Brown Citrus Aphid Research Articles

Characterization of two Bursicon genes and their association with wing development in the brown citrus aphid, Aphis citricidus

AbstractThe tanning hormone, Bursicon, is a neuropeptide secreted by the insect nervous system that functions as a heterodimer composed of Burs‐α and Burs‐β subunits. It plays a critical role in the processes of cuticle tanning and wing expansion in insects. In this study, we successfully identified the AcBurs‐α and AcBurs‐β genes in Aphis citricidus. The open reading frames of AcBurs‐α and AcBurs‐β were 480 and 417 bp in length, respectively. Both AcBurs‐α and AcBurs‐β exhibited 11 conserved cysteine residues. AcBurs‐α and AcBurs‐β were expressed during all developmental stages of A. citricidus and showed high expression levels in the winged aphids. To investigate the potential role of AcBurs‐α and AcBurs‐β in wing development, we employed RNA interference (RNAi) techniques. With the efficient silencing of AcBurs‐α (44.90%) and AcBurs‐β (52.31%), malformed wings were induced in aphids. The proportions of malformed wings were 22.50%, 25.84%, and 38.34% in dsAcBurs‐α‐, dsAcBur‐β‐, and dsAcBurs‐α + dsAcBur‐β‐treated groups, respectively. Moreover, feeding protein kinase A inhibitors (H‐89) also increased the proportion of malformed wings to 30.00%. Feeding both double‐stranded RNA and inhibitors (H‐89) significantly downregulated the wing development‐related genes nubbin, vestigial, notch and spalt major. Silence of vestigial through RNAi also led to malformed wings. Meanwhile, the exogenous application of 3 hormones that influence wing development did not affect the expression level of AcBursicon genes. These findings indicate that AcBursicon genes plays a crucial role in wing development in A. citricidus; therefore, it represents a potential molecular target for the control of this pest through RNAi‐based approaches.

Insect-specific RNA virus affects the stylet penetration activity of brown citrus aphid (Aphis citricidus) to facilitate its transmission.

Sap-sucking insects often transmit plant viruses but also carry insect viruses, which infect insects but not plants. The impact of such insect viruses on insect host biology and ecology is largely unknown. Here, we identified a novel insect-specific virus carried by brown citrus aphid (Aphis citricidus), which we tentatively named Aphis citricidus picornavirus (AcPV). Phylogenetic analysis discovered a monophyletic cluster with AcPV and other unassigned viruses, suggesting that these viruses represent a new family in order Picornavirales. Systemic infection with AcPV triggered aphid antiviral immunity mediated by RNA interference, resulting in asymptomatic tolerance. Importantly, we found that AcPV was transmitted horizontally by secretion of the salivary gland into the feeding sites of plants. AcPV influenced aphid stylet behavior during feeding and increased the time required for intercellular penetration, thus promoting its transmission among aphids with plants as an intermediate site. The gene expression results suggested that this mechanism was linked with transcription of salivary protein genes and plant defense hormone signaling. Together, our results show that the horizontal transmission of AcPV in brown citrus aphids evolved in a manner similar to that of the circulative transmission of plant viruses by insect vectors, thus providing a new ecological perspective on the activity of insect-specific viruses found in aphids and improving the understanding of insect virus ecology.

Bioinsecticide Activity of Neem Oil and Tobacco Extract Mixture Against Citrus Aphids and Green Scale on Citrus

The aim of this research is to determine the effectiveness of a mixture of neem (Azadirachta indica A. Juss.) and tobacco (Nicotiana tabacum L.) as bioinsecticides on the mortality of aphids (Toxoptera citricidus [Kirkaldy, 1907]) and the green scale (Coccus viridis [Green, 1889]). Data were analyze by variance and by Duncan Multiple Range Test (DMRT) at 5 % level of significance. The results showed that the bioinsecticide caused mortality of T. citricidus more than 50 % starting from 24 h after application (HAP) and at the 168 HAP the mortality 96 % to 100 %, not significantly different from the imidacloprid synthetic insecticide and significantly different from the control. The application of the bioinsecticide was less effective against C. viridis. The mortality of C. viridis is significantly different from the insecticide imidacloprid. The application of a bioinsecticide mixture of neem and tobacco extracts was effective against the brown citrus aphid T. citricidus, but less effective against the green scale C. viridis.

