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.

Automated 'lights-out' searching of all recovered fingerprints: Review of the current workflow for latent fingerprint processing in Queensland, Australia.

The process of linking an offender to a crime scene via their fingerprints has historically required significant human effort to compare latent fingerprints recovered from the scene with known fingerprints of a suspect. Increasing the speed of such comparisons, whilst maintaining accuracy and reliability and minimising error, is crucial for providing rapid intelligence to police investigators. One major opportunity for streamlining fingerprint examination is the adaptation of 'lights-out' technology to the comparison and matching of latent fingerprints. Here, we review the development, trial and validation process undertaken by the Queensland Police Service (QPS), Australia, to support implementation of a lights-out latent (LOL) workflow for automated latent fingerprint searching that is fully integrated with the existing case management systems. Targeted trials were undertaken using random selections of previously identified latent fingerprints that were searched using the LOL workflow against a local 10-print database. The results suggested that the LOL workflow could identify up to 44% of latent fingerprints with minimal human intervention and supported its implementation for all latent fingerprint comparisons in QPS casework. Review of LOL casework comparison outcomes for 2019 revealed that LOL-based identifications contributed approximately one quarter of all fingerprint identifications. Several procedural and technical factors that influenced the speed and efficiency of the LOL workflow are discussed, along with opportunities for improvement and future validation as an expert system.

A new molecular phylogeny for the Tanypodinae (Diptera: Chironomidae) places the Australian diversity in a global context.

The non-biting midge subfamily Tanypodinae (Diptera: Chironomidae) is species-rich, ecologically diverse, and near-globally distributed. Within the subfamily, aspects of generic and species-level taxonomy remain poorly understood, in particular the validity of assignment of Australian and New Zealand taxa to genera erected for northern hemisphere (Holarctic) fauna. Here, we place the austral diversity within this global context by extensive geographical and taxonomic sampling in concert with a multilocus phylogenetic approach. We incorporated sequence data for mitochondrial COI, and nuclear 28S and CAD, and conducted Bayesian and maximum likelihood phylogenetic inferences and Bayesian divergence time estimation. The resolved phylogeny supported many associations of Australian taxa with their proposed Holarctic congeners, with the exception of Apsectrotanypus Fittkau, and validates several taxa as endemic. Three of four New Zealand sampled taxa had their sister groups in Australia; New Zealand Monopelopia Fittkau was sister to a German congener. This included the first record of Procladius Kieffer from New Zealand. Most nodes connecting austral and Holarctic taxa clustered around the Cretaceous-Tertiary boundary (60-80 mya), whereas New Zealand-Australia nodes were generally slightly younger (53-57 mya). Together, these data contribute substantially to our understanding of the taxonomy, systematics and biogeography of the Australian Tanypodinae and more broadly to knowledge of Australia's aquatic insect biodiversity.

Contemporary issues in forensic science-Worldwide survey results.

Forensic science continues to be openly challenged and criticized. The aim of this study was to gain an understanding of forensic workplaces and the perceived current and potential future issues from forensic scientists via a detailed survey. An online survey was designed and disseminated to forensic practitioners and researchers worldwide. 544 participants from more than 20 countries took part in the survey. Participants personally rated ten forensic disciplines with subjective methodologies, responded on a five-point Likert scale to 22 statements that addressed subjectivity and objectivity, validation and proficiency testing and error and bias and answered demographic questions relating to their workplace type, level of experience and qualifications. Participants also commented freely on forensic issues specifically important to them. The purpose of this paper is to provide the survey results and consensuses captured on several key issues. Overall participants believed forensic science must be valid and reliable and supported development of objective methodologies, validation and further investigation into the application statistics, use of error rates and implications of cognitive bias. Participants raised consensus concerns with the provision of expert evidence and other broader issues. This information and understanding from the forensic front line are essential for forensic science moving forward to best address current challenges and criticisms not only of forensic evidence for the court but for applications of forensic science beyond the courtroom.

Bloodstain pattern analysis: Does experience equate to expertise?

Bloodstain pattern analysis (BPA) has long been accepted by courts as an area of expertise; however, that position has recently been challenged. The discipline has been criticized for limited empirical research into practitioner determination error rates and whether determinations require specialized knowledge/expertise, including whether practitioner experience level influences accuracy. This study attempted to address these knowledge gaps as they relate to bloodstain pattern recognition. The aims were twofold: to establish whether practitioners would outperform lay non-practitioners, and whether practitioner experience influenced accuracy and error in determinations. Comparisons of practitioner responses under three scenarios (forced, casework, and definitive) were also made to assess conservatism/certainty in pattern recognition. Participants (both BPA practitioners and non-practitioners) analyzed photographs of bloodstain patterns and made determinations of the broad bloodstain category and specific bloodstain pattern type. When forced to provide only a single response, practitioners identified bloodstain categories and patterns significantly more accurately than non-practitioners (p = 0.0001, p < 0.00001, respectively). Practitioner accuracy in bloodstain pattern recognition was positively associated with experience level (p = 0.0429) and this was consistent regardless of response scenario. Although no significant difference in practitioner accuracy was observed across response scenarios, practitioner conservatism/certainty varied significantly among the broad bloodstain category and specific pattern types. Overall, these results support bloodstain pattern recognition as an area of expertise and that practitioner experience positively influences accuracy. Based on these results, a series of recommendations were proposed aimed at further improving practices within the discipline to maximize accuracy and reliability of BPA evidence.

