Withania somnifera, commonly referred to as Indian Ginseng, is a medicinally important plant that can be found in many regions of the world, such as South Africa and India. Withanolides are key secondary metabolites responsible for conferring the plant its unique medicinal properties. The reaction of W. somnifera to salt stress, in particular the possible effects on its withanolide production, has yet to be explored. Under salt stress, germination efficiency dropped by 17.8% and caused physiological damage to emerging roots. A transcriptome analysis of salt-stressed leaves revealed the differential regulation of 3048 transcripts, with gene ontology analysis indicating an increased transcript abundance associated with phenylpropanoid, plant hormone signaling, detoxification response, concomitant with reduced growth, energy metabolism, and protein modification. Most withanolide pathway genes were significantly downregulated. Notably, 26 transcription factor families, particularly AP2/ERF, NAC, GRAS, and HB-WOX members, as well as 10 Late Embryogenesis-Abundant members, predominantly dehydrins, were significantly differentially regulated, suggesting their potential involvement in the salt stress response of W. somnifera. This study provides valuable insights into the molecular responses of W. somnifera to salt stress, highlighting potential targets for future research and subsequently for withanolide production, which has vast potential in the pharmaceutical industry.

The rapid and accurate identification of invasive pest insects at quarantine stations or ports of entry is crucial for preventing their establishment and spread in new territories. Here, we present an effective and rapid genetic identification technique utilising loop-mediated isothermal amplification (LAMP) to detect the presence of the peach fruit fly, Bactrocera zonata. The LAMP assay was designed to target the cytochrome oxidase subunit I (COI) gene region of B. zonata, ensuring high specificity and sensitivity, and to discriminate it from a range of other economically important Bactrocera species. By utilising isothermal conditions, LAMP eliminates the need for lengthy thermal cycling and enables rapid amplification of target DNA within a short period. Additionally, a rapid DNA extraction method combined with the use of an intercalating UV fluorescent dye allows for fast and reliable interpretation of results without the need for specialised equipment. Validation studies were conducted using known samples of B. zonata, as well as other Bactrocera species commonly encountered in quarantine settings including B. dorsalis, B. correcta, B. tryoni, B. oleae, B. cucurbitae and B. tau. The power of this LAMP assay lies in its specificity to accurately distinguish B. zonata from other closely related Bactrocera species. Moreover, the assay exhibits a remarkably short turnaround time, with results obtained in under an hour, facilitating timely decision-making at quarantine stations.

Atmospheric oxygen, which is essential for energy metabolism, can directly influence an animal's heat tolerance by affecting oxygen transport processes, especially in those living in oxygen-poor environments such as plant tissues, underground or aquatic environments. Yet, oxygen availability and heat tolerance are rarely studied together, limiting our ability to predict their combined effects on insect performance. This study examines the larval tolerance of a large xylophagous cerambycid beetle Cacosceles newmannii to combined hypoxic and thermal stress using performance assays (duration of righting response) coupled with metabolomic and transcriptomic analyses. Metabolomic profiling showed that most metabolites were downregulated in the body but upregulated in the haemolymph as stress increased. Transcriptomic profiles clustered primarily by temperature (25°C vs 35°C), independent of oxygen level. Cacosceles newmannii appeared capable of modulating its performance to reduce the energy costs and physiological damage induced by hypoxia. This suggested a high baseline hypoxia tolerance rather than a rapid plastic (induced) physiological hypoxia response, probably due to the species' endophytic lifestyle. Conversely, thermal stress led to a predictable increase in metabolic activity but did not markedly affect performance, triggering adjustments to maintain cellular functions while limiting the impact of stresses expected under conditions of high temperature, such as desiccation. In short, our study highlights the distinct metabolic pathways mobilised to cope with hypoxic versus thermal stress, emphasizing the importance of integrated approaches in understanding insect responses to environmental challenges. These findings have significant implications for understanding the ecology of the species, with applications for pest management and sustainable agriculture in the context of climate change.

