Case of mistaken identity: resolving the taxonomy between Trioza eugeniae Froggatt and T. adventicia Tuthill (Psylloidea: Triozidae).

The 'Eugenia psyllid' or 'Lilly pilly psyllid', widely recognized in Australia and in the USA as Trioza eugeniae Froggatt (Hemiptera: Triozidae), is not T. eugeniae, but rather T. adventicia Tuthill. In this study we assessed morphological comparisons of materials from throughout the native and introduced ranges and re-examined original descriptions of both taxa, together with Froggatt's type specimens of T. eugeniae. Furthermore, through DNA barcoding analyses, we confirmed the validity of both T. adventicia and T. eugeniae as separate species. We re-described both species to include additional characters not previously included and designated a lectotype for T. eugeniae. T. eugeniae has smaller fore wings that are slightly more elongate. These lack infuscation around veins R and R1, vein Rs is relatively longer, meeting the costa closer to the wing apex; with certain veins bearing long, fine divergent setae, a character not previously described. It has consistently three inner and one outer metatibial spurs. The male parameres appear narrowly pyriform with a weak dorsolateral lobe and weakly sclerotized apices. T. adventicia has larger fore wings that are slightly more ovate with dark infuscation around veins R and R1; vein Rs is relatively shorter, meeting the costa further from the wing apex, with veins lacking long, fine divergent setae. The usual configuration of two inner and one outer metatibial spurs, previously used to separate the two species, appears inconsistent. The male parameres appear a little more broadly pyriform with slightly more sclerotized apices. T. eugeniae refers to a distinct species which has a restricted distribution only in its native range in southern subcoastal New South Wales, Australia. T. adventicia refers to a separate species, with a natural distribution in eastern subcoastal Australia, but has been introduced widely in southern Australia, to New Zealand and the USA. This study elucidates a long history of misidentification of T. eugeniae in the nursery industry and in almost 30 years of literature on its biological control in the USA. Regardless, the biological control program, unknowingly, targeted the correct species of psyllid, T. adventicia, in its foreign exploration and importation of the appropriate parasitoid as a biocontrol agent in the USA. Despite being firmly entrenched in both the nursery trade and scientific literature, the name T. eugeniae is misapplied. While the acceptance of the valid name, T. adventicia, might be regarded as both problematic and protracted, this is the correct taxonomical attribution.

LAMP assay for the detection of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psylloidea: Psyllidae).

Diaphorina citri Kuwayama, also known as the Asian citrus psyllid (ACP), can vector the bacterium Candidatus Liberibacter asiaticus (CLas), agent of Huanglongbing (HLB): an incurable disease affecting citrus trees worldwide. In citrus growing regions where ACP and HLB are absent, such as Australia, the risk of an incursion and consequent economic damage to citrus industries make this psyllid one of the top-priority pests. Due to ACP's small dimensions and the generally poorly studied native psylloid fauna worldwide, morphological identification of this insect to distinguish it from harmless species is challenging, especially in the field, and with immature, partial or damaged specimens. To allow rapid and efficient detection of ACP in the field, we designed and optimised a new Loop-mediated isothermal amplification (LAMP) assay for the detection of D. citri based on the mitochondrial 16S locus. The optimised ACP 16S LAMP assay produced amplification from D. citri samples within 13.3 ± 3.6 min, with an anneal derivative of ~ 78.5 °C. A synthetic gBlock gene fragment was also developed to be used as positive control for the new LAMP assay with a different anneal derivative of ~ 83 °C. An existing commercially available LAMP assay for detection of the bacterium CLas was also tested in this study on ACP DNA. The ACP 16S LAMP assay we developed and tested here provides a valuable new in-field compatible tool that can allow early detections of ACP, enabling a quick biosecurity response, and could potentially be adopted by a wide range of users, from farmers to agronomists and from researchers to industry.

Insect phylogeny structures the bacterial communities in the microbiome of psyllids (Hemiptera: Psylloidea) in Aotearoa New Zealand.

