Medical and Veterinary Entomology

Dengue is a viral infection transmitted by mosquitoes of the Aedes genus, responsible for millions of cases each year. In Benin, several outbreaks have been reported, particularly in Abomey-Calavi, where the first dengue-related death was recorded in 2019. In the absence of a specific treatment, vector control remains the primary preventive measure. However, the emergence of insecticide resistance, notably through kdr mutations, could compromise its effectiveness. This study aims to identify the dengue virus serotypes circulating in the commune of Abomey-Calavi and to assess the frequency of kdr mutations V410L, V1016G, V1016I, F1534C, and S989P in Aedes aegypti and Aedes albopictus mosquitoes. Mosquitoes were collected in August 2024 from four arrondissement Togba, Calavi, Akassato, and Godomey through morning and afternoon spraying. After morphological identification, molecular analyses were conducted to detect the viral serotypes and kdr mutations. A total of 218 Aedes mosquitoes were collected, with a predominance of Aedes aegypti (68.8%) compared to Aedes albopictus (31.2%). The overall dengue positivity rate was 23.39% (95% CI: 17.9 - 29.6). The most frequent serotype was DENV-3 (12.4%), followed by DENV-2 (6.9%), DENV-1 (5%), and DENV-4 (0.09%). Six mosquitoes were co-infected with two serotypes, including three with DENV-1 and DENV-4. kdr mutations were detected in both species, with frequencies of 45% for V1016G, 44% for S989P, 40% for F1534C, 22% for V410L, and 19% for V1016I. Four mosquitoes carried three simultaneous mutations, while twenty-two carried two. Two mosquitoes co-infected with two serotypes also carried two different mutations. These results highlight the active circulation of the dengue virus and the presence of kdr mutations in Aedes mosquitoes in Abomey-Calavi. However, no significant association was observed between dengue infection and kdr mutations, and their distribution was independent of the viral serotype. These findings emphasize the need for regular monitoring of kdr mutations to adapt vector control strategies and limit the spread of dengue.

The New World screwworm, Cochliomyia hominivorax, was eradicated from North and Central America through the first application of the sterile insect technique. The sterile screwworm adult fly releases were mixed sex, but experience with other flies suggests that releasing only males could be up to five times more effective. Here we describe screwworm transgenic sexing strains (TSSs) with expected embryo lethality developed using the Tetracycline-off (Tet-off) system with the Lucilia cuprina nullo (DR6) or C. macellaria CG14427 (DR7) gene promoters and a tTA activated effector to promote female lethality. The TSSs expressed tTA highest in 2-3 h embryos and low in larvae and adults. However, most strains showed high expression in pupae. Evaluation on two doxycycline (Dox) regimens found that inclusion of Dox in the last larval feeding rescued females of the subsequent generation, likely by maternal Dox transfer to embryos. All TSSs produced only males on a reduced Dox feeding regimen but the female lethal period for the DR6 TSS was too late in development to save diet costs. Production parameters were met by all strains in colony, but strains had lower male fly survival than current production strains after removing Dox. In non-competitive mating success trials DR6 strains performed poorly, but DR7 performed equally to production strain males. However, males from all TSSs faired poorly in mating competition tests against production males. Our study highlights the importance of tightly-regulated gene promoters and suitable antibotics feeding schemes for the development and evaluation of TSSs based on Tet-off system.