When invasive populations establish in regions far from their origin, they may accumulate deleterious mutations that limit population viability and later expansion. Invasions stemming from such bridgehead populations may experience further sequential bottlenecks. However, deleterious mutations can be masked or eliminated when populations outbreed with other lineages. Here, we analyse global invasions of a species complex of persistently inbreeding ambrosia beetles, using genomic data (N=247) from invasive populations in Africa, North America and Australia, and from native populations in Asia. We mostly focus on one species of this complex (Euwallacea fornicatus) which poses a severe threat to tree species worldwide and is rapidly expanding its global range. We uncover a single lineage of this species across California, South Africa, and Western Australia, involving an invasive bridgehead and containing almost no nuclear genetic variation. In South Africa we identify a second lineage that has repeatedly hybridised with the first lineage. Genetic patterns in the native range indicate that such opportunistic outbreeding may be common. Despite lacking nuclear variation, the first lineage contained two CO1 haplotypes that were also observed in every hybrid lineage, pointing to heteroplasmy and possible hybrid origins of this lineage. Native populations had fewer missense mutations than invasive populations, indicating that opportunistic outbreeding may help purge fixed deleterious mutations when local lineage diversity is high. These findings highlight the importance of outbreeding even when inbreeding is common, and they demonstrate the biosecurity threat posed by subsequent gene flow into invasive populations.

When invasive populations establish in regions far from their origin, they risk accumulating harmful mutations (genetic load) that limit population viability and subsequent spread. This may be exacerbated by the multiple, sequential bottlenecks experienced when invasions stem from a bridgehead population. However, populations may be able to purge genetic load when they can outbreed with other lineages from subsequent invasions. Here, we analyse global invasions of a species complex of persistently inbreeding ambrosia beetles, using genomic data (N=247) from invasive populations in Africa, North America and Australia, and from native populations in Asia. We focus particularly on one species of this complex ( Euwallacea fornicatus ) which poses a catastrophic threat to tree species worldwide and is rapidly expanding its global range. We uncover a single lineage of this species across South Africa, California and Western Australia, derived from an invasive bridgehead and containing almost no nuclear genetic variation. In South Africa we identify a second lineage that has repeatedly hybridised with the first lineage. Genetic patterns in the native range indicate that such opportunistic outbreeding may be common. Although purifying selection was evident in all lineages, native populations had fewer missense mutations than invasive populations, suggesting that opportunistic outbreeding may help purge fixed deleterious mutations when local lineage diversity is high. These findings show how inbreeding depression can affect populations even where inbreeding is common, and they highlight the biosecurity threat posed by subsequent gene flow into invasive populations.

Little is known about how past and current thermal environment interact to determine dispersal in novel environments, and thus invasion potential of major agricultural pests. In this study, we tested experimentally how adult thermal history affects flight-related traits under diverse thermal conditions in three major agricultural pests of invasion concern: Bactrocera dorsalis, Bactrocera zonata and Ceratitis capitata. Across all species, the main factor affecting flight performance was body mass, with heavier individuals performing best. Both current and, to a lesser extent, past thermal environments affected flight performance and periodicity (flight interruptions), although their interaction was rarely significant. Furthermore, we show that 20 degrees C acclimation can have deleterious effects regardless of thermal conditions during flight, particularly in Bactrocera females, which had decreased flight performance and increased flight periodicity when acclimated at this temperature. The thermal environment during flight affected both flight periodicity and performance mainly in females of C. capitata and B. dorsalis, while only flight performance was affected in B. zonata males. When compared to warmer temperatures, flight at 20 degrees C tended to reduce flight performance, but flight periodicity increased at 30 degrees C compared to 20 degrees C. Overall, the flight performance of B. dorsalis was greater than that of C. capitata or B. zonata, regardless of the past and present thermal environment, but flight periodicity was lower in C. capitata. In all three species investigated, optimal flight performance occurs around 25 degrees C, and flies will therefore cover more distance around this temperature. The lack of interaction between past and current thermal environments in these three species indicates that flight-related traits may have low thermal plasticity. Finally, our results suggest that a reduction in thermal variability and average temperatures increasing temperate areas by a few degrees with climate change may facilitate movements of tropical tephritid pests in new areas at higher latitudes.Read the free for this article on the Journal blog.