The bacterial microbiome of psyllids has been studied for decades, with a strong focus on the primary and secondary endosymbionts capable of providing essential amino acids for the insects' diet and therefore playing a key role in the insects' ability to radiate on novel plant hosts. Here, we combine metabarcoding analysis of the bacterial communities hosted by psyllids with a multi-gene phylogenetic analysis of the insect hosts to determine what factors influence the bacterial diversity of the psyllids' microbiomes, especially in the context of the dispersal and evolutionary radiation of these insects in Aotearoa New Zealand. Using multi-gene phylogenetics with COI, 18S and EF-1α sequences from 102 psyllid species, we confirmed for the first time monophyly for all the six genera of native/endemic Aotearoa New Zealand psyllids, with indications that they derive from at least six dispersal events to the country. This also revealed that, after its ancestral arrival, the genus Powellia has radiated onto a larger and more diverse range of plants than either Psylla or Ctenarytaina, which is uncommon amongst monophyletic psyllids globally. DNA metabarcoding of the bacterial 16S gene here represents the largest dataset analysed to date from psyllids, including 246 individuals from 73 species. This provides novel evidence that bacterial diversity across psyllid species is strongly associated with psyllid phylogenetic structure, and to a lesser degree to their host plant association and geographic distribution. Furthermore, while the strongest co-phylogenetic signals were derived from the primary and secondary symbionts, a signal of phylosymbiosis was still retained among the remaining taxa of the bacterial microbiome, suggesting potential vertical transmission of bacterial lineages previously unknown to have symbiotic roles.

Non-destructive insect metabarcoding for surveillance and biosecurity in citrus orchards: recording the good, the bad and the psyllids.

Background: The Australian citrus industry remains one of the few in the world to be unaffected by the African and the Asian citrus psyllids, Trioza erytreae Del Guercio and Diaphorina citri Kuwayama, respectively, and the diseases their vectored bacteria can cause. Surveillance, early detection, and strict quarantine measures are therefore fundamental to safeguard Australian citrus. However, long-term targeted surveillance for exotic citrus pests can be a time-consuming and expensive activity, often relying on manually screening large numbers of trap samples and morphological identification of specimens, which requires a high level of taxonomic knowledge. Methods: Here we evaluated the use of non-destructive insect metabarcoding for exotic pest surveillance in citrus orchards. We conducted an 11-week field trial, between the months of December and February, at a horticultural research farm (SuniTAFE Smart Farm) in the Northwest of Victoria, Australia, and processed more than 250 samples collected from three types of invertebrate traps across four sites. Results: The whole-community metabarcoding data enabled comparisons between different trapping methods, demonstrated the spatial variation of insect diversity across the same orchard, and highlighted how comprehensive assessment of insect biodiversity requires use of multiple complimentary trapping methods. In addition to revealing the diversity of native psyllid species in citrus orchards, the non-targeted metabarcoding approach identified a diversity of other pest and beneficial insects and arachnids within the trap bycatch, and recorded the presence of the triozid Casuarinicola cf warrigalensis for the first time in Victoria. Ultimately, this work highlights how a non-targeted surveillance approach for insect monitoring coupled with non-destructive DNA metabarcoding can provide accurate and high-throughput species identification for biosecurity and biodiversity monitoring.

Disentangling bias for non-destructive insect metabarcoding.

A fast and reliable method for obtaining a species-level identification is a fundamental requirement for a wide range of activities, from plant protection and invasive species management to biodiversity assessments and ecological studies. For insects, novel molecular techniques such as DNA metabarcoding have emerged as a rapid alternative to traditional morphological identification, reducing the dependence on limited taxonomic experts. Until recently, molecular techniques have required a destructive DNA extraction, precluding the possibility of preserving voucher specimens for future studies, or species descriptions. Here we paired insect metabarcoding with two recent non-destructive DNA extraction protocols, to obtain a rapid and high-throughput taxonomic identification of diverse insect taxa while retaining a physical voucher specimen. The aim of this work was to explore how non-destructive extraction protocols impact the semi-quantitative nature of metabarcoding, which alongside species presence/absence also provides a quantitative, but biased, representation of their relative abundances. By using a series of mock communities representing each stage of a typical metabarcoding workflow we were able to determine how different morphological (i.e., insect biomass and exoskeleton hardness) and molecular traits (i.e., primer mismatch and amplicon GC%), interact with different protocol steps to introduce quantitative bias into non-destructive metabarcoding results. We discuss the relevance of taxonomic bias to metabarcoding identification of insects and potential approaches to account for it.