Dengue cases in Bangladesh have surged in recent years. The existing insecticide-based control program, implemented in parts of the country, is challenged by issues of insufficient household coverage and high levels of insecticide resistance in the primary dengue virus (DENV) vector, Aedes aegypti. A more sustainable, effective alternative could be the implementation of a Wolbachia-mediated disease management strategy. Infecting mosquitoes with Wolbachia can change their reproductive compatibility and their ability to transmit DENV. These new phenotypes can be exploited to suppress or replace wild-type Ae. aegypti populations. Such strategies require the development of well-characterised Wolbachia-infected strains with biological characteristics that are comparable with local mosquitoes. We created and characterised a Wolbachia-infected Ae. aegypti strain with a Dhaka wild-type genetic background, and compared its reproductive compatibility, maternal inheritance, fitness, and virus-blocking ability to the parental strains (Dhaka wild-type and wAlbB2-F4). The new Ae. aegypti strain wAlbB2-Dhaka demonstrated complete cytoplasmic incompatibility with the wild-type strain and complete maternal transmission, retaining levels of pyrethroid resistance of the Dhaka wild-type (70% survival to 10 times the dose of permethrin expected to kill susceptible mosquitoes). No significant fitness costs were detected during laboratory comparisons of fecundity, fertility, survival, mating competitiveness, or desiccation tolerance. Compared to the wild-type strain, wAlbB2-Dhaka mosquitoes had a significantly reduced number of DENV genome copies in the bodies (44.4%, p = 0.0034); two-fold reduction in dissemination to legs and wings (47.6%, p < 0.0001); and >13-fold reduction of DENV in saliva expectorates (proxy of transmission potential) (92.7%, p < 0.0001) 14 days after ingesting dengue-infected blood. Our work indicates that the wAlbB2-Dhaka strain could be used for Ae. aegypti suppression or replacement strategies for dengue management in Bangladesh. Author summaryBangladesh is currently grappling with a series of severe dengue outbreaks, resulting in more than 300 thousand infections, 1700 fatalities, and a significant influx of patients requiring hospitalisation in 2023. These outbreaks coincide with the emergence of high levels of insecticide resistance in the Ae. aegypti population, which is contributing to the failure of conventional, insecticidal control strategies. In search of more sustainable, effective dengue control strategies, approaches using the endosymbiotic bacteria Wolbachia that render mosquitoes resistant to arbovirus infection are being trialled in a number of countries. We transinfected Dhaka wild-type Ae. aegypti with the Wolbachia strain wAlbB2 by backcrossing it with a Wolbachia wAlbB2-infected Ae. aegypti strain and assessed the new strains suitability for field release. We compared the fitness and virus-blocking ability of the backcrossed strain with its parental strains. We demonstrated that the new strain, wAlbB2-Dhaka, is as fit as the Dhaka wild-type strain and blocked [~]92% of dengue virus transmission. This strain has great potential for Wolbachia-mediated dengue management strategies in Bangladesh.

Reference strains of Aedes aegypti, reared over numerous generations under laboratory conditions, are selected to fit laboratory conditions and commonly used in research due to their consistency and ease of handling. However, working exclusively with such strains may not fully reflect the traits of natural populations. While working on such strains is relevant in terms of reproducibility between labs, it is also important to perform some work on field-collected mosquitoes and their progeny to capture more representative biological and behavioural variation. In this study, we used the New Orleans reference strain as a control to adjust and evaluate methods to manipulate the F1 progeny of field-collected mosquitoes from Cayenne (French Guiana). To improve blood feeding rate, we tested the impact of several blood feeding systems for mosquitoes kept in a cage or in individual vials and adjusted starvation time. To monitor fertilization, we assessed if dissection buffer affects burst of spermatheca during dissection, whether mosquitoes were collected alive of shortly after death. The results described here may be helpful for studies on mosquito fitness, particularly on field-derived mosquitoes or on experiments requiring individual level monitoring. Author summaryAedes aegypti is the major vector of dengue virus, causing hundreds of millions of cases per year. To fight against disease transmission, mosquitoes are studied in the laboratory, notably using colonies of mosquitoes kept over decades in the laboratory. These reference strains have been selected to fit laboratory conditions and may thus not fully reflect the traits of natural populations. While working on such strains is relevant in a sake of consistency between labs and ease of handling, it is also important to work on field-collected mosquitoes and their progeny to capture more representative biological and behavioural variation. In this study, we adjusted and evaluated methods to manipulate the F1 progeny of field-collected mosquitoes from Cayenne (French Guiana), using the New Orleans reference strain as a control. To improve blood feeding rate, we tested the impact of several blood feeding systems for mosquitoes kept in a cage or in individual vials and adjusted starvation time. To monitor fertilization, we assessed if dissection buffer affects burst of spermatheca during dissection, whether mosquitoes were collected alive of shortly after death. The results described here may be helpful for studies on mosquito fitness, particularly on field-derived mosquitoes or on experiments requiring individual level monitoring.