Population dynamics of Citrus tristeza virus (CTV) in single aphid-transmitted sub-isolates of the South African GFMS12 isolate.

Grapefruit trees in South Africa have been cross protected against severe stem pitting genotypes of Citrus tristeza virus (CTV) since the 1920s using a mild strain initially called 'Nartia' but later referred to as grapefruit mild strain 12 (GFMS12).In the current study, the GFMS12 isolate was used as the source for single aphid transmissions (SAT) using Toxoptera citricida, commonly called the brown citrus aphid (BrCA). The BrCA-transmitted CTV sub-isolates were analyzed by the heteroduplex mobility assay (HMA), serological assays, genetic marker analysis (GMA), and selected sub-isolates were biologically indexed. Reverse transcription PCR of genomic regions was conducted using universal primers followed by cloning the PCR products, HMA and sequence analysis; nine genotypes of CTV were identified in the complex of GFMS12, including both severe and mild genotypes. A singleBrCAtransmitted up to six CTV genotypes simultaneously in one sub-isolate. The HMA was found to be a rapid, reliable tool for the identification of genotypes and can be useful in the development of CTV management strategies and budwood certification programs.

Identification of Endogenous Genes for Normalizing Titer Variation of Citrus Tristeza Virus in Aphids at Different Post-acquisition Feeding Times.

Citrus tristeza virus (CTV) is efficiently transmitted in a semi-persistent manner by the brown citrus aphid (Toxoptera citricida (Kirkaldy)). Currently, the most sensitive method for detecting plant viruses in insect vectors is reverse-transcription quantitative polymerase chain reaction (RT-qPCR). In this study, the elongation factor-1 alpha (EF-1α) gene and acidic p0 ribosomal protein (RPAP0) gene were confirmed to be suitable reference genes for RT-qPCR normalization in viruliferous T. citricida aphids using the geNorm, NormFinder, and BestKeeper tools. Then the relative CTV titer in aphids (T. citricida) at different post-acquisition feeding times on healthy plants was quantified by RT-qPCR using EF-1α and RPAP0 as reference genes. The relative CTV titer retained in the aphids gradually decreased with increasing feeding time. During the first 0.5 h of feeding time on healthy plants, the remaining CTV titer in aphids showed about 80% rapid loss for the highly transmissible isolate CT11A and 40% loss for the poorly transmissible isolate CTLJ. The relative CTV titer in aphids during more than 12 h post-acquisition times for CT11A was significantly lower than at the other feeding times, which is similar to the trend found for CTLJ. To our knowledge, this is the first report about the relative titer variation of CTV remaining in T. citricida at different post-acquisition feeding times on healthy plants.

Genome-wide analysis of long non-coding RNAs and their association with wing development in Aphis citricidus (Hemiptera: Aphididae)

Long non-coding RNAs (lncRNAs) play critical roles in the various physiological processes of insects. The wing is a successful adaptation allowing insects to escape from unfavorable environments, while information on lncRNAs related to wing development is limited. In this study, we constructed 12 libraries from two RNA-seq comparisons: 4th instar winged nymphs versus winged adults and 4th instar wingless nymphs versus wingless adults in the brown citrus aphid Aphis citricidus, to identify the wing development-associated lncRNAs. A total of 2914 lncRNAs were identified and 50 lncRNAs were differentially expressed during the 4th instar winged nymphs to winged adults transition, and 28 lncRNAs changed during the 4th instar wingless nymphs to wingless adults transition. The differentially expressed lncRNAs were grouped into six clusters according to the expression patterns in the combined two-winged morphs. lncRNA Ac_lnc54106.1 was up-regulated during 4th instar winged nymphs to winged adults transition, but a lack of change during the 4th instar wingless nymphs to wingless adults transition implied a critical role in the specific regulation of wing development. RNA interference of Ac_lnc54106.1 resulted in malformed wings. Targets prediction, expression patterns, and RNAi assay results showed that Ac_lnc54106.1 may target the PiggyBac transposable element-derived protein 4 (PGBD4) gene, decrease expression of the canonical wing development-related genes, and finally regulate wing development. The systematic identification of lncRNAs in an aphid increases our understanding of how non-coding RNA mediates the wing plasticity of insects.