Description of an Australian endemic species of Trioza (Hemiptera: Triozidae) pest of the endemic tea tree, Melaleuca alternifolia (Myrtaceae).

Psyllids, also known as jumping plant lice, are phloem feeding Hemiptera that often show a strict species-specific relationship with their host plants. When psyllid-plant associations involve economically important crops, this may lead to the recognition of a psyllid species as an agricultural or horticultural pest. The Australian endemic tea tree, Melaleuca alternifolia (Maiden & Betche) Cheel., has been used for more than a century to extract essential oils and, long before that, as a traditional medicine by Indigenous Australian people. Recently, a triozid species has been found to damage the new growth of tea trees both in Queensland and New South Wales, raising interest around this previously undocumented pest. Furthermore, adults of the same species were also collected from Citrus plantations, leading to potential false-positive records of the exotic pest Trioza erytreae (Del Guercio 1918), the African Citrus psyllid. Here we describe for the first time Trioza melaleucae Martoni sp. nov. providing information on its distribution, host plant associations and phylogenetic relationships to other Trioza species. This work enables both morphological and molecular identification of this new species, allowing it to be recognized and distinguished for the first time from exotic pests as well as other Australian native psyllids. Furthermore, the haplotype network analysis presented here suggests a close relationship between Trioza melaleucae and the other Myrtaceae-feeding Trioza spp. from Australia, New Zealand, and Taiwan.

On the complementarity of DNA barcoding and morphology to distinguish benign endemic insects from possible pests: the case of Dirioxa pornia and the tribe Acanthonevrini (Diptera: Tephritidae: Phytalmiinae) in Australia.

Fruit flies are considered economically important insects due to some species being agricultural pests. However, morphological identification of fruit fly adults and larvae can be difficult requiring a high level of taxonomic expertise, with misidentifications causing problematic false-positive/negative results. While destructive molecular techniques can assist with the identification process, these often cannot be applied where it is mandatory to retain a voucher reference specimen. In this work, we non-destructively (and partial-destructively) processed larvae and adults mostly belonging to the species Dirioxa pornia (Walker, 1849), of the poorly studied nonpest fruit fly tribe Acanthonevrini (Tephritidae) from Australia, to enable molecular identifications whilst retaining morphological vouchers. By retaining the morphological features of specimens, we confirmed useful characters for genus/species-level identification, contributing to improved accuracy for future diagnostics using both molecular and morphological approaches. We provide DNA barcode information for three species of Acanthonevrini known from Australia, which prior to our study was only available for a single species, D. pornia. Our specimen examinations provide new distribution records for three nonpest species: Acanthonevroides variegatus Permkam and Hancock, 1995 in South Australia, Acanthonevroides basalis (Walker, 1853) and D. pornia in Victoria, Australia; as well as new host plant records for D. pornia, from kangaroo apple, apricot and loquat.

Illuminating Insights into the Biodiversity of the Australian Psyllids (Hemiptera: Psylloidea) Collected Using Light Trapping.

The superfamily Psylloidea includes numerous species which play a key role in Australian ecology and biodiversity, as well as pests and biological control agents, and sometimes threatened species of conservation concern. Different psyllid sampling and collection techniques are usually performed depending on the nature and aim of the study: from the beating and sweeping of psyllid host plants for conservation and biodiversity assessment, to suction and sticky traps in agriculture. Due to a general lack of information on its efficacy for psyllids, however, light trapping has not usually been employed. Here we present the results obtained trapping psyllids using different light sources and we discuss the strengths and weaknesses of this technique to assess psyllid biodiversity. In particular, we highlight the strength of using this methodology paired with DNA barcoding, to cast some light on psyllid biodiversity. The results obtained here suggest that the psyllid fauna of Australia is heavily understudied and the number of undescribed species might be many times higher than previously expected. Additionally, we report, for the first time, the species Trioza adventicia Tuthill 1952, and Cryptoneossa triangula Taylor 1990 in the state of Queensland.

