Optimizing efficiencies in biosecurity surveillance and monitoring: testing colour and multi-lure traps on tomato potato psyllid, other plant-lice (Psylloidea) and stinkbugs (Pentatomoidea)

Cost efficiency in biosecurity surveillance is vital, and the ability to survey for multiple pest species using just one trap therefore highly appealing. The Psylloidea, or plantlice, contain significant horticultural pest species that act as vectors for a number of deleterious plant bacteriums. We examine the efficacy of using two different coloured sticky traps, and two lure types on the general Psylloidea and Pentatomoidea fauna, and a target extant pest psyllid; tomato potato psyllid (TPP) Bactericera cockerelli (Šulc). Specifically, we test the effect of lure (no lure, Asian citrus psyllid ACP lure, brown marmorated stink bug BMSB lure, combined lures), sticky trap color (green vs yellow), and sentinel plant (tomato vs citrus) on psyllid and stink bug species in 104 urban backyards across Perth, Australia. We found that tomato sentinel host plants and green traps significantly increased the capture rate of TPP, but that all lures decreased the capture of TPP. Green traps also increased the capture rate of all other Psylloidea. Although BMSB lures reduced TPP capture, these lures increased abundances of other Psylloidea and the pest stinkbug Plautia affinis (Dallas) on traps. Thus, our experiment demonstrates that increased efficiencies can be gained with combination traps and lures for particular groups, provided that they have been tested on focal organisms in the first instance, as reactions to non-target lures are unpredictable and species specific.

Seven species of whitefly new for Gibraltar, including fig whitefly Singhiella simplex (Singh) (Hemiptera: Aleyrodidae)

Seven species of whitefly (Hemiptera: Aleyrodidae) are recorded for the first time from Gibraltar, six of which are non-native introductions. The most notable species are: Bemisia tabaci (Gennadius) complex, which is a major economic agricultural and horticultural pest, and virus vector; and Singhiella simplex (Singh), which is native to S.E. Asia, feeding exclusively on Ficus spp., and a recent introduction to the Mediterranean. Singhiella simplex is also recorded for the first time from Mallorca (Spain) and Morocco.

Bactrocera tryoni (Queensland fruit fly).

B. tryoni, the Queensland fruit fly, is the most costly horticultural pest in Australia and has invaded several countries in the surrounding region (White and Elson-Harris, 1994). It has the potential to spread to many places around the world because of its wide climatic and host range (Meats 1989b; Sutherst et al., 2000) and a tendency to be carried by human travellers at the larval stage inside infested fruit. B. tryoni is a very serious pest of a wide variety of fruits throughout its range. Damage levels can be anything up to 100% of unprotected fruit.

Characterizing the Role of Orco Gene in Detecting Aggregation Pheromone and Food Resources in Protaetia brevitarsis Leiws (Coleoptera: Scarabaeidae)

An accurate olfactory system for recognizing semiochemicals and environmental chemical signals plays crucial roles in survival and reproduction of insects. Among all olfaction-related proteins, olfactory receptors (ORs) contribute to the conversion of chemical stimuli to electric signals and thereby are vital in odorant recognition. Olfactory receptor co-receptor (Orco), one of the most conserved ORs, is extremely essential in recognizing odorants through forming a ligand-gated ion channel complex with conventional ligand-binding odorant receptors. We have previously identified aggregation pheromone in Protaetia brevitarsis (Coleoptera: Scarabaeidae), a native agricultural and horticultural pest in East-Asia. However, to our best knowledge, its olfaction recognition mechanisms are still veiled. To illustrate how P. brevitarsis recognize aggregation pheromone and host plants, in the present study we cloned and sequenced the full-length Orco gene from P. brevitarsis antennae (named PbreOrco) and found that PbreOrco is highly conserved and similar to Orcos from other Coleoptera insects. Our real-time quantitative PCR (qRT-PCR) results showed that PbreOrco is mainly expressed in antenna. We also demonstrated that silencing PbreOrco using RNA interference through injecting dsOrco fragment significantly inhibited PbreOrco expression in comparison with injecting control dsGFP and subsequently revealed using electroantennogram and behavioral bioassays that decreasing PbreOrco transcript abundance significantly impaired the responses of P. brevitarsis to intraspecific aggregation pheromone and prolonged the time of P. brevitarsis spending on food seeking. Overall, our results demonstrated that PbreOrco is crucial in mediating odorant perception in P. brevitarsis.

Precise single base substitution in the shibire gene by CRISPR/Cas9-mediated homology directed repair in Bactrocera tryoni

Background Pest eradication using the Sterile Insect Technique (SIT) involves high-density releases of sterilized males that mate with wild females and ultimately suppress the population. Sterilized females are not required for SIT and their removal or separation from males prior to release remains challenging. In order to develop genetic sexing strains (GSS), conditional traits such as temperature sensitive lethality are required. Results Here we introduce a known Drosophila melanogaster temperature sensitive embryonic lethal mutation into Bactrocera tryoni, a serious horticultural pest in Australia. A non-synonymous point mutation in the D. melanogaster gene shibire causes embryonic lethality at 29 °C and we successfully used CRISPR/Cas9 technology to recreate the orthologous shibire temperature sensitive-1 (shits1) mutation in B. tryoni. Genotypic analyses over three generations revealed that a high fitness cost was associated with the shits1 mutant allele and shits1 homozygotes were not viable at 21 °C, which is a more severe phenotype than that documented in D. melanogaster. Conclusions We have demonstrated the first successful use of CRISPR/Cas9 to introduce precise single base substitutions in an endogenous gene via homology-directed repair in an agricultural pest insect and this technology can be used to trial other conditional mutations for the ultimate aim of generating genetic sexing strains for SIT.