Different Host Plants Distinctly Influence the Feeding Ability of the Brown Citrus Aphid Toxoptera citricida.

Simple SummaryThe brown citrus aphid, Toxoptera citricida, is an important pest of citrus and causes serious damage in the main production areas. Host plant resistance is an environmentally friendly method to manage aphid infestations and is becoming increasingly important as aphids develop greater resistance to insecticides. The aim of this study was to assess T. citricida resistance on seven widespread and common hosts using electropenetrography combined with a population development test. We showed that the feeding parameters of the brown citrus aphid differed significantly depending on the host plants. Toxoptera citricida spent more time in the pathway stage and less time in the phloem stage on Eureka, while the opposite was observed on Yuzu and Rough Lemon. Measurements of population development of aphids showed that on the Eureka, aphids developed more slowly. Our data suggest that different host plants distinctly influence the ability of T. citricida to feed. The brown citrus aphid did not prefer to feed on Eureka Lemon compared to the other six host plants.Piercing–sucking insects are important crop pests, and an understanding of their feeding behavior and population development plays a crucial role in studying insect population dynamics and crop resistance. In our study, we examined the probing behavior of the brown citrus aphid, Toxoptera citricida, using electropenetrography and assessed its population development after 8 days on seven host plants: Yuzu, Citrus junos Sieb. ex Tanaka; Rough Lemon, C. jambhiri Lush.; ‘Luofu’ kumquat, Fortunella margarita Swingle; ‘Olinda’ valencia orange, C. sinensis (L.) Osbeck; ‘Yanxiwanlu’ Ponkan, C. reticulata Blanco; ‘Rohde Red’ valencia orange, C. sinensis; and ‘Eureka’ lemon, C. limon (L.) Osbeck. The results demonstrated that probing by the brown citrus aphid differed significantly according to the target hosts. Toxoptera citricida produced significantly more pathway activities on Eureka than on Rough Lemon and Yuzu. Toxoptera citricida spent more time from the first probe to first salivation into phloem sieve elements on Eureka compared to Yuzu. In addition, the total duration of ingestion from sieve cells of each aphid in the phloem-feeding phase was shortest on Eureka, and this was significantly shorter than that on Yuzu, Rough Lemon, Luofu, and Olinda. The population number of T. citricida on Eureka after 8 days was significantly lower than that on the other hosts. Overall, Eureka was found to have obvious resistance to T. citricida, whereas Yuzu and Rough Lemon were susceptible host plants. These results provide a theoretical basis for exploring aphid-resistant fruit tree resources using resistant varieties.

First Report of Citrus tristeza virus Infecting Citrus Trees in Georgia, USA.

