Genomic evidence for role of inversion 3RP of Drosophila melanogaster in facilitating climate change adaptation.

Chromosomal inversion polymorphisms are common in animals and plants, and recent models suggest that alternative arrangements spread by capturing different combinations of alleles acting additively or epistatically to favour local adaptation. It is also thought that inversions typically maintain favoured combinations for a long time by suppressing recombination between alternative chromosomal arrangements. Here, we consider patterns of linkage disequilibrium and genetic divergence in an old inversion polymorphism in Drosophila melanogaster (In(3R)Payne) known to be associated with climate change adaptation and a recent invasion event into Australia. We extracted, karyotyped and sequenced whole chromosomes from two Australian populations, so that changes in the arrangement of the alleles between geographically separated tropical and temperate areas could be compared. Chromosome-wide linkage disequilibrium (LD) analysis revealed strong LD within the region spanned by In(3R)Payne. This genomic region also showed strong differentiation between the tropical and the temperate populations, but no differentiation between different karyotypes from the same population, after controlling for chromosomal arrangement. Patterns of differentiation across the chromosome arm and in gene ontologies were enhanced by the presence of the inversion. These data support the notion that inversions are strongly selected by bringing together combinations of genes, but it is still not clear if such combinations act additively or epistatically. Our data suggest that climatic adaptation through inversions can be dynamic, reflecting changes in the relative abundance of different forms of an inversion and ongoing evolution of allelic content within an inversion.

Polymorphism in the neurofibromin gene, Nf1, is associated with antagonistic selection on wing size and development time in Drosophila melanogaster.

In many invertebrates, body size shows genetically based clines, with size increasing in colder climates. Large body size is typically associated with prolonged development times. We consider variation in the CNS-specific gene neurofibromin 1 (Nf1) and its association with body size and development time. We identified two major Nf1 haplotypes in natural populations, Nf1-insertion-A and Nf1-deletion-G. These haplotypes are characterized by a 45-base insertion/deletion (INDEL) in Nf1 intron 2 and an A/G synonymous substitution (locus L17277). Linkage disequilibrium (LD) between the INDEL and adjacent sites is high but appears to be restricted within the Nf1 gene interval. In Australia, the frequency of the Nf1-insertion-A haplotype increases with latitude where wing size is larger, independent of the chromosomal inversion In(3R)Payne. Unexpectedly, the Nf1-insertion-A haplotype is negatively associated with wing size. We found that the Nf1-insertion-A haplotype is enriched in females with shorter development time. This suggests that the Nf1 haplotype cline may be driven by selection for development time rather than size; females from southern (higher latitude) D. melanogaster populations maintain a rapid development time despite being relatively larger, and the higher incidence of Nf1-insertion-A in Southern Australia may contribute to this pattern, whereas the effects of the Nf1 haplotypes on size may be countered by other loci with antagonistic effects on size and development time. Our results point to the potential complexity involved in identifying selection on genetic variants exhibiting pleiotropic effects when studies are based on spatial patterns or association studies.

The pest sap beetle Carpophilus (Myothorax) truncatus Murray, 1864 (Coleoptera: Nitidulidae)-a new synonymy and a related new species of Carpophilus.

Carpophilus truncatus Murray 1864, is a species of sap beetle which has been recorded from many countries worldwide, and has become recognised as an important pest of nuts. In this study, we present a re-description of C. truncatus including diagnostic photographic images of the adults and larvae, and demonstrate that Carpophilus jarijari Powell & Hamilton, 2019 is a junior subjective synonym of C. truncatus. Information about the species' distribution in Australia is updated. DNA barcode sequence data for C. truncatus is reviewed and augmented to enable differentiation from other morphologically similar Carpophilus species that are associated with nuts as hosts, including the cosmopolitan Carpophilus dimidiatus (Fabricius, 1792), for which C. truncatus has sometimes been misidentified. This analysis revealed that existing reference DNA sequences of "C. dimidiatus" consist of three highly genetically divergent lineages, representing three species: the cosmopolitan C. dimidiatus, the widespread C. truncatus, and a newly described species, Carpophilus imitatus sp. nov., known from south-eastern Asia and Australia.

