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Background Diarrhoeal disease is the third leading cause of death in children under 5 years old with domestic flies acting as important mechanical vectors of diarrhoeal pathogens. To assess the effectiveness of a novel house design, “Star home”, and improved toilets in reducing the abundance of domestic flies, potential carriers of diarrhoeal pathogens, a randomized controlled trial was carried out in rural Tanzania. Methods Domestic fly populations were monitored in 28 randomly selected Star homes and 28 traditional thatched roofs and mud-walled houses over 2 years from January 2022 to December 2023. Flies were sampled in kitchens and toilets using baited-fly traps from 07.00 h to 17.30 h every 7 weeks. To assess the production of flies from toilets, traps were placed over drop holes to collect emerging flies. Duration of external door openings to the kitchens was recorded with data loggers. Findings Of the 1527 flies collected, 76% were Chrysomya putoria , 16% Musca domestica and 8% Sarcophaga spp. In kitchen collections, there were 46% fewer C. putoria flies [adjusted mean rate ratio (RR) = 0.54] and 69% fewer Sarcophaga spp. (RR = 0.31) in Star homes compared to traditional houses. There was no difference in the abundance of M. domestica in the two study groups. In toilets, there was 49% fewer C. putoria (RR = 0.51), but no difference was observed for other domestic fly species. No flies emerged from Star home toilets compared with a mean of 4.2 flies/trap/day in traditional toilets. During the day, the external doors od Star homes were open for an average of 13.0 min/h less than in traditional houses. Conclusions Star homes reduced the abundance of domestic flies, apart from houseflies, in the kitchen and there were fewer C. putoria , a putative vector of diarrhoeal diseases, in Star home toilets compared to traditional houses. Changing the design of buildings can contribute to a decline in domestic flies and may lead to a reduction in diarrhoeal diseases. Graphical Abstract

Zoonotic cutaneous leishmaniasis (ZCL), caused by Leishmania major , is a neglected tropical disease affecting impoverished populations. Current treatments are limited by cost, resistance, and side effects, highlighting the need for affordable, sustainable interventions. Lucilia sericata larvae, used in maggot therapy, effectively treat chronic wounds through debridement, antimicrobial activity, and healing promotion. This study explores how L. sericata processes L. major and proposes its potential application in ZCL treatment. The life cycles of L. sericata and L. major were maintained in laboratory conditions. Larval-parasite interactions were tested across substrates [hen liver, rat spleen, Roswell Park Memorial Institute (RPMI) 1640 cell culture medium] and time intervals (30–240 minutes). Extracorporeal effects were evaluated using trypan blue exclusion and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays; intracorporeal interactions via microscopy and nested-PCR targeting L. major rRNA genes. L. sericata excretion/secretion products and microbiota exhibited strong anti-leishmanial activity. Promastigotes were deformed within 1 hour post-exposure (hpe), fully inactivated at 4 hpe, and lysed by 6 hpe. In RPMI medium, the treatment group ( L. sericata  +  L. major ) showed significant reductions in active parasites and viable cells compared to controls after 4 hours. Microscopy revealed no parasites in larval guts, but PCR detected L. major DNA in all specimens, suggesting partial digestion. This study demonstrates that L. sericata can eliminate L. major through intra- and extra-oral digestion, supporting its potential as a biotherapeutic agent for ZCL-associated wounds. These findings offer a foundation for developing larval therapy protocols in dermatology. Further studies in animal models and clinical trials are required to validate this approach for managing ZCL.

Diptera are one of the four megadiverse insect orders, with great environmental, ecological, forensic and medical-sanitary relevance. The Jardim Botânico do Rio de Janeiro, located in an urban area of Rio de Janeiro, Brazil, constitutes an important refuge, supporting the conservation of several species. Through the knowledge of the diversity of Calliphoridae and Mesembrinellidae in this location, it will be possible to identify the behavior of native species along with the invasive genus Chrysomya , and to evaluate the influence of abiotic factors (temperature, relative humidity and pluviosity) on insect capture. Eight traps containing sardines were set and a total of 36,035 Diptera specimens were collected, of which 35,890 were of the Calliphoridae family and 145 of the Mesembrinellidae family. The average abiotic variables recorded were: Temperature 25,6 ºC (± 3,17), humidity 68% (±9,33%) and rainfall 3.42 mm (± 7,99). The total abundance of Calliphoridae and Mesembrinellidae peaked during January 2015, with the highest temperature. Among the variables and abundance and richness indices, significant results were only observed between temperature and abundance. None of the variables showed significant correlation with Calliphoridae and Mesembrinellidae richness. The presence of some species of Calliphoridae and Mesembrinellidae indicates the area’s good conservation status as they only occur in highly preserved forest areas. Through the knowledge of their richness and abundance, new legislation can be developed to help conservation efforts in deeply modified environments.

