Bishwo Prasad Mainali

The entomopathogenic fungus Metarhizium pingshaense infects diverse insect host species. We present an annotated draft genome of M. pingshaense (Commonwealth Scientific and Industrial Research Organisation [CSIRO] strain M-1000) isolated from a Spodoptera species individual, thereby contributing to future research of M. pingshaense as a potential biological control agent.

Bacillus thuringiensis (Bt) endotoxins are often considered environmentally friendly pest control tools. However, the development of resistance to Bt toxins and emergence of exotic pests necessitate the characterisation of new Bt isolates. This study aims to identify and characterise novel Bt toxins and bioactive compounds that may be utilised to mitigate the impact of Spodoptera frugiperda (fall armyworm, FAW), a polyphagous agricultural pest species that has recently established populations in over 80 countries including Australia. Eight Bt isolates were used in bioassays to ascertain toxicity to FAW neonates. Six Bt isolates (Bt_01‐02 and Bt_05‐08) exhibited potential insecticidal activities. Three isolates including Bt_01 and Bt_07‐08 caused 100% mortality, while Bt_02, Bt_05, and Bt_06 induced 71.27 ± 21.17, 98.44 ± 2.21 and 92.19 ± 11.05% mortality, respectively. Genome analysis was conducted to characterise the toxins and secondary metabolite gene clusters of each isolate. Four isolates (Bt_01, Bt_06, Bt_07, Bt_08) contained the Cry1Na‐partial and Cry1I toxins, while Bt_05 contained Cry2A, Cry1H and Cry1‐like amino acid sequences. In addition, the gene cluster for zwittermicin A, a crystal toxin enhancer, was present in all isolates. However, no known toxins or insecticidal compounds were identified in Bt_02 despite inducing high mortality. The pathogenicity of Bt_02 was also tested against two Australian native pest species: Helicoverpa armigera conferta and H. punctigera. This includes both the susceptible and Cry1Ac‐resistant (Hp9‐3784) lines of H. punctigera. Bt_02 caused 74.88 ± 19.82% mortality in H. armigera, 95.65 ± 6.15% mortality in H. punctigera and 90.91 ± 12.86% mortality in Hp9‐3784. This suggests that Bt_02 may possess unknown toxins or bioactive compounds responsible for its effectiveness against three species of lepidopteran pests including those that exhibited Cry1Ac resistance.

Previously, we assessed the pathogenicity of eleven endemic entomopathogenic fungi (EPF), including six Beauveria isolates, four Metarhizium isolates, and one M. pingshaense, against the agricultural pest Spodoptera frugiperda (fall armyworm, FAW). We found that four Beauveria and one Metarhizium isolates were effective, with Beauveria isolates B-0571 and B-1311 exhibiting high mortality within 24 h post-spore application. This study aimed to identify and characterise the entomopathogenesis mechanisms of these isolates as potential FAW biocontrol agents. All Beauveria isolates were determined as B. bassiana, the Metarhizium isolates as two M. robertsii, one M. majus, and an unknown Metarhizium species. Despite the high mortality from B-0571 and B-1311 isolates, scanning electron microscopy showed no fungal spore germination on dead larvae 24 h after spore application. Four insecticide compound gene clusters, i.e., bassianolide, beauvericin, beauveriolide, and oosporein, were identified and characterised in all B. bassiana isolates. These compounds are hypothesised to contribute to the high early mortality rates in FAWs. Identifying and characterising gene clusters encoding these insecticide compounds in B-0571 and B-1311 will contribute to a better understanding of the entomopathogenicity of these isolates that will be vital to developing these EPF isolates as sustainable alternative FAW biocontrol agents.

Previously, we assessed the pathogenicity of eleven endemic entomopathogenic fungi (EPF) including six Beauveria isolates, four Metarhizium isolates, and one M. pingshaense, against the agricultural pest Spodoptera frugiperda (fall armyworm, FAW). We found that four Beauveria and one Metarhizium isolates were effective, with Beauveria isolates B-0571 and B-1311 exhibited high mortality within 24 hours post spore application. This study aimed to identify and characterise entomopathogenesis mechanisms of these isolates as potential FAW biocontrol agents. All Beauveria isolates were determined as B. bassiana, the Metarhizium isolates as two M. robertsii, one M. majus, and an unknown Metarhizium species. Despite the high mortality from B-0571 and B-1311 isolates, scanning electron microscopy showed no fungal spore germination on dead larvae 24 hours after spore application. Four insecticide compound gene clusters, i.e., bassianolide, beauvericin, beauveriolide, and oosporein, were identified and characterised in all B. bassiana isolates. These compounds are hypothesised to contribute to the high early mortality rates in FAW. Identifying and characterising of gene clusters encoding these insecticide compounds in B-0571 and B-1311 will contribute to better understanding the entomopathogenicity of these isolates that will be vital to developing these EPF isolates as sustainable alternative FAW biocontrol agents.