Resolving an 87-year-old taxonomical curiosity with the description of Psylla frodobagginsi sp. nov. (Hemiptera: Sternorrhyncha: Psyllidae), a second distinct Psylla species on the New Zealand endemic plant kowhai.

A recent DNA-based assessment of the psyllid fauna of New Zealand recorded high genetic variation between populations that were expected to belong to the same psyllid species. Among these, a number of populations of the kowhai psyllid Psylla apicalis (Ferris & Klyver, 1932), from a kowhai species, Sophora microphylla Aiton (Fabaceae), presented high genetic variability. This gave new endorsement of an 87-year-old observation made by the entomologists Ferris and Klyver who, when describing the kowhai psyllid, from Sophora tetraptera J.S. Muell., suggested that morphological variations could support more than one species. Accordingly, the morphological assessment conducted here, together with the genetic information now available, resulted in the description of Psylla frodobagginsi sp. nov. as a second New Zealand endemic psyllid species hosted by S. microphylla.

Acizzia errabunda sp. nov. and Ctenarytaina insularis sp. nov.: Descriptions of two new species of psyllids (Hemiptera: Psylloidea) discovered on exotic host plants in New Zealand.

A recent molecular-based assessment of the psyllid fauna of New Zealand reported two genetically distinct, undescribed psyllid taxa on host plants not native to that country. Here, a morphological examination confirmed species-level variation that resulted in the description of two new psyllid species: Acizzia errabunda sp. nov. (Hemiptera: Psyllidae) from Acacia baileyana F. Muell and Ctenarytaina insularis sp. nov. (Hemiptera: Aphalaridae) from Syzygium smithii (Poir.) Nied. Furthermore, the examination of specimens from entomological collections and from observations recorded on an online database enabled a better understanding of the distribution and host plant associations of these psyllid species. The description of A. errabunda is based on material collected in both New Zealand and Australia from the same plant species, A. baileyana, whereas the psyllid C. insularis has been found to be present in Brunei and New Zealand on S. smithii and in New Caledonia on Melaleuca quinquenervia (Cav.) S. T. Blake.

Assessing subspecies status of leopards (Panthera pardus) of northern Pakistan using mitochondrial DNA.

Despite being classified as critically endangered, little work has been done on leopard protection in Pakistan. Once widely present throughout this region, leopards are now sparsely distributed, and possibly extinct from much of their previously recorded habitat. While leopards show morphological and genetic variation across their species range worldwide, resulting in the classification of nine different subspecies, the leopard genetic structure across Pakistan is unknown, with previous studies including only a very limited sampling. To clarify the genetic status of leopards in Pakistan we investigated the sequence variation in the subunit 5 of the mitochondrial gene NADH from 43 tissue samples and compared it with 238 sequences available from online databases. Phylogenetic analysis clearly separates the Pakistani leopards from the African and Arabian clades, confirming that leopards from Pakistan are members of the Asian clade. Furthermore, we identified two separate subspecies haplotypes within our dataset: P. p. fusca (N = 23) and P. p. saxicolor (N = 12).

An annotated checklist of the Cook Islands psyllids with keys to the species and two new records (Hemiptera, Psylloidea).

An annotated checklist of the psyllids of the Cook Islands is presented. The presence of Syntomozatahuata (Klyver, 1932) and Triozaalifumosa Klyver, 1932 in the archipelago, based on new material collected, is reported for the first time. This is the first record from these islands of the genus Syntomoza and the family Liviidae. An identification key to the psyllid species known from the Cook Islands is provided, and their origin and provenance are discussed in relation to their biogeographic implications.

Hybridogenesis and a potential case of R2 non-LTR retrotransposon horizontal transmission in Bacillus stick insects (Insecta Phasmida).