Insecticide resistance is a serious threat to our ability to control mosquito vectors which transmit pathogens including malaria parasites and arboviruses. Understanding the underlying mechanisms is an essential first step in tackling the challenges presented by resistance. This study aimed to functionally characterise the carboxylesterase, CCEae3A, the elevated expression of which has been implicated in temephos resistance in Aedes aegypti and Aedes albopictus larvae. Using our GAL4/UAS expression system, already established in insecticide-sensitive Anopheles gambiae mosquitoes, we produced transgenic An. gambiae mosquitoes that express an Ae. aegypti CCEae3A ubiquitously. This new transgenic line permits examination of CCEae3A expression in a background which does not express the gene and allows comparison with existing An. gambiae GAL4-UAS lines. Insecticide resistance profiling of these transgenic An. gambiae larvae indicated significant increases in resistance ratio for three organophosphate insecticides, temephos (5.98), chloropyriphos (6.64) and fenthion (3.18) when compared to the parental strain. Cross resistance to adulticides from three major insecticide classes: organophosphates (malathion, fenitrothion and pirimiphos methyl), carbamates (bendiocarb and propoxur) and pyrethroid (alpha-cypermethrin) was also detected. Resistance to certain organophosphates and carbamates validates conclusions drawn from previous expression and phenotypic data. However, detection of resistance to pirimiphos methyl and alphacypermethrin has not previously been formally associated with CCEae3A, despite occurring in Ae. aegypti strains where this gene was upregulated. Our findings highlight the importance of characterising individual resistance mechanisms, thereby ensuring accurate information is used to guide future vector control strategies. Author SummaryInsecticides are vital disease control tools against pathogen-transmitting mosquitoes. However, they are becoming less effective as mosquitoes develop resistance. Among the molecular changes that contribute to resistance, increased production of enzymes that break down/sequester the insecticide is common. In Ae. aegypti mosquitoes, which spread many arboviruses, over-expression of the carboxylesterase enzyme, CEae3A, has been associated with resistance to certain insecticides used for vector control, particularly organophosphate compounds. However, multiple resistance enzymes/mechanisms are likely to be present in resistant mosquitoes at the same time. To examine the effect of CCEae3A expression in isolation, we utilised the An. gambiae mosquito with its convenient access to GAL4/UAS technology to regulate gene expression. This enabled production of CCEae3A in a normally insecticide-sensitive mosquito strain, permitting expression without interference from other resistance mechanisms. As anticipated, resistance to organophosphates was observed in larvae expressing CCEae3A. In adults, resistance was also found against compounds from organophosphate, carbamate and pyrethroid insecticide classes, including two compounds for which there had been no previous association. As well directly linking CCEae3A expression to specific insecticide resistance, this transgenic line can be included in a panel expressing alternative enzymes to screen new insecticidal compounds for liability to existing resistance mechanisms.

Tsetse are the insects responsible for transmitting African trypanosomes, which cause sleeping sickness in humans and animal trypanosomiasis in wildlife and livestock. Knowing the age of these flies is important when assessing the effectiveness of vector control programs and modelling disease risk. However, current methods to assess fly age are labour-intensive, slow, and often inaccurate as skilled personnel are in short supply. Mid-infrared spectroscopy (MIRS), a fast and cost-effective tool to accurately estimate several biological traits of insects, offers a promising alternative. This is achieved by characterising the biochemical composition of the insect cuticle using infrared light coupled with machine learning algorithms to estimate the traits of interest. We tested the performance of MIRS in estimating tsetse sex and age for the first time using spectra obtained from their cuticle. We used 541 insectary-reared Glossina m. morsitans of two different age groups for males (5 and 7 weeks) and three age groups for females (3 days, 5 weeks, and 7 weeks). Spectra were collected from the head, thorax, and abdomen of each sample. Machine learning models differentiated between male and female flies with a 96% accuracy and predicted the age group with 94% and 87% accuracy for males and females, respectively. The key infrared regions important for discriminating sex and age classification were characteristic of lipid and protein content. Our results support the use of MIRS as a fast and accurate way to identify tsetse sex and age with minimal pre-processing. Further validation using wild-caught tsetse can pave the way for this technique to be implemented as a routine surveillance tool in vector control programmes. Author summaryMale and female tsetse transmit the parasites that cause sleeping sickness in humans and nagana in livestock. To control these diseases, knowing the age of these flies is important, as it helps evaluate the efficacy of control measures and assess disease risk. However, current age-grading methods are laborious, often unreliable, and in the case of male tsetse, highly inaccurate. This study explores a novel approach that uses mid-infrared spectroscopy (MIRS) to estimate the age of individual tsetse. Machine learning can detect signatures in MIRS that help identify the composition of a flys cuticle, which differs between sexes and changes as they age. We trained machine learning models that distinguished male from female flies with 96% accuracy and predicted the correct age group with 94% accuracy for males and 87% accuracy for females. MIRS offers a fast and reliable way to identify tsetse sex and age with minimal preparation. If this method is successfully validated with wild flies, it holds the potential to vastly increase the accuracy of the way we monitor and combat these disease-carrying insects, thus offering significant advantages in our efforts to control them.