The Polyphagous Shot Hole Borer (PSHB; Euwallacea fornicatus, Coleoptera: Curculionidae: Scolytinae) is an invasive and destructive tree pest. To assess whether thermal acclimation influences E. fornicatus locomotion performance (i.e., induced plastic responses) that may influence invasion potential, beetles were acclimated to three temperatures (18 °C, 25 °C, and 32 °C), and four locomotion traits were measured across six temperatures (13 °C, 18 °C, 23 °C, 28 °C, 33 °C and 38 °C) per acclimation group to construct thermal performance curves, capturing critical thermal minimum (Tmin), critical thermal maximum (Tmax), thermal breadth (Tbr), optimal performance rate (Umax). Substantial plasticity of performance curves was found in E. fornicatus. Generally, cold (18 °C) acclimation increased the thermal range of several locomotor performance traits without affecting performance levels, thereby supporting the colder-is-better hypothesis. To assess the consequences of these plastic responses, using the thermal performance curves established here, movement rates of E. fornicatus in an at-risk orchard area in South Africa were predicted across seasons while considering artificial warm and cold spells. Cold-acclimated beetles exhibited the highest cumulative distance traveled in both summer and winter, while warm-acclimated beetles had the lowest. Therefore, short-term thermal variation significantly influenced E. fornicatus locomotion performance, with cold acclimation notably improving dispersal across a wide range of thermal conditions. These findings highlight the importance of considering recent thermal history when predicting E. fornicatus invasion potential. By integrating these data with microclimatic conditions and functional models, this study offers valuable insights for predicting E. fornicatus spread, informing targeted management strategies, and refining spatially explicit risk assessments to mitigate the impacts of this invasive pest.

Withania somnifera, commonly referred to as Indian Ginseng, is a medicinally important plant that can be found in many regions of the world, such as South Africa and India. Withanolides are key secondary metabolites responsible for conferring the plant its unique medicinal properties. The reaction of W. somnifera to salt stress, in particular the possible effects on its withanolide production, have yet to be explored. Under salt stress, germination efficiency dropped by 17.8% and caused physiological damage to emerging roots. A transcriptome analysis of salt stressed leaves revealed the differential regulation of 3 048 transcripts, with gene ontology analysis indicating an increased detoxification response concomitant with reduced growth, energy metabolism and protein modification. Notably, 26 transcription factor families, particularly AP2/ERF, NAC, GRAS and HB-WOX members as well as 10 Late Embryogenesis-Abundant members, predominantly dehydrins, were significantly differentially regulated, suggesting their potential involvement in the salt stress response of W. somnifera. Although the withanolide pathway genes showed limited response to the salt treatment, three genes in the pathway (HMGS, FPPS and WsCYP98A) were significantly downregulated. This study provides valuable insights into the molecular responses of W. somnifera to salt stress, highlighting potential targets for future research and subsequently for withanolide production which has vast potential in pharmaceutical industry.

Knowing how environmental conditions affect performance traits in pest insects is important to improve pest management strategies. It can be informative for monitoring, but also for control programs where insects are mass-reared, and field-released. Here, we investigated how adult thermal acclimation in sterile Bactrocera dorsalis affects dispersal and recapture rates in the field using a mark-release-recapture method. We also considered how current abiotic factors may affect recapture rates and interact with thermal history. We found that acclimation at 20 or 30 °C for 4 d prior to release reduced the number of recaptures in comparison with the 25 °C control group, but with no differences between groups in the willingness to disperse upon release. However, the deleterious effects of acclimation were only detectable in the first week following release, whereafter only the recent abiotic conditions explained recapture rates. In addition, we found that recent field conditions contributed more than thermal history to explain patterns of recaptures. The two most important variables affecting the number of recaptures were the maximum temperature and the average relative humidity experienced in the 24 h preceding trapping. Our results add to the handful of studies that have considered the effect of thermal acclimation on insect field performance, but notably lend support to the deleterious acclimation hypothesis among the various hypotheses that have been proposed. Finally, this study shows that there are specific abiotic conditions (cold/hot and dry) in which recaptures will be reduced, which may therefore bias estimates of wild population size.

The false codling moth (FCM; Thaumatotibia leucotreta, Meyrick; Lepidoptera: Tortricidae) is a highly polyphagous, major agricultural pest indigenous to sub-Saharan Africa. With growing international trade, there is an increasing concern about introducing this pest into other countries. In South Africa, FCM poses a risk to multiple crops, and is currently suppressed through a combination of chemical, microbial, cultural, augmentative biological control, and the sterile insect technique. Compared with other lepidopteran agricultural pests, such as codling moth Cydia pomonella, genetic and other -omic resources for FCM have not been as well developed and/or not made publicly available to date. The need to develop genomic resources to address questions around insecticide resistance, chemosensory capabilities, and ultimately, develop novel control methods (e.g. gene editing) of this pest is highlighted. In this study, an adult male was sequenced using long-read PacBio Sequel II reads and Illumina NextSeq short reads and assembled using a hybrid assembly pipeline and Pilon error correction. Using the chromosome-level genome assembly of Cy. pomonella, we performed comparative analysis, arranged FCM scaffolds to chromosomes, and investigated genetic variation related to insecticide resistance and chemosensory capabilities. This work provides a platform upon which to build future genomic research on this economically important agricultural pest.