Citrus tristeza virus (CTV) [genus Closterovirus; family Closteroviridae] is one of the most important, economically devastating viruses of citrus worldwide. On citrus trees grafted onto sour orange rootstock, typical CTV symptoms include dieback and defoliation, stunting, curling and chlorotic leaves, stem-pitting, and pinholes below the bud union on the inner face of the bark (Moreno et al. 2008). This single-stranded, positive-sense RNA virus is most efficiently transmitted by the brown citrus aphid (Toxoptera citricida), but it can also be transmitted by other aphid species and through grafting of infected plant material onto healthy plants (Moreno et al 2008; Herron et al. 2006). In Fall 2020, leaf material for virus testing was collected from 13 navel orange trees (Citrus × sinensis) grafted onto Poncirus trifoliata rootstocks (including 'Flying Dragon') located in a citrus research orchard in Tifton, GA. Trees ranged in age from 2 to 10 years, with the younger trees having been grafted from cuttings taken from the older trees. The oldest of these trees was derived from cuttings taken in 2009 from an orange tree growing locally in a residential yard in Tifton; this parent tree was more than 15 years old when these cuttings were obtained and was no longer available for sampling as of 2020. Symptoms or other visible signs of disease had not been noted on any of the tested trees, and trees were chosen for testing prior to the further dissemination of this plant material. The presence of CTV was verified via molecular and serological testing. CTV infection was initially confirmed in 8 of 13 tested samples using the ImmunoStrip® for CTV assay (Agdia® Inc., Elkhart, IN, cat no: ISK 78900/0025) according to the manufacturer's instructions. RNA was extracted from leaf material collected from the 13 sampled trees using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Following cDNA synthesis, samples were tested for the presence of CTV by reverse-transcription PCR using primer pair AR18F (5'-ATGTCAGGCAGCTTGGGAAATT-3') and AR18R (5'-TTCGTGTCTAAGTCRCGCTAAACA-3') which produces a 511 bp amplicon (Roy et al., 2005). PCR reactions confirmed the presence of CTV, with the same eight samples that had previously tested positive via Immunostrip® producing PCR fragments of the expected size. Amplified products from two of these samples were then sequenced using Sanger sequencing (Retrogen Inc, San Diego, CA, USA) and subjected to BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for further identification. Sequence analysis revealed that the obtained partial sequences (MW540805) from the p18 gene of both isolates were 100% identical to one another and shared 100% identity to corresponding sequences from CTV strain N4 (MK779711.1). To the best of our knowledge, this is the first report of CTV infecting citrus plants in Georgia. CTV could pose an imminent threat to the emerging citrus industry in Georgia if it were to become established in commercial citrus plantings either via the dissemination of infected plant material or via vector transfer of the virus under field conditions. While the brown citrus aphid is not known to be widespread in Georgia at this time, other CTV vectors are prevalent including the cotton aphid (Aphis gossypii) and the black citrus aphid (T. aurantia). Georgia citrus growers and plant propagators should be aware of this virus and take appropriate control measures to prevent the spread of this viral diseas.

Citrus tristeza virus P33 Protein is Required for Efficient Transmission by the Aphid Aphis (Toxoptera) citricidus (Kirkaldy).

Plant viruses are threatening many valuable crops, and Citrus tristeza virus (CTV) is considered one of the most economically important plant viruses. CTV has destroyed millions of citrus trees in many regions of the world. Consequently, understanding of the transmission mechanism of CTV by its main vector, the brown citrus aphid, Aphis (Toxoptera) citricidus (Kirkaldy), may lead to better control strategies for CTV. The objective of this study was to understand the CTV–vector relationship by exploring the influence of viral genetic diversity on virus transmission. We built several infectious clones with different 5′-proximal ends from different CTV strains and assessed their transmission by the brown citrus aphid. Replacement of the 5′- end of the T36 isolate with that of the T30 strain (poorly transmitted) did not increase the transmission rate of T36, whereas replacement with that of the T68-1 isolate (highly transmitted) increased the transmission rate of T36 from 1.5 to 23%. Finally, substitution of p33 gene of the T36 strain with that of T68 increased the transmission rate from 1.5% to 17.8%. Although the underlying mechanisms that regulate the CTV transmission process by aphids have been explored in many ways, the roles of specific viral proteins are still not explicit. Our findings will improve our understanding of the transmission mechanisms of CTV by its aphid vector and may lead to the development of control strategies that interfere with its transmission by vector.

Limited Occurrence of Tristeza in Yuzu and Possible Tristeza-free Cultivation in Northernmost Commercial Yuzu-producing Areas of Japan

Citrus tristeza virus (CTV) commonly infects yuzu (Citrus junos) in most parts of the citrus-growing areas in Japan. This study examined the occurrence of CTV in the northernmost commercial citrusproducing areas of Japan. Colonies of the brown citrus aphid (Toxoptera citricidus) were detected within the areas investigated. However, 23 of the 41 trees that were examined by RT-PCR and both or either subject to stem-pitting observation and immunostrip testing were free of CTV. Several CTVfree trees were apparently more than 60 years old. A field trial revealed that all eight uninoculated seedlings tested negative for CTV at 52 months after transplanting. These results suggest that CTVfree yuzu production is possible in the Rikuzen-Takata and Ofunato areas of Japan.