Dipterans of the Calliphoridae and Mesembrinellidae families are of high relevance in the Atlantic Forest of Rio de Janeiro, and it is important to examine their diversity and abundance in the different ecological areas of this biome over a time interval. This study aimed to study the diversity and abundance of Calliphoridae and Mesembrinellidae by evaluating the sexual variation and the influence of abiotic factors (average temperature, relative humidity and total precipitation) on the capture of insects collected during the four seasons of the year. Four traps were installed in each ecological area containing 300 grams of beef liver as attractive bait, which remained exposed for 48 hours in each season during the period between autumn 2021 and summer 2022. The collected dipterans were sacrificed, sent to the Laboratório de Estudos de Dípteros (LED-UNIRIO), and taxonomically identified. The Kruskal-Wallis and Wilcoxon tests were used to examine the influence of the four seasons on the abundance, and the Spearman correlation was used to relate abundance to abiotic variables. A total of 2,826 dipterans were collected during the four seasons of the year, represented by nine species of the Calliphoridae family and ten of the Mesembrinellidae family. During the summer, a numerically larger amount of insects was collected, but the Kruskal-Wallis test (chi-square = 5.2781, p = 0.1525) showed there was no significant difference between the abundance of the species collected and the seasons. Spearman’s correlation showed that most species did not show a significant correlation between their respective abundances and the analyzed abiotic factors. The Wilcoxon test indicated that there is a significant difference between the abundance of females and males, with females being significantly more abundant than males, however the difference is statistically greater within the Calliphoridae family (W = 60.49, p = 5.8x10 -12 ) in relation to the Mesembrinellidae family (W = 1231.5, p = 0.019).

The oriental latrine fly, Chrysomya megacephala (Diptera: Calliphoridae), is a medically important synanthropic blow fly species characterized by its necrophagy and coprophagy, often observed near carrion and animal feces. Notably, C. megacephala always arrives at carcass earlier than other species. To elucidate the underlying mechanisms behind the host choice in C. megacephala , we present the chromosome-scale genome assembly for this species. The genome size is 816.79 Mb, with a contig N50 of 1.60 Mb. The Hi-C data were anchored to six chromosomes, accounting for 99.93% of the draft assembled genome. Comparative genomic analysis revealed significant expansions in pathways of ligand-gated ion channel activity, passive transmembrane transporter activity, and protein methyltransferase activity, which may be closely associated with host localization and oviposition. After identifying 69 odor-binding proteins (OBPs) in the assembled genome, phylogenetic analysis showed that DmelOBP99b and CmegOBP99b exhibited high homology. Transcriptome analysis demonstrated that the relative expression of CmegOBP99b was consistently the highest during the metamorphosis, and RT-qPCR further confirmed the similar results. Additionally, CmegOBP99b exhibited a strong binding affinity to DMDS (dimethyl disulfide) as determined by molecular docking. To determine the protein expression level of CmegOBP99b in various body parts, we prepared recombinant CmegOBP99b protein and anti- CmegOBP99b polyclonal antibodies. Western blot analysis showed that CmegOBP99b was significantly expressed in the female’s head compared to other parts, which is consistent with RT-qPCR results. Therefore, CmegOBP99b may be the primary odor-binding protein responsible for olfactory recognition and the behavioral coordination of C. megacephala . This study not only provides valuable insights into the molecular mechanisms of oviposition localization in C. megacephala but also facilitates further research into the genetic diversity and phylogeny of the Calliphoridae family.

Studies under constant temperatures are the most common to estimate the Postmortem Interval (PMI). It is imperative that forensic sciences have data from studies carried out in the field. Therefore, this work aims to: (1) evaluate the parameters (weight, length, development time) associated with the life cycles of Lucilia ochricornis (Wiedemann) (Diptera: Calliphoridae) and Lucilia purpurascens (Walker) under experimental conditions in the field considering fluctuating temperatures, and (2) compare these results with those known and published by the same authors for cultures realized in the laboratory under constant temperatures; which will permit us to contrast the most widely used existing methodologies for forensic application in estimating the minimum postmortem interval (PMImin). For each season of the year, cultures of both species were made in the field, collecting information on temperature, humidity, and photoperiod to perform laboratory cultures, later comparing: development time, length, weight, and Accumulated Degree-Hours (ADH) in both types of cultures. Methods for estimating the PMI were obtained and validated with the information of the cultures grown in the field. The two types of cultures showed differences between each other for both species. The forensic use methods to estimate PMI were enhanced and their precision increased when maximum larval length data were used, and it was also concluded that feeding larval stages are the most accurate to be used in making estimates because the larva is growing. The estimation of the PMI through the use of necrophagous flies development remains reliable for obtaining the PMImin.