A comparison of three shoe sole impression lifting methods at high substrate temperatures.

Footwear impressions are a common form of evidence found at crime scenes, and the accurate recovery and recording of such impressions is critical for shoe sole comparison and identification. The lifting of shoe sole impressions from hot surfaces (>30°C/86°F) and in hot environments has received little attention in the literature, particularly in relation to the recovery of class and randomly acquired characteristics (RACs) required for accurate comparisons. This study addressed this knowledge gap by comparing the performance of three common impression lifters (gelatin, adhesive, and vinyl static cling film) at recovering shoe sole impressions in dust from hot flooring substrates. Dry origin dust shoe sole impressions were made on ceramic tile, galvanized metal, and laminated wood flooring using a shoe that possessed two RACs and five class characteristics present on the sole. Substrates were left in direct full sun for five hours during a summer day prior to lifting. Performance was measured by the proportion of RACs and class characteristics visible in each lifted impression. Results demonstrated that the vinyl static cling film tested performed poorly across all substrates, particularly for metal (23.8% marks recovered), including notable shrinkage of the lifted impression. In contrast, adhesive (~96% marks recovered over all substrates), and to a lesser extent gelatin (~85%), lifts were highly successful on hot substrates. These data suggest that adhesive lifts can consistently and accurately recover shoe sole impressions from hot substrates. This study contributes critical information for crime scene examiners to improve and expand evidence recovery in hot environments.

Application of forward-looking infrared (FLIR) imaging from an unmanned aerial platform in the search for decomposing remains.

Traditional methods of cadaver detection in outdoor environments include manual ground search, cadaver dogs, and manned aerial reconnaissance during daylight. These methods have limitations; however, a potential low-cost alternative may be to employ thermal imaging equipment mounted on an unmanned aerial vehicle (UAV) to detect heat emitted by insect and bacterial activity on the decomposing remains. No studies have addressed the influence of wrappings on detection of maggot mass thermal signatures nor assessed thermal detection of smaller body fragments. We addressed these knowledge gaps by utilizing a two-phase experimental approach to explore thermal detection of carcasses using UAV-mounted infrared imaging. In Phase 1, pig body fragments were deposited on the surface or shallow buried. In Phase 2, whole pig carcasses were deposited in four conditions: on the surface uncovered, wrapped in plastic or carpet, or buried. Our results demonstrated that observable heat emissions from remains corresponded to peak insect activity during active decay and could be readily detected in uncovered whole carcasses and fragments. Although plastic and carpet wrappings partly impeded detection of insect heat signatures, these materials were clearly detectable themselves because of their contrast to the background ground surface. Thermal signatures of buried partial remains and disturbed gravesoil were also observed; however, the buried whole carcass transitioned to adipocere prior to the decay stage and without any insect colonization or heat signature. These data can inform operational implementation of this technique to complement existing search strategies to offer a robust, low-cost alternative for use where scene characteristics allow.

Variation in decomposition stages and carrion insect succession in a dry tropical climate and its effect on estimating postmortem interval.

Insects have an important role in minimum postmortem interval (PMImin) estimation. An accurate PMImin estimation relies on a comprehensive study of the development and succession of local carrion insects. No published research on carrion insect succession exists for tropical north Queensland. To address this, we aimed to obtain preliminary observational data concerning the rate of decomposition and insect succession on pig carcasses in Townsville and compare these with other regions of Australia and overseas. Adult insects were collected daily from three pig carcasses for 30 d during summer and identified to family level. Observations of decomposition rate were made each day and progression through the stages of decomposition were recorded. Adult insects were identified to family and their presence/absence used as a proxy for arrival at/departure from the remains, respectively. These preliminary data highlight several interesting trends that may be informative for forensic PMImin estimation. Decomposition was rapid: all carcasses were at the dry/remains stage by Day 5, which was substantially quicker than all other regions in the comparison. Differences were also observed in the presence/absence of insect families and their arrival and departure times. Given the rapid progression through early decomposition, we argue that later-arriving coleopteran taxa may be more forensically informative in tropical Australia, in contrast with temperate regions where Diptera appear most useful. This research contributes preliminary observational data to understanding insect succession patterns in tropical Australia and demonstrates the critical need for comprehensive local succession data for each climatic region of Australia to enable accurate PMImin estimation. These data will inform future research targeted at gaining a more comprehensive understanding of insect succession in the Australian tropics.Key points:We obtained preliminary observational data concerning the rate of decomposition and insect succession on pig carcasses in tropical Australia.Decomposition was rapid: all carcasses were at the dry/remains stage by Day 5.Coleopteran taxa may be more forensically informative in tropical Australia than dipterans.

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini).