Alternative insecticides in the control of the brown citrus aphid Toxoptera citricida (Hemiptera: Aphididae)

The objective of this study was to evaluate the efficacy of alternative insecticides to control the brown citrus aphid Toxoptera citricida (Hemiptera: Aphididae). The treatments consisted of neem oil Azadirachta indica (Sapindales: Meliaceae); smoke syrup Nicotiana tabacum (Solanales: Solanaceae); coconut soap solution (commercial product); black pepper extract Piper nigrum (Piperales: Piperaceae); chemical insecticide Decis® 25 EC; and distilled water. Ten adult insects were placed on a filter paper in a Petri dish, applying 1.5 mL of each treatment to each dish, kept in a BOD-type air-conditioned chamber at a temperature of 25 ± 1 °C and a photophase of 12 hours. The experimental design used was completely randomized, using six treatments with six repetitions, consisting of 10 aphids. Survival assessment were performed at 6, 12, 18 and 24 hours after application of treatments. The alternative insecticides coconut soap and the smoke syrup caused the mortality of 45% and 38.3%, respectively, of the T. citricida at 24 hours after application, showing promising in the control of this pest.

Hormesis in the Brown Citrus Aphid, Toxoptera citricida (Kirkaldy) (Hemiptera: Aphididae) Exposed to Sublethal Doses of Imidacloprid

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a major pest that transmits phloem-limited, gram negative bacteria including ‘Candidatus’ liberibacter asiaticus (Clas), causing huanglongbing. Management of this pest relies primarily on insecticides, such as the neonicotinoid imidacloprid, that may affect secondary pests including the brown citrus aphid, Toxoptera citricida (Kirkaldy) (Hemiptera: Aphididae). Here, we report on direct toxicity and sublethal dose effects of imidacloprid on T. citricida nymphs and adults following direct contact and ingestion. We also examined transgenerational dose-response and hormetic effects following exposure of T. citricida to a sublethal concentration of imidacloprid. Toxicity of imidacloprid was similar for nymphs (0.6 ng per µL) and adults (1 ng per µL) at 72 h. Fecundity and finite rate of increase were greater for populations exposed to systemic and foliar treatments at a sublethal concentration (0.1 ng per µL) compared with untreated controls. Development times of first instar nymphs in the F1 generation and third instar nymphs in the F2 generation were significantly greater for individuals treated with the sublethal dose than an untreated control. Survival of second instar T. citricida on plants treated with a sublethal concentration also was significantly greater than controls. There also was a significant increase in fecundity of F1 and F2 individuals after sublethal treatment compared with controls. Our results indicated that a sublethal concentration of imidacloprid increased the reproductive performance and induced possible physiologically stimulative (hormetic) effects in T. citricida. Hormesis in secondary pests should be considered when developing a management program for pathogen vectors such as D. citri.

The miR-9b microRNA mediates dimorphism and development of wing in aphids

Wing dimorphism is a phenomenon of phenotypic plasticity in aphid dispersal. However, the signal transduction for perceiving environmental cues (e.g., crowding) and the regulation mechanism remain elusive. Here, we found that aci-miR-9b was the only down-regulated microRNA (miRNA) in both crowding-induced wing dimorphism and during wing development in the brown citrus aphid Aphis citricidus We determined a targeted regulatory relationship between aci-miR-9b and an ABC transporter (AcABCG4). Inhibition of aci-miR-9b increased the proportion of winged offspring under normal conditions. Overexpression of aci-miR-9b resulted in decline of the proportion of winged offspring under crowding conditions. In addition, overexpression of aci-miR-9b also resulted in malformed wings during wing development. This role of aci-miR-9b mediating wing dimorphism and development was also confirmed in the pea aphid Acyrthosiphon pisum The downstream action of aci-miR-9b-AcABCG4 was based on the interaction with the insulin and insulin-like signaling pathway. A model for aphid wing dimorphism and development was demonstrated as the following: maternal aphids experience crowding, which results in the decrease of aci-miR-9b. This is followed by the increase of ABCG4, which then activates the insulin and insulin-like signaling pathway, thereby causing a high proportion of winged offspring. Later, the same cascade, "miR-9b-ABCG4-insulin signaling," is again involved in wing development. Taken together, our results reveal that a signal transduction cascade mediates both wing dimorphism and development in aphids via miRNA. These findings would be useful in developing potential strategies for blocking the aphid dispersal and reducing viral transmission.

First Report of a Mesonivirus and Its Derived Small RNAs in an Aphid Species Aphis citricidus (Hemiptera: Aphididae), Implying Viral Infection Activity.