Anopheles stephensi is rapidly expanding across Africa, posing new challenges for malaria control. Its biting time patterns, however, remain poorly characterized, raising uncertainty about the effectiveness of bed nets against this invasive vector. To address this gap, we investigated diel biting activity, feeding propensity, and flight behavior using complementary behavioral assays on females reared from wild-caught larvae in Hawassa City, southern Ethiopia. Flight monitoring assays revealed that An. stephensi exhibited pronounced activity at dusk, beginning between 18:00 and 19:00 with the onset of scotophase, and little to no activity during the photophase. Blood-feeding propensity, defined as the proportion of mosquitoes taking a blood meal when offered, peaked during the early scotophase (18:00-22:00) at 33.3-51.7%, but was markedly reduced during daylight hours (0-16.7%). Human landing catches in large-cage enclosures confirmed this early evening activity: 83.5% of total landings occurred between 18:00 and 22:00 with a sharp peak at 18:00-19:00, corresponding to a mean biting rate of 17.8 bites per person per hour. These findings demonstrate that invasive An. stephensi primarily seek hosts and bite during the early evening, a time when people are often unprotected by bed nets. This behavior reduces the protective impact of conventional net-based interventions and underscores the need for African National Malaria Control Programs to deploy complementary measures such as spatial repellents and larval source management to mitigate early-evening transmission. Moreover, this study highlights the utility of integrated behavioral assays for estimating biting time, offering approaches that can be extended to other vector species across Africa.

BackgroundReports on adult Hyalomma ticks in certain regions of the Carpathian Basin date back to the 19th century. These ticks were thought to emerge from nymphs dropping from birds, then molting to adults. Although the role of migratory birds in carrying ticks of this genus is known from all parts of Europe, in most countries no contemporaneous multiregional surveillance of bird-associated ticks was reported which could allow the recognition of hotspots in this context. MethodsIxodid ticks were collected from birds at seven ringing stations in Hungary, including both the spring and autumn migration period in 2022. Ixodes and Haemaphysalis species were identified morphologically, whereas Hyalomma species molecularly. ResultsFrom 38 passeriform bird species 957 ixodid ticks were collected. The majority of developmental stages were nymphs (n=588), but 353 larvae and 16 females were also present. On most birds (n=381) only a single tick was found and the maximum number of ticks removed from the same bird was 30. Tick species were identified as Ixodes ricinus (n=598), Ixodes frontalis (n=18), Ixodes lividus (n=6), Haemaphysalis concinna (n=322), and D. reticulatus (n=1). All twelve Hyalomma sp. ticks (11 engorged nymphs and an unengorged larva) were identified as Hyalomma rufipes based on three mitochondrial markers. This species was only found in the Transdanubian region and along its southeastern border. The Common Blackbird (Turdus merula) and the European Robin (Erithacus rubecula) were the two main hosts of I. ricinus and I. frontalis, whereas H. concinna was almost exclusively collected form long-distance migrants. The predominant hosts of H. rufipes were reed-associated bird species, the Sedge Warbler (Acrocephalus schoenobaenus) and the Bearded Reedling (Panurus biarmicus), both harboring these ticks at the end of June (i.e., the nesting period) in southwestern Hungary. ConclusionsThis study provides ornithological explanation for the regional, century-long presence of adult Hyalomma ticks under continental climate in the Transdanubian Region of the Carpathian Basin. More importantly, the autochthonous occurrence of a H. rufipes population was revealed for the first time in Europe, based on the following observations: (1) the bird species infested with H. rufipes are not known to migrate during their nesting period; (2) one larva was not yet engorged; (3) the larva and the nymphs must have belonged to different local generations; and (4) all H. rufipes found in the relevant location were identical in their haplotypes based on three maternally inherited mitochondrial markers, probably reflecting founder effect. This study also demonstrated that the species of ticks carried by birds were significantly different between collection sites even within a geographically short distance (200 km). Therefore, within a country multiregional monitoring is inevitable to assess the overall epidemiological significance of migratory birds in importing exotic ticks, and also in maintaining newly established tick species.

