A diagnostic LAMP assay for rapid identification of an invasive plant pest, fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae)

Fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae), is a highly polyphagous invasive plant pest that has expanded its global geographic distribution, including recently into much of Australia. Rapid diagnostic tests are required for identification of FAW to assist subsequent management and control. We developed a new loop-mediated isothermal amplification (LAMP) assay based on the mitochondrial cytochrome c oxidase subunit I (COI) gene for accurate and timely diagnosis of FAW in the field. The specificity of the new assay was tested against a broad panel of twenty non-target noctuids, including eight other Spodoptera species. Only S. frugiperda samples produced amplification within 20 min, with an anneal derivative temperature of 78.3 ± 0.3 °C. A gBlock dsDNA fragment was developed and trialled as a synthetic positive control, with a different anneal derivative of 81 °C. The new FAW LAMP assay was able to detect FAW DNA down to 2.4 pg, similar to an existing laboratory-based real-time PCR assay. We also trialled the new FAW assay with a colorimetric master mix and found it could successfully amplify positive FAW samples in half the time compared to an existing FAW colorimetric LAMP assay. Given the high sensitivity and rapid amplification time, we recommend the use of this newly developed FAW LAMP assay in a portable real-time fluorometer for in-field diagnosis of FAW

A novel diagnostic gene region for distinguishing between two pest fruit flies: Bactrocera tryoni (Froggatt) and Bactrocera neohumeralis (Hardy) (Diptera: Tephritidae)

Bactrocera tryoni and Bactrocera neohumeralis are morphologically similar sibling pest fruit fly species that possess different biological attributes, geographic distributions, and host ranges. The need to differentiate between the two species is critical for accurate pest status assessment, management, biosecurity, and maintenance of reference colonies. While morphologically similar, adults may be separated based on subtle characters; however, some characters exhibit intraspecific variability, creating overlap between the two species. Additionally, there is currently no single molecular marker or rapid diagnostic assay that can reliably distinguish between B. neohumeralis and B. tryoni; therefore, ambiguous samples remain undiagnosed. Here we report the first molecular marker that can consistently distinguish between B. tryoni and B. neohumeralis. Our diagnostic region consists of two adjacent single nucleotide polymorphisms (SNPs) within the pangolin (pan) gene region. We confirmed the genotypes of each species are consistent across their distributional range, then developed a tetra-primer amplification refractory mutation system (ARMS) PCR assay for rapid diagnosis of the species. The assay utilizes four primers in multiplex, with two outer universal primers, and two internal primers: one designed to target two adjacent SNPs (AA) present in B. tryoni and the other targeting adjacent SNPs present in B. neohumeralis (GG). The assay accurately discriminates between the two species, but their SNP genotypes are shared with other nontarget tephritid fruit fly species. Therefore, this assay is most suited to adult diagnostics where species confirmation is necessary in determining ambiguous surveillance trap catches; maintaining pure colony lines; and in Sterile Insect Technique management responses

Lamprolonchaea

Additional <i>Lamprolonchaea</i> species in Australia <p>(Figs. 45–50)</p> <p> A number of other <i>Lamprolonchaea</i> species have been recorded from Australia (Pitkin 1996); all of these species differ from <i>L. brouniana</i> in possessing a smooth frons. Below we provide a key to known Australian <i>Lamprolonchaea</i> based upon published species descriptions:</p>Published as part of <i>Blacket, Mark J. & Malipatil, Mallik B., 2010, Redescription of the Australian metallic-green tomato fly, Lamprolonchaea brouniana (Bezzi) (Diptera: Lonchaeidae), with notes on the Australian Lamprolonchaea fauna, pp. 31-51 in Zootaxa 2670</i> on page 47, DOI: <a href="http://zenodo.org/record/199123">10.5281/zenodo.199123</a&gt