We report a new positive-sense single-stranded RNA (ss RNA+) virus from the brown citrus aphid Aphis citricidus. The 20,300 nucleotide (nt)-long viral genome contains five open-reading frames and encodes six conserved domains (TM2, 3CLpro, TM3, RdRp, Zm, and HEL1). Phylogenetic analysis and amino acid sequence analysis revealed this virus might belong to an unassigned genus in the family Mesoniviridae. The presence of the virus was also confirmed in the field population. Importantly, analysis of the virus-derived small RNAs showed a 22-nt peak, implying that viral infection triggers the small interfering RNA pathway as antiviral immunity in aphids. This is the first report of a mesonivirus in invertebrates other than mosquitoes.

Comparative Insight into the Bacterial Communities in Alate and Apterous Morphs of Brown Citrus Aphid (Hemiptera: Aphididae).

Wing polyphenism (alate and apterous morphs) in aphids is a trade-off between dispersal and reproduction. How bacterial communities are associated with wing polyphenism in aphids is still not clearly understood. This study used 16S rRNA sequencing to examine the differences in diversity of the bacterial community between alate and apterous morphs in Aphis citricidus, the main vector of the Citrus tristeza virus. Eighty-one operational taxonomic units (OTUs) belonging to 37 orders, 34 classes, and 13 phyla were identified from all samples. Among these OTUs, Wolbachia (79.17%), Buchnera (17.64%), and Pseudomonas (2.99%) were the dominant bacterial genera. The diversity of symbionts varied between the two morphs; apterous morphs had more bacterial diversity (69 OTUs belonging to 45 families, 21 classes, and 12 phyla) than alate morphs (45 OTUs belonging to 36 families, 15 classes, and 10 phyla). In addition, the abundance of five OTUs was significantly different between two morphs. Among these OTUs, two Pseudomonas species (Pseudomonas_brenneri [OTU21] and unclassified_Pseudomonas [OTU13]) represented a high proportion (3.93% and 2.06%) in alate morphs but were present in low abundance (0.006% and 0.002%) in apterous morphs. RT-qPCR showed consistent results with high-throughput DNA sequencing. The preliminary survey showed the difference in composition and frequency of bacteria between alate and apterous morphs. Thus, the results contribute to anew insight of microorganisms that may be involved in wing dimorphism and helpful for controlling the dispersal of this pest through artificial elimination or reinfection of bacterial symbionts or targeting symbiosis-related host genes by RNA interference in future.

Inability of the brown citrus aphid (Toxoptera citricida) to transmit citrus psorosis virus under controlled conditions

Might Toxoptera citricida (BrCA) be a citrus psorosis virus (CPsV) vector? We examined CPsV transmission by the BrCA throughout two experiments. In experiment 1, 4 CPsV-infected plants bearing BrCA colonies were introduced in separated cages with 12 healthy 'Madame Vinous' sweet orange (MV) seedlings in each one (48 in total). In experiment 2, 5 BrCAs collected from each CPsV-infected plant were transferred into 3 MV seedlings for each one (12 in total) and left for a 72-h inoculation period. Both experiments were replicated once. No psorosis symptoms or CPsV detection evidences a BrCA inability for CPsV transmission under our controlled conditions.

The mitochondrial genome of the brown citrus aphid Aphis (Toxoptera) citricidus: Insights into the repeat regions in aphids and phylogenetic implications

We sequenced the complete mitochondrial (mt) genome of brown citrus aphid, Aphis (Toxoptera) citricidus (Kirkaldy) and compared it with that of other aphids. The mt genome of A. citricidus is a circular molecule of 16,763 bp with 84.0% AT content, containing 13 protein-coding genes (PCGs), 2 rRNAs, 22 tRNAs, a control region (CR) and an additional repeat region between trnE and trnF. Like other aphids, A. citricidus was slightly A-skewed (0.073) and strongly C-skewed (−0.309). Most PCGs used standard ATW start codons and TAA stop codons except nad4, which terminates with an incomplete codon T. All tRNAs and rRNA could be folded as typical secondary structures, except for lacking a dihydrouridine (DHU) arm in tRNASer (AGN). The repeat region possessed the most copy number of repeat motif (~7.5 times) among the reported aphid mt genomes, like in Acyrthosiphon pisum. Both Bayesian inference (BI) and Maximum likelihood (ML) analyses suggested that Toxoptera citricidus should revert to Aphis citricidus. The subfamily Aphidinae is monophyletic, but the Eriosomatinae is non-monophyletic. This study provides new insight into the evolution of aphid mt genomes, as well as useful information for resolving aphid phylogenetic questions.