The global application of Transeius montdorensis (Acari: Phytoseiidae) as a biological control agent across various protected crops has proven effective against a range of insect pests like thrips and whiteflies, as well as pest mites like broad mites and russet mites. Optimization of rearing T. montdorensis under laboratory conditions is crucial for further studies of this species to improve their application in Integrated Pest Management (IPM) programs. Here, we evaluated the development and reproduction of T. montdorensis when fed on four different diets, including cattail pollen ( Typha latifolia ), living dried fruit mites ( Carpoglyphus lactis ), frozen C. lactis eggs, and a mixed diet of frozen C. lactis eggs and T. latifolia pollen. Females consuming the mixed diet exhibited superior total fecundity and daily oviposition rate, along with the highest intrinsic rate of increase (r _m ) and net productive rate (R ₀ ) among all diets tested. The immature period was significantly longer for mites on a diet of living C. lactis compared to those on other diets. Importantly, utilizing frozen C. lactis eggs and T. latifolia pollen mitigates the risk of infestation and contamination from the living dried fruit mites, which is important for laboratory and field settings when releasing the predator colonies. Our findings not only present an optimized rearing method for predatory mites under laboratory conditions but also suggest potential broader applications for enhancing the effectiveness and sustainability of biological control strategies across various agroecosystems and reducing dependency on chemical pesticides.

The development of effective pest management strategies for Spodoptera frugiperda is a high priority for crop protection across its invasive ranges. Here, we examined six Beauveria and five Metarhizium fungal isolates against this pest. Two Beauveria isolates (B-0571, B-1311) induced high mortality toward 3rd and 6th instar caterpillars and adults. For B-0571 mortality was 82.81 ± 5.75%, 61.46 ± 6.83%, and 93.75 ± 3.61%, and 73.72 ± 2.51%, 71.88 ± 5.41%, and 97.92 ± 2.08% for B-1311, with deaths in caterpillars largely occurring under 24 h (3rd instar control 0.74 ± 0.33%, B-0571 73.96 ± 7.85% and B-1311 62.08 ± 3.67%; 6th instar control 0%, B-0571 66.67% ± 11.02% and B-1311 62.5% ± 9.55%). Infection from both Beauveria isolates fully prevented reproduction in surviving S. frugiperda females. In contrast, all five Metarhizium isolates tested and the remaining four Beauveria isolates exhibited lower virulence. The discovery of two highly virulent Beauveria fungal isolates to S. frugiperda opens avenues to develop novel biological control tools against this highly invasive pest.

The Queensland fruit fly (Q‐fly), Bactrocera tryoni (Froggatt), is a major horticultural pest in Australia. Sterile insect technique (SIT) is increasingly used in area‐wide integrated management of Q‐fly, as well as for eradication of outbreaks in Q‐fly‐free regions. Quality control (QC) procedures are important to monitor and maintain high standards of mass‐reared insects for SIT. Effective use of QC procedures, and the setting of meaningful standards, requires a clear understanding of the short‐term factors that can cause changes in key parameters, such as failures in production systems, and also of the longer‐term effects of domestication on the performance of mass‐reared colonies. Artificial rearing conditions impose substantial selection pressures that can lead to changes in performance, and hence changes in the expected baseline values in QC assays. We investigated the effects of domestication on QC parameters of old and young Q‐fly colonies originating from three distant geographical locations in Australia: Sydney (Generation >100 and 7), Brisbane (Generation 59 and 14) and Cairns (Generation 29 and 9). Standard QC parameters including development time, pupal recovery, pupal weight, adult emergence, sex ratio, flight ability, fecundity, egg hatchability and longevity were assessed. Regardless of their origin, older Q‐fly colonies had lower egg hatchability, shorter developmental time, higher fecundity, higher survival under stress and greater longevity. We emphasize the importance of understanding the effects of continued domestication on QC measures when setting standards for assessment of fruit fly strains used in SIT programmes, particularly when using strains that are regularly replaced or infused with wild stock.