Forensic entomology is a branch of science that uses insects as evidence in medicolegal investigations as they could provide valuable information regarding the cause and time since death specially in the case of buried bodies. The present study was the first attempt in India to compare the decomposition and associated insects on pig carcasses above the ground and those buried at two different depths (40 and 80 cm) during spring season over two consecutive years. Two pigs were buried at each depth and one pig carcass was placed above the ground. One of the buried carcasses was exhumed frequently and other one was kept undisturbed to investigate the decomposition rate and insect access to the carcass. The rate of decomposition was found to be faster above the ground as compared to the buried environment. Also, the carcass buried at 80 cm depth exhibited slower decomposition rates and had limited insect activity as compared to the one buried at 40 cm. Species within the Calliphoridae and Sarcophagidae families were dominant on the surface-exposed carcass, whereas Phoridae were more prevalent on the buried carcasses even at a depth of 80 cm. The findings of the study highlights the significant influence of burial depth and exhumation on decomposition rate and insect arrival and colonization.

The screwworm fly, Cochliomyia hominivorax , is a significant pest in Brazil, posing threats to agriculture and public health. Laboratory rearing is crucial for developing control strategies like the sterile insect technique (SIT); but the effects of multi-generational rearing on the fly’s biological parameters are not fully understood. This study investigated the impact of rearing C. hominivorax for 12 inbred generations on its morphometrics, reproductive capacity, and life expectancy. Specimens were collected at distinct developmental stages across 12 generations for morphometric analysis, and key viability traits were assessed through biological assays. Data were analyzed using linear mixed-effects models and Kaplan–Meier survival analysis. The results revealed that morphometric parameters and key reproductive traits (oviposition, egg weight, hatchability, and larval survival) remained stable across all generations ( p  > 0.05). However, a significant decline in adult life expectancy was observed (log-rank test, χ 2  = 338.257, p  < 0.001), becoming pronounced from the fifth generation onwards. Significant sexual dimorphism was also confirmed: males were larger and heavier (Mann–Whitney U  = 9884.5, p  < 0.001), while females exhibited significantly greater longevity (log-rank test, χ 2  = 44.538, p  < 0.001). Continuous laboratory rearing of C. hominivorax appears robust for maintaining physical and reproductive traits, but negatively impacts fitness-related traits like longevity, likely due to inbreeding. These findings provide a critical quantitative baseline for quality control in mass-rearing programs and highlight the need for strategies to manage genetic diversity in laboratory colonies to ensure their effectiveness for research and SIT applications.

Background The blowfly Lucilia cuprina is a destructive parasite of sheep that causes flystrike or myiasis. Larvae consume the animal’s living flesh, producing large wounds that can lead to death. The main aim of this study was to identify genes that may play important roles in the behavior and physiology of L. cuprina larvae. Methods An RNA-Seq analysis of RNA from whole larvae at different developmental stages and third-instar head and gut tissues was used to identify sensory receptors and other genes relevant to the physiology of L. cuprina larvae. In addition, CRISPR/Cas9 gene editing was used to obtain a loss-of-function mutation for the L. cuprina odorant coreceptor gene ( LcupOrco ). The response of mutant larvae and adult females to fresh and rotten meat at different temperatures was evaluated. Results The RNA-Seq analysis suggested that odorant (OR), gustatory, ionotropic, and Pickpocket receptors may not play a central role in the L. cuprina larval sensory signaling and digestive systems. Rather, ATP-binding cassettes (ABCs) were highly enriched in head and gut RNA, and odorant-binding proteins (OBPs) only in the head. To confirm that ORs are not essential for larval detection of rotten beef, diet-choice assays were performed including larvae and adults homozygous for a null mutation in LcupOrco . While the attraction of adult females to rotten beef was disrupted, LcupOrco mutant larvae showed no change in diet preference. Conclusions The expression pattern of the ABC and OBP gene families suggests a central role in the sensory system of the L. cuprina larva for these receptors. Behavioral assays showed that ORs are essential for the adult female response to rotten beef, but not for larval behavior. These findings are consistent with high levels of expression of LcupOrco in the adult female antenna but very low expression in larvae. Graphical abstract

Ecological conditions are known to change the expression of mutualisms though the causal agents driving such changes remain poorly understood. Here we show that temperature stress modulates the harm threatened by a common enemy, and thereby induces a phoretic mite to become a protective mutualist. Our experiments focus on the interactions between the burying beetle Nicrophorus vespilloides , an associated mite species Poecilochirus carabi and their common enemy, blowflies, when all three species reproduce on the same small vertebrate carrion. We show that mites compete with beetle larvae for food in the absence of blowflies, and reduce beetle reproductive success. However, when blowflies breed on the carrion too, mites enhance beetle reproductive success by eating blowfly eggs. High densities of mites are especially effective at promoting beetle reproductive success at higher and lower natural ranges in temperature, when blowfly larvae are more potent rivals for the limited resources on the carcass.