The black soldier fly, Hermetia illucens, is mass-reared throughout the world to convert organic waste into ingredients for pet and livestock feed, as well as frass and other biological products. To promote the optimization of breeding regimes carried out by industrial black soldier fly operations, it is critical to better understand adult reproductive anatomy and its potential relationship with fertile egg production. However, in this species, little is known about how reproductive anatomy changes with respect to increases in body size. Hence, this study investigated the nutritional static allometric relationship between the external genitalia of adult male black soldier flies and their body size. Size differences were induced top-down by increasing the larval rearing density, which has a negative effect on adult body size. For each of 3 rearing densities, a random sample of 30 adults was selected, and measurements taken three times each for the head, thorax, parameral sheath, and gonostylus. Fitting a generalized linear log-log model to the data revealed that for every 10.0% increase in body size (thorax length), genital length (parameral shealth) only increased by 1.8%. The resulting allometric slope of genitalia to thorax size was 0.195, indicating a pattern of hypoallometry. The presence of hypoallometric genitalia in a domestic population suggests individuals should be able to copulate regardless of differences in body size, which is consistent with most other insects. Moreover, this finding implies that black soldier fly genitalia were hypoallometric prior to their domestication and continues to persist within captive black soldier fly populations. To confirm, future work should investigate the direct impacts of hypoallometric genitalia on fitness, especially in flies which have been genetically edited or artificially selected to be increasingly large. LAY SUMMARYAdult male black soldier fly shown to have similar sized genitals despite differences in body size. SHORT SUMMARYThe black soldier fly, Hermetia illucens, is an economically important insect mass-reared throughout the globe; however, a large knowledge gap exists in terms of its reproductive anatomy and physiology. This study examined the relationship of male genitalia to body size, finding a 10% increase in body size corresponded with a 1.8% increase in genitalia size, meaning the structures are hypoallometric. This finding is important because it indicates large and small flies have similar sized genitalia, which may allow differently-sized individuals to copulate.

BackgroundInsecticides used in vector control mostly rely on vectors being exposed through contact with treated surfaces, yet little is known about the amount picked up by the insect. Measuring this amount is relevant not only for determining the actual doses that are lethal to the mosquito, but also for understanding effects on the physiology and vector competence of mosquitoes. Insecticides at sub-lethal doses can affect both parasites developing inside mosquitoes and mosquito microbiota, hence it is important to understand the processes by which parasites are exposed to insecticide inside the insect. These doses will inevitably depend on the amount of insecticide that mosquitoes pick up when they come into contact with treated nets. MethodsThree to five days old non-blood fed female Anopheles coluzzii mosquitoes were exposed to a long-lasting insecticidal net (PermaNet 2.0 containing 55 mg/m2 deltamethrin), using a wire ball frame, for 0.5-5.0 minutes. Our in-house developed colorimetric test was used to visually detect the amount of deltamethrin on different parts of the mosquito (legs, heads, thoraxes, abdomens) following exposure to the net. The amount of insecticide picked up by mosquitoes from the net over a range of exposure times was measured using a high-performance liquid chromatography with diode array detection (HPLC-DAD). ResultsThe colorimetric test, designed to only detect the type 2 pyrethroids (i.e deltamethrin, -cypermethrin and {lambda}-cyhalothrin) on fabrics (e.g. ITNs) and sprayed walls, was successfully used for the first time to detect deltamethrin on mosquitoes following exposure to the net. The confirmatory HPLC-DAD analysis determined that after 2 min exposure up to 12 ng of deltamethrin adhered to mosquitoes following exposure to PermaNet 2.0 (mean = 5.2 ng/mosquito, SE = 1.9) and that the final dose depends on the length of exposure time. ConclusionsThis study demonstrated the potential of a screening (type 2 pyrethroid colorimetric test) and a confirmatory test (HPLC-DAD) to determine the amount of insecticide that adheres to mosquitoes on contact with treated surfaces. This has implications for a precise lethal dose determination and detection of specific insecticide that causes the greatest mosquito mortality in circumstances where mixtures of insecticides may be used to maximise effectiveness of interventions.