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

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.

Development of a cost-effective, morphology-preserving method for DNA isolation from bulk invertebrate trap catches: Tephritid fruit flies as an exemplar.

Insect identification and preservation of voucher specimens is integral to pest diagnostic and surveillance activities; yet bulk-trapped insects are a diagnostic challenge due to high catch numbers and the susceptibility of samples to environmental damage. Many insect trap catches rely on examination of morphological characters for species identifications, which is a time consuming and highly skilled task, hence there is a need for more efficient molecular approaches. Many bulk DNA extraction methods require destructive sampling of specimens, resulting in damaged, or fully destroyed, voucher specimens. We developed an inexpensive, rapid, bulk DNA isolation method that preserves specimens as pinned vouchers to a standard that allows for post-extraction morphological examination and inclusion in insect reference collections. Our protocol was validated using a group of insects that are time-consuming to identify when trapped in large numbers-the dacine fruit flies (Diptera: Tephritidae: Dacinae). In developing our method, we evaluated existing protocols against the following criteria: effect on morphology; suitability for large trap catches; cost; ease of handling; and application to downstream molecular diagnostic analyses such as real-time PCR and metabarcoding. We found that the optimum method for rapid isolation of DNA extraction was immersing flies in a NaOH:TE buffer at 75°C for 10 minutes, without the need for proteinase K or detergents. This HotSOAK method produced sufficient high-quality DNA whilst preserving morphological characters suitable for species-level identification with up to 20,000 flies in a sample. The lysates performed well in down-stream analyses such as loop-mediated isothermal amplification (LAMP) and real-time PCR applications, while for metabarcoding PCR the lysate required an additional column purification step. Development of this method is a key step required for upscaling our capacity to accurately detect insects captured in bulk traps, whether for biodiversity, biosecurity, or pest management objectives.

Molecular basis of adaptive shift in body size in Drosophila melanogaster: functional and sequence analyses of the Dca gene.

Latitudinal body size clines in animals conforming to Bergmann's rule occur on many continents but isolating their underlying genetic basis remains a challenge. In Drosophila melanogaster, the gene Dca accounts for approximately 5-10% of the natural wing size variation (McKechnie SW, Blacket MJ, Song SV, Rako L, Carroll X, Johnson TK, Jensen LT, Lee SF, Wee CW, Hoffmann AA. 2010. A clinally varying promoter polymorphism associated with adaptive variation in wing size in Drosophila. Mol Ecol. 19:775-784). We present here functional evidence that Dca is a negative regulator of wing size. A significant negative latitudinal cline of Dca gene expression was detected in synchronized third instar larvae. In addition, we clarified the evolutionary history of the three most common Dca promoter alleles (Dca237-1, Dca237-2, and Dca247) and showed that the insertion allele (Dca247), whose frequency increases with latitude, is associated with larger wing centroid size and higher average cell number in male flies. Finally, we showed that the overall linkage disequilibrium (LD) was low in the Dca promoter and that the insertion/deletion polymorphism that defines the Dca alleles was in strong LD with two other upstream sites. Our results provide strong support that Dca is a candidate for climatic adaptation in D. melanogaster.

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.

Loop-mediated isothermal amplification (LAMP) assays for detection of the New Guinea fruit fly Bactrocera trivialis (Drew) (Diptera: Tephritidae).