Opiine braconids are parasitoids of the immature stages of frugivorous tephritids. The female wasp lays her eggs into the eggs or larvae of the fruit fly host, where the immature wasp develops before emerging as a next‐generation adult from the now dead host pupal case. In support of a new generation of Australian fruit fly parasitoid research, this paper comprehensively reviews what is known about the Australian fruit fly infesting opiines. Based on the most recent taxonomic revision 11 fruit fly infesting opiine species are documented to occur in Australia, but we consider as doubtful the record forDiachasmimorpha longicaudataand consider the record forFopius illusoriusto be tentative without further collections. We identify that the systematics and taxonomy of the Australian native fruit fly infesting opiines are in urgent need of further work. The history of fruit fly biological control in Australia is comprehensively reviewed, including the export of native Australian opiines for fruit fly control elsewhere in the world. Australia was actively involved in three major classical biological control programmes against fruit flies from the turn of the 1900s until the mid‐1960s. Despite the introduction of 11 opiine species, plus numerous other natural enemies, onlyFopius arisanusestablished in eastern Australia, where in South‐east Queensland it can now cause between 30 and 40% mean parasitism. In addition to the exoticF. arisanus, the native speciesDiachasmimorpha kraussiiandDiachasmimorpha tryonialso cause fruit fly parasitism in agriculturally important crops: both species have also been liberated widely outside of Australia for fruit fly control. Other Australian opiines have not been reared from flies infesting commercial crops and appear biologically restricted to the fruits and environs of Australian east‐coast rainforests. The biology literature forD. tryoniandD. kraussiiis comprehensively reviewed, while forF. arisanus, already reviewed elsewhere, key literature only is covered. Forward looking, we consider the potential for inoculative or inundative releases of opiines in areas where they do not currently occur to be good, while conservation biological control may help to increase the impacts of parasitoids in areas where they are already established.

In Sterile Insect Technique (SIT) programs, the released sterile insects must attain sexual maturity at an early adult age so that a large proportion survive to mature and contribute to reducing reproduction of pest populations. Previous field studies based on release of adult Queensland fruit fly, Bactrocera tryoni (Froggatt) (‘Q‐fly’), at 2 days of age found a significant increase in the recapture of mature male Q‐flies when they were provided methoprene or raspberry ketone (RK) before release. SIT for Q‐flies has subsequently adopted release at 5 days of age; there is now a contextual need to re‐assess the merit of methoprene and RK supplements. We assessed field abundance and dispersal of mature sterile male Q‐flies that had been held for 5 days on pre‐release standard diets of sugar and yeast hydrolysate (3:1) (control) with and without methoprene or RK supplements. Overall, the proportion of control‐ and RK‐treated flies recaptured was higher than the proportion of methoprene‐treated flies recaptured. We found no evidence that either methoprene or RK supplements yielded improvements over the control diet alone concerning abundance and dispersal of the male Q‐flies. Laboratory studies indicate that while the doses of methoprene (control + methoprene at 0.05% or 0.5%) and RK (control + RK at 1.25% or 5%) used in field releases at 2 days of age did not affect male longevity when sustenance was provided, these doses resulted in reduced longevity when no sustenance was provided. Furthermore, all doses of methoprene and RK used in field releases at 5 days of age reduced male longevity regardless of sustenance. At 5 days of age, the flies are already in an advanced state of maturity when released and in this context, it appears that additional pre‐release treatments of methoprene and RK provide no further improvement and may even be detrimental when nutrition is scarce.

The sterile insect technique (SIT) is a sustainable pest management tool based on the release of millions of sterile insects that suppress reproduction in targeted populations. Success of SIT depends on survival, maturation, dispersal, and mating of released sterile insects. Laboratory and field cage studies have demonstrated that dietary supplements of methoprene and raspberry ketone (RK) promote sexual maturation of adult Queensland fruit fly, Bactrocera tryoni (Froggatt), and may hence shorten the delay between release and maturity in the field. We investigated the effects of methoprene and RK dietary supplements on field abundance of sexually mature sterile Q-flies relative to untreated flies fed only sugar and yeast hydrolysate before release at 2 d of age. Compared with untreated flies, more methoprene- and RK-treated flies were recaptured in cuelure traps to which only sexually mature males are attracted. At distances of 100 and 200 m from the release point, recapture rates were higher for methoprene- and RK-treated flies than for untreated flies, but at 300 m recapture rates were low and were similar for treated and untreated flies. Rainfall, relative humidity, wind speed, and wind direction did not affect recapture rates, but temperature was positively correlated with recapture rates for all treatments. There was a strong correlation between the number of sterile and wild flies caught in traps, indicating co-location in the field. Dietary supplements of methoprene and RK can substantially increase abundance of sexually mature sterile male Q-flies in the field following release as 2-d-old immature adults.