Characterization of a new bunyavirus and its derived small RNAs in the brown citrus aphid, Aphis citricidus.

High-throughput sequencing is widely used for virus discovery, and many RNA viruses have been discovered and identified. A new negative-sense single-stranded RNA virus was identified in the brown citrus aphid and named Aphis citricidus bunyavirus. The genome consists of large (7037nt), medium (3462nt), and small (1163nt) segments. Phylogenetic analysis and amino acid sequences identities of this virus with other bunyaviruses suggest that it is a new species belonging to the family Phenuiviridae. The small interfering RNA pathway could be involved against the infection of this virus in brown citrus aphid as supported by the viral derived small RNAs. The discovery of this virus illustrates the diversity of RNA viruses and contributes to the classification of bunyaviruses.

An analysis of the feeding behavior of three stages ofToxoptera citricidabyDCelectrical penetration graph waveforms

AbstractWith the purpose of studying the feeding behavior of the brown citrus aphid pest,Toxoptera citricida(Kirkaldy) (Hemiptera: Aphididae), we compared stylet probing behaviors of third and fourth instars and adults onCitrus unshiuMarc (Rutaceae) seedlings using the electrical penetration graph (EPG) technique.EPGwaveforms exhibited the full suite of stylet behaviors – stylet pathway, intracellular stylet puncture, phloem salivation (E1), sieve ingestion (E2), and xylem sap ingestion activities, plus the non‐penetration (Np) waveform. Before the phloem phase, the number of probes was significantly higher for third‐instar nymphs than for adults. Overall duration of Np events by adults was significantly lower than the duration of third and fourth instars. The number of short probes of the fourth instars was significantly higher than that of the adults. In the phloem phase, adults made more frequent and longer E1 events than the third and fourth instars. Third instars made more frequent but shorter E2 events, whereas adults made fewer but longer events. These results showed adults gained nutrients by increasing feeding time during phloem ingestion. Thus, the probability of phloem‐associated virus acquisition and transmission ofT. citricidawas higher in adults than in nymphs.

Titer Variation of Citrus Tristeza Virus in Aphids at Different Acquisition Access Periods and Its Association with Transmission Efficiency.

Tristeza, caused by citrus tristeza virus (CTV; Closterovirus, Closteroviridae), is of significant economic importance. Tristeza epidemics have caused severe declines in productivity, and even death, of millions of citrus trees on sour orange rootstock in many regions all over the world. In the field, CTV is most efficiently vectored by the brown citrus aphid (Toxoptera citricida (Kirkaldy)) in a semipersistent manner. The transmission efficiency of the vector is influenced by its acquisition access period (AAP) for CTV. A real-time RT-PCR assay using SYBR Green fluorescent dye was used to estimate the CTV titers in groups of 15 aphids under AAPs after 0.5 to 48 h for three CTV isolates (CT11A, CT16-2, and CTLJ). Similar trends for CTV titer in viruliferous aphids were displayed for the three isolates. The maximum CTV titer was at AAP 6 h for isolates CT11A and CT16-2, and at 4 h for isolate CTLJ. During the AAPs from 0.5 to 6 h, the mean CTV titer of CT16-2 increased from 7.8 × 104 to 1.71 × 107 copies per 15 aphids, and was correlated with an increase in transmission rate from 20 to 90.9%. This suggests that the transmission efficiency is positively correlated with viral titer in the insect from 0.5 h until 6 h AAPs. While a downward trend in CTV titer was observed after a 6-h AAP, the transmission rate remained higher than 90% up to 48 h. These results indicate that factors other than the virus titer in the vector contribute to successful transmission under long acquisition conditions. This is the first detailed quantitative analysis of CTV in its main vector species following different AAPs and its association with transmission efficiency, and should enhance our understanding of T. citricida-CTV interactions.