Ticks are medically important vectors of pathogens, many of which are zoonotic or impact domestic animal and/or wildlife health. Climate change, landscape use, and increasing interactions between domestic animals, wildlife, and humans have resulted in changes in tick-host dynamics and the emergence of novel pathogens worldwide. Therefore, it is crucial to describe the host and geographic ranges of vector species to assess disease risk, particularly in areas where research is lacking. This task necessitates adopting a One Health approach. In sub-Saharan Africa, previous work on ticks has focused primarily on those species most relevant to domestic livestock or humans, highlighting a significant knowledge gap concerning species of ticks that infest peri-domestic animals in rural areas. The objective of this study was to investigate the species diversity of ticks on peri-domestic dogs in rural areas of Chad, Africa. From 2019-2022, we collected 3,412 ixodid ticks from 435 peri-domestic dogs from 23 villages in Chad, Africa during both dry and wet seasons. Ticks were identified to species using morphological techniques and/or molecular analyses of the 16S rRNA, 12S rRNA, and cytochrome oxidase I gene regions. We identified 11 species of ticks from dogs including Amblyomma variegatum, Amblyomma marmoreum, Haemaphysalis leachi, a Haemaphysalis sp., Hyalomma impressum, Hyalomma truncatum, Rhipicephalus decoloratus, Rhipicephalus guilhoni, Rhipicephalus muhsamae, Rhipicephalus linnaei (=R. sanguineus tropical lineage), and a Rhipicephalus sp. Several of these tick species are known vectors for important canine and zoonotic pathogens and some are more commonly associated with cattle hosts. Our results show that sampling ticks from peri-domestic dogs provides an opportunity to examine vectors that may infest domestic animals, agricultural animals, wildlife, and humans as hosts in an understudied area. HighlightsO_LITick-borne diseases are significant concern across sub-Saharan Africa C_LIO_LILimited data on ticks of animals and people in Chad, Africa C_LIO_LIA high diversity of tick species infested domestic dogs in Chad C_LIO_LIMany detected tick species associated with important pathogens C_LI

Aedes (Aedimorphus) vexans (Meigen, 1830) is a globally distributed mosquito species commonly associated with temporary flooded habitats, including those in the South Pacific region. While its presence has been documented in several Pacific archipelagos, its occurrence on Tahiti island had remained unconfirmed. A retrospective examination of historical mosquito collections held at the Institut de Recherche pour le Developpement (IRD, France) and the Institut Louis Malarde (ILM, Tahiti) revealed evidence of Ae. vexans on Tahiti dating back as early as 1993. This historical detection was corroborated by targeted field surveys conducted between 2023 and 2025, which confirmed the species presence at multiple sites, particularly in the southern part of the island. To support these observations, COI gene sequences from newly collected specimens were analyzed alongside reference Ae. vexans sequences from GenBank. The Tahitian specimens fall unambiguously within the Pacific clade of Ae. vexans and show no genetic divergence that would support their assignment to Ae. nocturnus or Ae. nipponii. Similarly, sequences published under these names in GenBank do not form distinct lineages within Ae. vexans. This study provides the first confirmed record of Ae. vexans on Tahiti, based on both morphological identification and molecular evidence. The findings underscore the need to integrate molecular tools into entomological monitoring to improve species detection and prevent misidentifications in island environments where vector diversity is often underestimated.