Accurate species-level identifications underpin many aspects of basic and applied biology; however, identifications can be hampered by a lack of discriminating morphological characters, taxonomic expertise or time. Molecular approaches, such as DNA "barcoding" of the cytochrome c oxidase (COI) gene, are argued to overcome these issues. However, nuclear encoding of mitochondrial genes (numts) and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications. One insect group for which these molecular and morphological problems are significant are the dacine fruit flies (Diptera: Tephritidae: Dacini). We addressed these issues associated with COI barcoding in the dacines by first assessing several "universal" COI primers against public mitochondrial genome and numt sequences for dacine taxa. We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region. Our new primers were tested alongside universal primers on a selection of dacine species, including both fresh preserved and decades-old dry specimens. Additionally, Bactrocera tryoni mitochondrial and nuclear genomes were compared to identify putative numts. Four numt clades were identified, three of which were amplified using existing universal primers. In contrast, our new primers preferentially amplified the "true" mitochondrial COI barcode in all dacine species tested. The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable. Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.

Improved Area of Origin Estimation for Bloodstain Pattern Analysis Using 3D Scanning.

An important component of crime scene reconstruction is bloodstain pattern analysis (BPA). Where BPA concerns impact patterns, estimating the area of origin is critical information for scene reconstruction. Traditionally, this is achieved by measuring individual bloodstains and performing trigonometric calculations; however, 3D scanning has been proposed as a viable alternative for overcoming logistical and practical concerns with the manual method. Therefore, this project aimed to establish whether the FARO Focus 3D scanner and FARO Zone 3D software can improve the accuracy of area of origin estimates relative to the manual method. We created a series of eight bloodstain impact patterns and performed paired analysis using the two methods to estimate areas of origin for each pattern. Our data suggested that FARO-derived estimates were generally more accurate than using the manual method. FARO-estimated heights of origin areas were generally closer to the true distance. Both methods underestimated the distance from the wall for most patterns originating 150mm or greater from the wall, but overestimated distances for patterns originating closer to the wall. The degree to which distances were underestimated increased significantly the further the blood source was from the wall and was greater for FARO-derived estimates. The results of this research contribute to the validation of these instruments for operational implementation for BPA and should be considered alongside the practical benefits of 3D scanning relative to manual methods. Further, 3D scanning can provide reliable BPA reconstruction documentation for technical review and court presentation.

Differential gene expression of Australian Cricotopus draysoni (Diptera: Chironomidae) populations reveals seasonal association in detoxification gene regulation.

Understanding the molecular mechanisms of organismal response to human-derived ecosystem change is recognised as a critical tool in monitoring and managing impacts, especially in freshwater systems. Fundamental to this approach is to determine the genes involved in responding to ecosystem change and detect modifications to their expression and activity in natural populations. Potential targets for this approach include well-known detoxification genes that are upregulated in response to stress. Here, we tested whether expression of such genes varied in association with differences in ecosystem health and could be detected in the field. We sampled populations of the freshwater midge, Cricotopus draysoni, from two geographically proximate sites in southeast Queensland, Australia, which differed in their ecosystem health, at multiple time points. We assessed transcriptome-level differential gene expression and predicted greatest differential expression between sites, associated with organismal responses to local physico-chemical factors. In contrast, we observed a clear and dramatic difference in gene expression - including of known detoxification genes - between time points, specifically between periods at the start and end of the austral summer rainfall when in-stream water levels are most different. These data suggest that these waterways experience greatest pollution load when water levels are high following rainfall events.

Nomenclatural corrections to Australian species of Cricotopus (Wulp) (Diptera; Chironomidae).

Our attention has been drawn to lapsi and errors in a recent publication in this journal concerning Cricotopus Wulp (Diptera: Chironomidae) (Drayson et al., 2015).

Taxonomic review of the chironomid genus Cricotopus v.d. Wulp (Diptera: Chironomidae) from Australia: keys to males, females, pupae and larvae, description of ten new species and comments on Paratrichocladius Santos Abreu.

The Australian species of the Orthocladiinae genus Cricotopus Wulp (Diptera: Chironomidae) are revised for larval, pupal, adult male and female life stages. Eleven species, ten of which are new, are recognised and keyed, namely Cricotopus acornis Drayson & Cranston sp. nov., Cricotopus albitarsis Hergstrom sp. nov., Cricotopus annuliventris (Skuse), Cricotopus brevicornis Drayson & Cranston sp. nov., Cricotopus conicornis Drayson & Cranston sp. nov., Cricotopus hillmani Drayson & Cranston, sp. nov., Cricotopus howensis Cranston sp. nov., Cricotopus parbicinctus Hergstrom sp. nov., Cricotopus tasmania Drayson & Cranston sp. nov., Cricotopus varicornis Drayson & Cranston sp. nov. and Cricotopus wangi Cranston & Krosch sp. nov. Using data from this study, we consider the wider utility of morphological and molecular diagnostic tools in untangling species diversity in the Chironomidae. Morphological support for distinguishing Cricotopus from Paratrichocladius Santo-Abreu in larval and pupal stages appears lacking for Australian taxa and brief notes are provided concerning this matter.