Crystal structure of Epiphyas postvittana pheromone binding protein 3

The insect olfactory system operates as a well-choreographed ensemble of molecules which functions to selectively translate volatile chemical messages present in the environment into neuronal impulses that guide insect behaviour. Of these molecules, binding proteins are believed to transport hydrophobic odorant molecules across the aqueous lymph present in antennal sensilla to receptors present in olfactory sensory neurons. Though the exact mechanism through which these proteins operate is still under investigation, these carriers clearly play a critical role in determining what an insect can smell. Binding proteins that transport important sex pheromones are colloquially named pheromone binding proteins (PBPs). Here, we have produced a functional recombinant PBP from the horticultural pest, Epiphyas postvittana (EposPBP3), and experimentally solved its apo-structure through X-ray crystallography to a resolution of 2.60 Å. Structural comparisons with related lepidopteran PBPs further allowed us to propose models for the binding of pheromone components to EposPBP3. The data presented here represent the first structure of an olfactory-related protein from the tortricid family of moths, whose members cause billions of dollars in losses to agricultural producers each year. Knowledge of the structure of these important proteins will allow for subsequent studies in which novel, olfactory molecule-specific insecticides can be developed.

Towards best-practice management of mistletoes in horticulture

Mistletoe is increasingly being reported as a horticultural pest, infecting many species grown commercially for fruit, nuts, and other food products. Unlike mistletoe impacts on forestry, the published research on mistletoe in horticulture is scant, with management guidelines reliant on anecdotes, un-replicated trials on unrelated species, and often in different countries and growing systems. We have integrated the existing work to summarize information on the most effective control strategies for mistletoe in horticulture, and call attention to the paucity of empirical research. Despite grower interest in growth regulators and herbicides, limited trials suggest chemical treatment of mistletoe is ineffective, consistent with findings from forestry and ornamental trees. Although labour-intensive, ongoing mechanical removal is the most effective strategy to minimize mistletoe impacts but, without information available on effects of mistletoe infection on yield or tree mortality, cost-effectiveness calculations are not possible. Given the range of herbivores that consume mistletoe tissues, biological control may be useful, both to prevent initial infection and also reduce impacts on infected hosts in commercial plantations. To catalyse more research on mistletoes in horticulture, we articulate six priorities for further work, emphasizing the utility of tree crops as model systems to address questions regarding mistletoe ecology and host-parasite dynamics more broadly.

RNA Interference-Mediated Knockdown of Male Fertility Genes in the Queensland Fruit Fly Bactrocera tryoni (Diptera: Tephritidae)

The Queensland fruit fly, Bactrocera tryoni, is Australia’s most important horticultural pest. The Sterile Insect Technique (SIT) has been used to control this species for decades, using radiation to sterilize males before field-release. This method of sterilization can potentially reduce the insects’ abilities to compete for mates. In this study, RNA interference (RNAi) techniques were examined for their potential to sterilize male B. tryoni without adversely affecting mating competitiveness. B. tryoni adults were injected or fed double-stranded RNAs (dsRNAs) targeting spermatogenesis genes (tssk1, topi and trxt); quantitative reverse-transcriptase PCR analyses confirmed that transcript levels were reduced 60–80% for all three genes following injections. Feeding produced a significant gene knockdown for tssk1 and trxt after three days, but interestingly, two genes (trxt and topi) produced an excess of transcripts after 10 days of feeding. Despite these fluctuations in transcript levels, all three dsRNAs impacted the fecundity of treated males, with tssk1- and topi-dsRNA-treated males producing 75% fewer viable offspring than the negative controls. Mating competition assays demonstrated that dsRNA-treated males can actively compete with untreated males. These findings suggest that RNAi technology could serve as an alternative to radiation as a means of sterilizing these insects in an SIT program.

Minor components modulate sensitivity to the pheromone antagonist Z11-14:Ac in male lightbrown apple moth, Epiphyas postvittana (Lepidoptera: Tortricidae) in the field

Epiphyas postvittana is a major horticultural pest in many countries, including New Zealand. Recently, two minor components (E)-11-tetradecen-1-ol and (E)-11-hexadecenyl acetate were found to increase the attraction of a previously identified two-component sex pheromone (95:5 blend of (E)-11-tetradecenyl acetate [E11-14:Ac] and (E,E)-9,11-tetradecadienyl acetate [E9,E11-14:Ac]) of Epiphyas postvittana. We hypothesised that the interaction between these minor components and the behavioural antagonist (Z)-11-tetradecenyl acetate (Z11-14:Ac) would modulate its antagonistic effect. The effect of increasing the ratio (0.5—10%) of Z11-14:Ac was tested in traps baited with E11-14:Ac and E9,E11-14:Ac (95:5), with or without the addition of the two minor compounds. Catch decreased as the percentage of Z11-14:Ac increased (χ2=108.74; d.f.=9; P<0.001). Overall, more moths were caught in traps baited with four- vs two-component lures (χ2=9.81; d.f.=1; P=0.002); in pair-level comparisons, significant differences in catch number between the two- and four-component lures were observed when the E11-14:Ac : Z11-14:Ac ratio was 99:1 (P=0.031) or 98:2 (P=0.047). The addition of the two minor components mitigated the reduced catch effect due to lower proportions of E11-14:Ac and the presence of 1—2% antagonist.