The cue-lure-responding New Guinea fruit fly, Bactrocera trivialis, poses a biosecurity risk to neighbouring countries, e.g., Australia. In trapping programs, lure caught flies are usually morphologically discriminated from non-target species; however, DNA barcoding can be used to confirm similar species where morphology is inconclusive, e.g., Bactrocera breviaculeus and B. rufofuscula. This can take days-and a laboratory-to resolve. A quicker, simpler, molecular diagnostic assay would facilitate a more rapid detection and potential incursion response. We developed LAMP assays targeting cytochrome c oxidase subunit I (COI) and Eukaryotic Translation Initiation Factor 3 Subunit L (EIF3L); both assays detected B. trivialis within 25 min. The BtrivCOI and BtrivEIF3L assay anneal derivatives were 82.7 ± 0.8 °C and 83.3 ± 1.3 °C, respectively, detecting down to 1 × 101 copies/µL and 1 × 103 copies/µL, respectively. Each assay amplified some non-targets from our test panel; however notably, BtrivCOI eliminated all morphologically similar non-targets, and combined, the assays eliminated all non-targets. Double-stranded DNA gBlocks were developed as positive controls; anneal derivatives for the COI and EIF3L gBlocks were 84.1 ± 0.7 °C and 85.8 ± 0.2 °C, respectively. We recommend the BtrivCOI assay for confirmation of suspect cue-lure-trapped B. trivialis, with BtrivEIF3L used for secondary confirmation when required.

Polymorphism in the couch potato gene clines in eastern Australia but is not associated with ovarian dormancy in Drosophila melanogaster.

Natural selection can generate parallel latitudinal clines in traits and gene frequencies across continents, but these have rarely been linked. An amino acid (isoleucine to lysine, or I462K) polymorphism of the couch potato (cpo) gene in Drosophila melanogaster is thought to control female reproductive diapause cline in North America (Schmidt et al. 2008, Proc Natl Acad Sci USA, 105, 16207-16211). Here, we show that under standard diapause-inducing conditions (12 °C and short photoperiod) (Saunders et al. 1989, Proc Natl Acad Sci USA, 86, 3748-3752), egg maturation in Australian flies is delayed, but not arrested at previtellogenic stages. At 12 °C, the phenotypic distribution in egg development was bimodal at stages 8 and 14 and showed a strong nonlinear pattern on the east coast of Australia, with incidence of egg maturation delay (ovarian dormancy) increasing both toward tropical and temperate climates. Furthermore, we found no evidence for an association between the cpo I462K polymorphism and ovarian dormancy at either 12 or 10 °C (when egg maturation was often delayed at stage 7). Owing to strong linkage disequilibrium, the latitudinal cline in cpo allele frequencies was no longer evident once variation in the In(3R)P inversion polymorphism was taken into account. Our results suggest that the standard diapause-inducing conditions (12 °C and short photoperiod) were not sufficient to cause the typical previtellogenic developmental arrest in Australian flies and that the cpo I462K polymorphism does not explain the observed delay in egg development. In conclusion, ovarian dormancy does not show a simple latitudinal cline, and the lack of cpo-dormancy association suggests a different genetic basis to reproductive dormancy in North America and Australia.

Ecologically relevant measures of tolerance to potentially lethal temperatures.

The acute thermal tolerance of ectotherms has been measured in a variety of ways; these include assays where organisms are shifted abruptly to stressful temperatures and assays where organisms experience temperatures that are ramped more slowly to stressful levels. Ramping assays are thought to be more relevant to natural conditions where sudden abrupt shifts are unlikely to occur often, but it has been argued that thermal limits established under ramping conditions are underestimates of true thermal limits because stresses due to starvation and/or desiccation can arise under ramping. These confounding effects might also impact the variance and heritability of thermal tolerance. We argue here that ramping assays are useful in capturing aspects of ecological relevance even though there is potential for confounding effects of other stresses that can also influence thermal limits in nature. Moreover, we show that the levels of desiccation and starvation experienced by ectotherms in ramping assays will often be minor unless the assays involve small animals and last for many hours. Empirical data illustrate that the combined effects of food and humidity on thermal limits under ramping and sudden shifts to stressful conditions are unpredictable; in Drosophila melanogaster the presence of food decreased rather than increased thermal limits, whereas in Ceratitis capitata they had little impact. The literature provides examples where thermal limits are increased under ramping presumably because of the potential for physiological changes leading to acclimation. It is unclear whether heritabilities and population differentiation will necessarily be lower under ramping because of confounding effects. Although it is important to clearly define experimental methods, particularly when undertaking comparative assessments, and to understand potential confounding effects, thermotolerance assays based on ramping remain an important tool for understanding and predicting species responses to environmental change. An important area for further development is to identify the impact of rates of temperature change under field and laboratory conditions.