Horizontal transfer (HT) is an event in which the genetic material is transferred from one species to another, even if distantly related, and it has been demonstrated as a possible essential part of the lifecycle of transposable elements (TEs). However, previous studies on the non-LTR R2 retrotransposon, a metazoan-wide distributed element, indicated its vertical transmission since the Radiata-Bilateria split. Here we present the first possible instances of R2 HT in stick insects of the genus Bacillus (Phasmida). Six R2 elements were characterized in the strictly bisexual subspecies B. grandii grandii, B. grandii benazzii and B. grandii maretimi and in the obligatory parthenogenetic taxon B. atticus. These elements were compared with those previously retrieved in the facultative parthenogenetic species B. rossius. Phylogenetic inconsistencies between element and host taxa, and age versus divergence analyses agree and support at least two HT events. These HT events can be explained by taking into consideration the complex Bacillus reproductive biology, which includes also hybridogenesis, gynogenesis and androgenesis. Through these non-canonical reproductive modes, R2 elements may have been transferred between Bacillus genomes. Our data suggest, therefore, a possible role of hybridization for TEs survival and the consequent reshaping of involved genomes.

Elongation Factor-1α Accurately Reconstructs Relationships Amongst Psyllid Families (Hemiptera: Psylloidea), with Possible Diagnostic Implications.

The superfamily Psylloidea (Hemiptera: Sternorrhyncha) lacks a robust multigene phylogeny. This impedes our understanding of the evolution of this group of insects and, consequently, an accurate identification of individuals, of their plant host associations, and their roles as vectors of economically important plant pathogens. The conserved nuclear gene elongation factor-1 alpha (EF-1α) has been valuable as a higher-level phylogenetic marker in insects and it has also been widely used to investigate the evolution of intron/exon structure. To explore evolutionary relationships among Psylloidea, polymerase chain reaction amplification and nucleotide sequencing of a 250-bp EF-1α gene fragment was applied to psyllids belonging to five different families. Introns were detected in three individuals belonging to two families. The nine genera belonging to the family Aphalaridae all lacked introns, highlighting the possibility of using intron presence/absence as a diagnostic tool at a family level. When paired with cytochrome oxidase I gene sequences, the 250 bp EF-1α sequence appeared to be a very promising higher-level phylogenetic marker for psyllids.

An annotated checklist of the psyllids of New Zealand (Hemiptera: Psylloidea).

A checklist of extant species of Psylloidea in New Zealand is presented. The list is structured according to the latest taxonomic classification of families, subfamilies and genera. It includes 99 species, 71 of which are formally described and named, along with information on the host plants and the natural enemies as aspects that are either species-specific or assist in their recognition. An updated distribution of each species is given based on literature records and material held in the major New Zealand entomological collections and databases, including from very recent field surveys. A new record for New Zealand is Phellopsylla formicosa.

Dead element replicating: degenerate R2 element replication and rDNA genomic turnover in the Bacillus rossius stick insect (Insecta: Phasmida).

R2 is an extensively investigated non-LTR retrotransposon that specifically inserts into the 28S rRNA gene sequences of a wide range of metazoans, disrupting its functionality. During R2 integration, first strand synthesis can be incomplete so that 5' end deleted copies are occasionally inserted. While active R2 copies repopulate the locus by retrotransposing, the non-functional truncated elements should frequently be eliminated by molecular drive processes leading to the concerted evolution of the rDNA array(s). Although, multiple R2 lineages have been discovered in the genome of many animals, the rDNA of the stick insect Bacillus rossius exhibits a peculiar situation: it harbors both a canonical, functional R2 element (R2Brfun) as well as a full-length but degenerate element (R2Brdeg). An intensive sequencing survey in the present study reveals that all truncated variants in stick insects are present in multiple copies suggesting they were duplicated by unequal recombination. Sequencing results also demonstrate that all R2Brdeg copies are full-length, i. e. they have no associated 5' end deletions, and functional assays indicate they have lost the active ribozyme necessary for R2 RNA maturation. Although it cannot be completely ruled out, it seems unlikely that the degenerate elements replicate via reverse transcription, exploiting the R2Brfun element enzymatic machinery, but rather via genomic amplification of inserted 28S by unequal recombination. That inactive copies (both R2Brdeg or 5'-truncated elements) are not eliminated in a short term in stick insects contrasts with findings for the Drosophila R2, suggesting a widely different management of rDNA loci and a lower efficiency of the molecular drive while achieving the concerted evolution.