Nitidulidae Latreille 1802

<p>Key to adults of Nitidulidae</p> <p>species commonly associated with nut orchards in south eastern Australia and some closely related species* (adapted, in part, from Leschen & Marris 2005 and Connell 1991)</p> <p>1 Abdomen usually with at least three exposed tergites (Fig. 8C,I)................................................ 2</p> <p>- Abdomen with two exposed tergites (Fig. 8D–H)............................................................ 3</p> <p> 2 Body with lateral margins slightly curved (Fig. 8I), dorso-ventrally convex; pronotal margin (in lateral view) with bead twice as thick along anterior half as along posterior half (Fig. 8B); elytra with punctures scattered randomly............................................................................................. <i>Urophorus humeralis</i> (Fabricius)</p> <p> - Body with lateral margins more or less parallel-sided (Fig. 8C), strongly flattened dorso-ventrally; pronotal margin with thickness of bead even throughout (Fig. 8A); elytra with punctures in distinct rows, parallel along elytra length................................................................................................. <i>Brachypeplus</i> spp.</p> <p>3 Mesosternal disc with raised carinae bearing oblique lateral arms (Fig. 9A)........................................4</p> <p>- Mesosternal disc without carinae (Fig. 9B)................................................................. 5</p> <p> 4 Elytra with posterior half bearing distinct pale orange markings (M-shaped) and orange patches on anterior lateral humeral angles (Fig. 8F).................................................................... <i>C. hemipterus</i> (Linnaeus)</p> <p> - Elytra dark brown, black without any distinctive orange colour patterns although sometimes with faint humeral angles (Fig. 8J)................................................................................ <i>C. obsoletus</i> (Erichson)</p> <p> 5 Metaventrite with large axillary space, axillary line almost straight and extending to at least half way to 2/3 length along metepisternum (Fig. 9C)......................................................... <i>C. marginellus</i> Motschulsky</p> <p> <i>-</i> Metaventrite with small axillary space, line curved reaching ¼ length along metepisternum or axillary space not clearly present (Fig. 9D).............................................................................................6</p> <p> 6 Prosternum weakly punctate or granulate (Fig. 9H), often convex and shiny in appearance; body often ventrally bicoloured, metaventrite usually darker than prosternum and abdomen but may be unicoloured; female 9th tergite truncate with median apical tubercle (Fig. 9E)................................................................ <i>C. davidsoni</i> Dobson</p> <p> - Prosternum moderately to distinctly punctate (Figs 1D,I, 9G); body unicoloured ventrally; female 9 th tergite without distinct median tubercle...................................................................................... 7</p> <p>7 Male metatibia, not constricted at base, gradually widened towards apex (Fig. 9I).................................. 8</p> <p>- Male metatibia distinctly and abruptly constricted along basal 1/3 to ¼, widened towards apex (Figs 1E,J, 7E).......... 10</p> <p> 8 Prosternum and hypomeron distinctly punctate (Fig. 9G); 3 rd antennomere about 1.3x length of 2 nd antennomere........................................................................................... <i>C. dimidiatus*</i> (Fabricius)</p> <p> - Prosternum distinctly to moderately punctate or granulate, hypomeron weakly punctate (Figs 1D,I, 3C,G, 4C); 3 rd antennomere less than 1.3x length of 2 nd antennomere (Fig. 1C,H)......................................................... 9</p> <p> 9 Body chestnut brown with no distinctive pattern (Fig. 8G); male mandibles asymmetrical, right mandible distinctly elbowed (right angled) (Fig. 9F); prosternum entirely punctate........................................ <i>C. mutilatus</i> Erichson</p> <p> - Body generally reddish brown, elytra golden brown but usually with a distinctive dark brown band across posterior margin (Fig. 8H); male mandibles are symmetrical; prosternum punctate medially but laterally only weakly punctate or granulate............................................................................................. <i>C. nepos*</i> Murray</p> <p> 10 Body length 2.8 to 3.4 mm (> 2.5 mm); body dark brown/ black, anterior lateral margin of elytra with a pale brown/ orange patch (Figs 1A,F, 3A,E); male paramere, is distinctly truncate apically, outer apical angle with slight tooth, mesal margin distinctly angled around mid-length (Fig. 2B,E)...................................................... <i>C. truncatus</i> Murray</p> <p> - Body length small, around 2.0 to 2.4 mm; body uniformly brown (not usually black and no paler patches on elytra) (Fig. 7A,H); male paramere roundly truncate, (not distinctly truncate) outer apical margin somewhat rounded, mesal margin slightly concave around mid-length but not distinctly angled (Fig. 7F)........................................... <i>C. imitatus</i> sp. nov.</p> <p> Note: Only <i>Carpophilus truncatus</i> was found damaging nuts in the current study. All other species in this key were collected in pheromone lures within almond and pistachio orchards, or are closely related species (as marked with an asterisk), which could be confused with <i>C. truncatus</i>.</p>Published as part of <i>Semeraro, Linda, Blacket, Mark J., Rako, Lea & Cunningham, John Paul, 2023, The pest sap beetle Carpophilus Myothorax truncatus Murray, 1864 (Coleoptera Nitidulidae) - a new synonymy and a related new species of Carpophilus, pp. 51-74 in Zootaxa 5301 (1)</i> on pages 54-55, DOI: 10.11646/zootaxa.5301.1.2, <a href="http://zenodo.org/record/8016457">http://zenodo.org/record/8016457</a&gt