A LAMP (loop-mediated isothermal amplification) test for rapid identification of Khapra beetle (Trogoderma granarium).

BACKGROUND: Khapra beetle (Trogoderma granarium Everts) is a significant pest of food products around the world, causing great losses of stored grain and produce, with export restrictions imposed on countries with established beetle populations. Khapra beetle is a high-priority exotic invertebrate pest in many countries requiring a rapid quarantine/biosecurity response when incursions occur. To address this, we developed a novel Khapra LAMP (loop-mediated isothermal amplification) assay using a portable real-time fluorometer and an additional 18S ribosomal DNA (18S) insect control LAMP assay for confirmation of the presence of insect DNA. Both LAMP tests can be performed either in a portable real-time fluorometer or using simple, visual colorimetric technique. RESULTS: Both the Khapra and 18S LAMP tests amplify positive samples within ≤ 25 min, with an anneal derivative temperature of 77.7 ± 0.7 °C for Khapra LAMP test and 88.0 ± 1.0 °C for 18S. The new Khapra LAMP assay is sensitive to very low levels of DNA (1.02 × 10-6  ng µL-1 ). Additionally, we developed a gBlock double stranded DNA fragment for use as positive Khapra control with a different anneal derivative of 80 °C. Both assays are simple to use in the field and are capable of amplifying DNA from target beetles, even when samples are partially degraded which is typically found during surveillance activities. By screening a broad panel of Dermestidae species we demonstrate that our new assay is species-specific, with no detections of false positives. Also, we evaluated multiple DNA extraction methods, with both QuickExtract and HotSHOT extraction methods proving suitable for in-field use. CONCLUSION: The novel Khapra and 18S LAMP assays should improve speed, accuracy and confidence of detection of Khapra beetle at incursion points and aid rapid biosecurity responses in any country affected, especially as the assays described here are portable and easy to implement in the field conditions where resources are limited. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effects of small Hsp genes on developmental stability and microenvironmental canalization.

BACKGROUND: Progression of development has to be insulated from the damaging impacts of environmental and genetic perturbations to produce highly predictable phenotypes. Molecular chaperones, such as the heat shock proteins (HSPs), are known to buffer various environmental stresses, and are deeply involved in protein homeostasis. These characteristics of HSPs imply that they might affect developmental buffering and canalization. RESULTS: We examined the role of nine Hsp genes using the GAL4/UAS-RNAi system on phenotypic variation of various morphological traits in Drosophila melanogaster. The stability of bristle number, wing size and wing shape was characterized through fluctuating asymmetry (FA) and the coefficient of variation (CV), or among-individual variation. Progeny of the GAL4/Hsp-RNAi crosses tended to have reduced trait means for both wing size and wing shape. Transcriptional knockdown of Hsp67Bc and Hsp22 significantly increased FA of bristle number, while knockdown of Hsp67Ba significantly increased FA and among-individual variation of wing shape but only in males. Suppression of Hsp67Bb expression significantly increased among-individual variation of bristle number. The knockdown of gene expression was confirmed for Hsp67Ba, Hsp67Bc, Hsp22, and Hsp67Bb. Correlation between FA and CV or among-individual variation of each trait is weak and not significant except for the case of male wing shape. CONCLUSION: Four small Hsp genes (Hsp22, Hsp67Ba, Hsp67Bb and Hsp67Bc) showed involvement in the processes of morphogenesis and developmental stability. Due to possible different functions in terms of developmental buffering of these small Hsps, phenotypic stability of an organism is probably maintained by multiple mechanisms triggered by different environmental and genetic stresses on different traits. This novel finding may lead to a better understanding of non-Hsp90 molecular mechanisms controlling variability in morphological traits.