The factors that influence parasite associated bacterial microbial diversity and the geographic distributions of bacteria are not fully understood. In an effort to gain a deeper understanding of the relationship between the bacterial diversity of Ctenocephalides fleas and host species and the external environment, we conducted a metagenetic analysis of 107 flea samples collected from 8 distinct sampling sites in South Africa. Pooled DNA samples mostly comprising of 2 or 3 individuals sampled from the same host, and belonging to the same genetic cluster, were sequenced using the Ion PGM™ Hi-Q™ Kit and the Ion 316™ Chip v2. Differences were detected in the microbiome compositions between Ctenocephalides felis , Ctenocephalides canis and Ctenocephalides connatus. Although based on a small sample, C. connatus occurring on wildlife harboured a higher bacterial richness when compared to C. felis on domestic animals. Intraspecific differences in the microbial OTU diversity were detected within C. f. felis that occurred on domestic cats and dogs. Different genetic lineages of C. f. felis were similar in microbial compositions but some differences exist in the presence or absence of rare bacteria. Rickettsia and Bartonella OTU's identified in South African cat fleas differ from those identified in the USA and Australia. Intraspecific microbial compositions also differ across geographic sampling sites. Generalized dissimilarity modelling showed that temperature and humidity are potentially important environmental factors explaining the pattern obtained.

BACKGROUND:Thermal history may induce phenotypic plasticity in traits that affect performance and fitness. One type of plastic response triggered by thermal history is acclimation. Because flight is linked to movement in the landscape, trapping and detection rates, and underpins the success of pest management tactics, it is particularly important to understand how thermal history may affect pest insect flight performance. We investigated the tethered-flight performance of Ceratitis capitata, Bactrocera dorsalis and Bactrocera zonata (Diptera: Tephritidae), acclimated for 48 h at 20, 25 or 30 °C and tested at 25 °C. We recorded the total distance, average speed, number of flight events and time spent flying during 2-h tests. We also characterized morphometric traits (body mass, wing shape and wing loading) that can affect flight performance. RESULTS:The main factor affecting most flight traits was body mass. The heaviest species, B. dorsalis, flew further, was faster and stopped less often in comparison with the two other species. Bactrocera species exhibited faster and longer flight when compared with C. capitata, which may be associated with the shape of their wings. Moreover, thermal acclimation had sex- and species-specific effects on flight performance. Flies acclimated at 20 °C stopped more often, spent less time flying and, ultimately, covered shorter distances. CONCLUSION:Flight performance of B. dorsalis is greater than that of B. zonata and C. capitata. The effects of thermal acclimation are species-specific. Warmer acclimation temperatures may allow pest fruit flies to disperse further and faster. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The growing demands on protein producers and the dwindling available resources have made Hermetia illucens (the black soldier fly, BSF) an economically important species. Insights into the genome of this insect will better allow for robust breeding protocols, and more efficient production to be used as a replacement of animal feed protein. The use of microRNA as a method to understand how gene regulation allows insect species to adapt to changes in their environment, has been established in multiple species. The baseline and life stage expression levels established in this study, allow for insight into the development and sex-linked microRNA regulation in BSF. To accomplish this, microRNA was extracted and sequenced from 15 different libraries with each life stage in triplicate. Of the total 192 microRNAs found, 168 were orthologous to known arthropod microRNAs and 24 microRNAs were unique to BSF. Twenty-six of the 168 microRNAs conserved across arthropods had a statistically significant (p < 0.05) differential expression between Egg to Larval stages. The development from larva to pupa was characterized by 16 statistically significant differentially expressed microRNA. Seven and 9 microRNA were detected as statistically significant between pupa to adult female and pupa to adult male, respectively. All life stages had a nearly equal split between up and down regulated microRNAs. Ten of the unique 24 miRNA were detected exclusively in one life stage. The egg life stage expressed five microRNA (hil-miR-m, hil-miR-p, hil-miR-r, hil-miR-s, and hil-miR-u) not seen in any other life stages. The female adult and pupa life stages expressed one miRNA each hil-miR-h and hil-miR-ac respectively. Both male and female adult life stages expressed hil-miR-a, hil-miR-b, and hil-miR-y. There were no unique microRNAs found only in the larva stage. Twenty-two microRNAs with 56 experimentally validated target genes in the closely related Drosophila melanogaster were identified. Thus, the microRNA found display the unique evolution of BSF, along with the life stages and potential genes to target for robust mass rearing. Understanding of the microRNA expression in BSF will further their use in the crucial search for alternative and sustainable protein sources.