Carpophilus (Myothorax) truncatus Murray 1864

<i>Carpophilus</i> (<i>Myothorax</i>) <i>truncatus</i> Murray 1864 <p>(Figs 1–5)</p> <p> <i>Carpophilus truncatus</i> <b>Murray 1864:</b> 381 [original description]; <b>Williams</b> <i>et al.</i> <b>1983</b>: 11 [World catalogue of Nitidulidae]; <b>Nagel</b> <i>et al.</i> <b>1989</b>: [ecology, pollinator of Annonanceae]; <b>Kirejtshuk 1996</b> [illustration of male genitalia and taxonomy]; <b>Leschen & Marris 2005</b>: 23, 38, 40 [redescription, male features illustrated]; <b>Jelínek & Audisio, 2007</b>: 467 [listed in Catalogue of <i>Carpophilus</i> species from the Palaearctic region, synonymies]; <b>Brown 2009</b>: 15, 20, 22, 30, 38 [distribution in the Pacific region, pest status, diagnostic features].</p> <p> <i>Carpophilus floridanus</i> Fall 1910 (synonymised with misidentified <i>C. pilosellus</i>, Gillogly 1962; treated as a synonym of <i>C. truncatus</i> in Jelínek & Audisio, 2007)</p> <p> <i>Carpophilus halli</i> Dobson 1954 (synonymised with misidentified <i>C. pilosellus</i>, Connell 1963; treated as a synonym of <i>C. truncatus</i> in Jelínek & Audisio, 2007)</p> <p> <i>Carpophilus jarijari</i> Powell and Hamilton 2019, <b>syn. nov.</b></p>Published as part of <i>Semeraro, Linda, Blacket, Mark J., Rako, Lea & Cunningham, John Paul, 2023, The pest sap beetle Carpophilus Myothorax truncatus Murray, 1864 (Coleoptera Nitidulidae) - a new synonymy and a related new species of Carpophilus, pp. 51-74 in Zootaxa 5301 (1)</i> on page 55, DOI: 10.11646/zootaxa.5301.1.2, <a href="http://zenodo.org/record/8016457">http://zenodo.org/record/8016457</a&gt

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

Abstract 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

LAMP (Loop-mediated isothermal amplification) assay for rapid identification of Varroa mites

Abstract Varroa mites are serious pests of European honeybees (Apis mellifera). For detection of Varroa mite, a new molecular LAMP-based assay has been developed, which retains the body of the mite intact for morphological identification. Six novel Varroa LAMP primers were designed from existing DNA sequences of the COI locus to target V. destructor and V. jacobsoni, providing the ability to tell them apart from other non-target beehive associated mite and insect species. This LAMP assay is specific in detecting these Varroa species and has been tested on specimens originating from multiple countries. It produces amplification of V. destructor and V. jacobsoni in 16 ± 3.4 min with an anneal derivative of 78 ± 0.5 °C whilst another Varroa species,V. underwoodi, showed late amplification. A gBlock gene fragment, used here as a positive control has a different anneal derivative of 80 °C. Three non-destructive DNA extraction methods (HotShot, QuickExtract and Xtract) were tested and found to be suitable for use in the field. The LAMP assay was sensitive to very low levels of Varroa DNA, down to 0.24 picogram (~ 1 × 10 copies/µL of Varroa gBlock). This is a new molecular tool for rapid and accurate detection and identification of Varroa mites for pest management, in areas where these mites do not occur