and are not important alternative hosts of the pathogen for transmission to citrus. However, under a combination of conditions highly favorable to Las infection and transmission and in the absence of effective quarantine procedures, these plants could eventually serve as carriers of Las to regions currently free from HLB.

Citrus flush shoot ontogeny modulates biotic potential of Diaphorina citri.

The biology and behaviour of the psyllid Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Liviidae), the major insect vector of bacteria associated with huanglongbing, have been extensively studied with respect to host preferences, thermal requirements, and responses to visual and chemical volatile stimuli. However, development of the psyllid in relation to the ontogeny of immature citrus flush growth has not been clearly defined or illustrated. Such information is important for determining the timing and frequency of measures used to minimize populations of the psyllid in orchards and spread of HLB. Our objective was to study how flush ontogeny influences the biotic potential of the psyllid. We divided citrus flush growth into six stages within four developmental phases: emergence (V1), development (V2 and V3), maturation (V4 and V5), and dormancy (V6). Diaphorina citri oviposition and nymph development were assessed on all flush stages in a temperature controlled room, and in a screen-house in which ambient temperatures varied. Our results show that biotic potential of Diaphorina citri is not a matter of the size or the age of the flushes (days after budbreak), but the developmental stage within its ontogeny. Females laid eggs on flush V1 to V5 only, with the time needed to commence oviposition increasing with the increasing in flush age. Stages V1, V2 and V3 were most suitable for oviposition, nymph survival and development, and adult emergence, which showed evidence of protandry. Flush shoots at emerging and developmental phases should be the focus of any chemical or biological control strategy to reduce the biotic potential of D. citri, to protect citrus tree from Liberibacter infection and to minimize HLB dissemination.