Complexity of the cold acclimation response in Drosophila melanogaster.

Insects can increase their resistance to cold stress when they are exposed to non-lethal conditions prior to the stress; these plastic responses are normally described only in terms of immediate effects on mortality. Here we examine in Drosophila melanogaster the short- and longer-term effects of different conditions on several measures of cold resistance, but particularly chill coma recovery. Short-term exposure to sublethal temperature (cold hardening) did not decrease chill coma recovery times even though it decreased mortality. Exposure to 12 degrees C for 2 days (acclimation) decreased chill coma recovery times for a range of stressful temperatures when flies were cultured at 25 degrees C, but did not usually affect recovery times when flies were cultured at 19 degrees C. In contrast, 2-day exposure to 12 degrees C decreased mortality regardless of rearing temperature. Rearing at 19 degrees C decreased mortality and chill coma recovery time relative to rearing at 25 degrees C. Acclimation increased the eclosion rate of eggs from stressed females, but did not affect development time or size of the offspring. These results indicate that plastic responses to cold in D. melanogaster are complex when resistance is scored in different ways, and that effects can extend across generations.

Adult heat tolerance variation in Drosophila melanogaster is not related to Hsp70 expression.

Expression of heat-inducible Hsp70 is considered closely linked to thermotolerance in Drosophila melanogaster and other ectotherms. However, intra-specific variation of Hsp70 expression levels and its relationship to heat resistance has only been investigated in a few studies. Although in Drosophila larvae Hsp70 expression may be a key determinant of heat tolerance, the evidence for this in adults is equivocal. We therefore examined heat-induced Hsp70 expression and several measurements of adult heat tolerance in three independent collections of D. melanogaster, measured in three laboratories and using slightly different protocols. Expression levels of Hsp70 were quantified using ELISA or Western blots on extracts from adult females. Both Hsp70 and heat tolerance exhibited substantial within-population variation as previously reported. However, in all experiments there were no significant correlation between Hsp70 expression and laboratory assays of adult heat tolerance commonly used in Drosophila. When combining data across three studies we had high power to detect associations but the results showed that variation in Hsp70 expression is only likely to explain a small proportion of variation in adult heat tolerance. Therefore, although Hsp70 expression is a major component of the cellular heat stress response, its influence on intra-specific heat tolerance variation may be life-stage specific.

Candidate genes and thermal phenotypes: identifying ecologically important genetic variation for thermotolerance in the Australian Drosophila melanogaster cline.

Clinal variation in traits often reflects climatic adaptation; in Drosophila melanogaster clinal variation provides an opportunity to link variation in chromosomal inversions, microsatellite loci and various candidate genes to adaptive variation in traits. We undertook association studies with crosses from a single population of D. melanogaster from eastern Australia to investigate the association between genetic markers and traits showing clinal variation. By genotyping parents and phenotyping offspring, we minimized genotyping costs but had the power to detect association between markers and quantitative traits. Consistent with prior studies, we found strong associations between the clinal chromosomal inversion In(3R)Payne and markers within it, as well as among these markers. We also found an association between In(3L)Payne and one marker located within this inversion. Of the five predicted associations between markers and traits, four were detected (increased heat, decreased cold resistance and body size with the heat shock gene hsr-omega S, increased cold resistance with the inversion In(3L)Payne), while one was not detected (heat resistance and the heat shock gene hsp68). In a set of eight exploratory tests, we detected one positive association (between hsp23a and heat resistance) but no associations of heat resistance with alleles at the hsp26, hsp83, Desat 2, alpha-Gpdh, hsp70 loci, while cold resistance was not associated with Frost and Dca loci. These results confirm interactions between hsr-omega and thermal resistance, as well as between In(3L)Payne and cold resistance, but do not provide evidence for associations between thermal responses and alleles at other clinically varying marker genes.