The ambrosia beetle Euwallacea fornicatus (Polyphagous Shot Hole Borer; PSHB), native to Asia, was documented in South Africa for the first time in 2012. Death of susceptible host trees is caused by blocking of xylem tissues by the mutualistic plant-pathogenic fungus, Fusarium euwallaceae and extensive tunnelling by the beetles into the sapwood. Within a few years, PSHB has spread from its putative entrance point in the coastal province of KwaZulu-Natal to nearly every other province in South Africa. This study serves as a preliminary assessment of dispersal pathways and population genetic relationships of PSHB in South Africa. PSHB individuals were collected from five provinces across South Africa. In addition, data on PSHB from three provinces in its native range in China and invasive PSHB from California were also generated here and supplemented by sequence data of PSHB available from GenBank. Comparisons of Cytochrome Oxidase Subunit I (COI) sequences of PSHB in South Africa revealed a nearly homogenous population. The majority of individuals have the same haplotype as is present in California, Israel and Vietnam (H33). A second haplotype was present in only two localities in KwaZulu-Natal and the Western Cape. This haplotype is also present in Vietnam and China (H38). The placement of the two haplotypes identified within South Africa, into different haplogroups suggests more than one invasion event. This pilot project justifies the use of more comprehensive genomic tools to finely map the relationships, global invasion pathways and within-country dispersal patterns of PSHB to better inform management of this invasive species.

The black soldier fly (BSF), Hermetia illucens, is a promising candidate for the emerging insect farming industry with favourable characteristics for both bioremediation and production of animal delivered nutritive and industrial compounds. The genetic management of commercial colonies will become increasingly important for the sustainability of the industry. However, r-selected life history traits of insects pose challenges to conventional animal husbandry and breeding approaches. In this study, the long-term genetic effects of mass-rearing were evaluated as well as mating systems in the species to establish factors that might influence genetic diversity, and by implication fitness and productivity in commercial colonies. Population genetic parameters, based on microsatellite markers, were estimated and compared amongst two temporal wild sampling populations and four generations (F28, F48, F52, and F62) of a mass-reared colony. Furthermore, genetic relationships amongst mate pairs were evaluated and parentage analysis was performed to determine the oc-currence of preferential mate choice and multiple paternity. The mass-reared colony showed a reduction in genetic diversity and evidence for inbreeding with significant successive generational genetic differentiation from the wild progenitor population. Population-level analysis also gave the first tentative evidence of positive assortative mating and genetic polyandry in BSF. The homoge-neity of the mass-reared colony seems to result from a dual action caused by small effective popu-lation size and increased homozygosity due to positive assortative mating. However, the high ge-netic diversity in the wild and a polyandrous mating system might suggest the possible restoration of diversity in mass-reared colonies through augmentation with the wild population.

We present the first sequencing results after separation of the X chromosome of Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae), the largest known X chromosome described to date, using flow cytometry. The X chromosome of D. noxia is 0.1824 pg (1C) and an estimated 178.4 Mb (1C) in size. Mapping confirmed that the X chromosome contains 13,799 protein coding genes, but with a slight bias towards GC richness when compared to the complete D. noxia genome.

Diuraphis noxia Kurdjumov, 1913 (Hemiptera: Aphididae), commonly known as the Russian wheat aphid, is a devastating pest of wheat and barley. Although fourteen sources of resistance (Dn genes) have been identified to date, none have been cloned. In this study a mapping population of 373 F3/4 segregating wheat plants and ‘Chinese Spring’ 7D deletion lines were screened with AFLP, SSR and EST markers. From the marker screens, a 122.8 cM genetic map was constructed placing the Dn1 resistance gene on chromosome 7DS near the centromere, in bin 7DS5-0.36-0.62 proportionally to the physical ‘Chinese Spring’ deletion 7D map. The map contains 38 new AFLP markers, one microsatellite (Xgwm111) and two EST markers. Of all the markers, AFLP E-ACT/M-CTG_0270.84 showed the closest linkage to Dn1 and were mapped at 3.5 cM, whereas EST markers RGA2-29_30 and SSH-RGA2 were mapped at 15.3 cm and 15.9 cM, respectively, from Dn1. An AFLP marker, E-ACA/M-CAG-257, located 21.4 cM from the Dn1 resistance gene, was successfully cloned and sequenced. The marker sequence was identified to belong to the interspersed repeat class of the subfamily CMC-EnSpm.