BackgroundTsetse flies (Glossina sp.) are vectors of Trypanosoma brucei subspecies that cause human African trypanosomiasis (HAT). Capturing and screening tsetse is critical for HAT surveillance. Classically, tsetse have been microscopically analysed to identify trypanosomes, but this is increasingly replaced with molecular xenomonitoring. Nonetheless, sensitive T. brucei-detection assays, such as TBR-PCR, are vulnerable to DNA cross-contamination. This may occur at capture, when often multiple live tsetse are retained temporarily in the cage of a trap. This study set out to determine whether infected tsetse can contaminate naive tsetse with T. brucei DNA via faeces when co-housed. Methodology/Principle FindingsInsectary-reared teneral G. morsitans morsitans were fed an infectious T. b. brucei-spiked bloodmeal. At 19 days post-infection, infected and naive tsetse were caged together in the following ratios: (T1) 9:3, (T2) 6:6 (T3) 1:11 and a control (C0) 0:12 in triplicate. Following 24-hour incubation, DNA was extracted from each fly and screened for parasite DNA presence using PCR and qPCR. All insectary-reared infected flies were positive for T. brucei DNA using TBR-qPCR. However, naive tsetse also tested positive. Even at a ratio of 1 infected to 11 naive flies, 91% of naive tsetse gave positive TBR-qPCR results. Furthermore, the quantity of T. brucei DNA detected in naive tsetse was significantly correlated with cage infection ratio. With evidence of cross-contamination, field-caught tsetse from Tanzania were then assessed using the same screening protocol. End-point TBR-PCR predicted a sample population prevalence of 24.8%. Using qPCR and Cq cut-offs optimised on insectary-reared flies, we estimated that prevalence was 0.5% (95% confidence interval [0.36, 0.73]). Conclusions/SignificanceOur results show that infected tsetse can contaminate naive flies with T. brucei DNA when co-caged, and that the level of contamination can be extensive. Whilst simple PCR may overestimate infection prevalence, quantitative PCR offers a means of eliminating false positives. Author SummaryTsetse flies (Glossina sp.) are vectors of Trypanosoma brucei parasites that cause human African trypanosomiasis, also known as sleeping sickness. As part of disease surveillance, tsetse can be captured in traps and checked for parasite presence. The molecular screening of disease vectors (such as mosquitoes, ticks and blackflies) for the presence of pathogen DNA has gained popularity in recent years. However, DNA contamination may occur at capture when live vectors are retained for a limited period in a trap cage. To explore this, we conducted experiments, initially with laboratory-reared tsetse and then field-caught tsetse from Tanzania. Our results show that infected tsetse can contaminate uninfected tsetse with T. brucei DNA when retained together in a trap cage, and that the level of contamination can be extensive. Infected tsetse consistently shed T. brucei DNA in their faeces, which in turn contaminates other tsetse. This can produce false-positive results, leading to inaccurate reporting of infection prevalence. These findings impact not only trypanosomiasis surveillance, but may also have ramifications for the xenomonitoring of other vector-borne neglected diseases. Future work should explore whether pathogen DNA contamination routes exist in other vector species and, if so, the methods to mitigate DNA contamination in entomological traps.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/700930v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@177a6b4org.highwire.dtl.DTLVardef@6186a3org.highwire.dtl.DTLVardef@ce6196org.highwire.dtl.DTLVardef@168bf43_HPS_FORMAT_FIGEXP M_FIG C_FIG The kangaroo soft tick, Australpavlovskyella gurneyi (Warburton, 1926), is found in sandy depressions ( wallows), under desert shade trees, formed by the activity of the red kangaroo, Osphranter rufus, resting under shade trees (https://youtu.be/AYLoqqPsifc). The field biology of the tick was examined on Moralana Station in arid mid-north, South Australia, between February 1969 and March 1971. The age of kangaroo dung in wallows showed that kangaroos visited wallows regularly during the hot summer and infrequently during the cooler months. All nymphal instars and adults were present at all times of the year in kangaroo wallows, but only a small proportion of the ticks present was trapped on any one occasion. Ticks were abundant in large kangaroo wallows under trees with dense shade, but scarce under smaller trees with sparse shade. The short-lived larvae were present only during spring and early summer, indicating that the long-lived female ticks bred only during spring and early summer. Laboratory tests showed that field-collected adult female ticks entered reproductive diapause from January to August (mid-summer to late-winter). Ticks placed in kangaroo wallows survived for at least one year without food. On Moralana Station, the population of first-instar nymphs increased in summer and subsequently the population of second-instar nymphs increased in early autumn, indicating that a life cycle could be completed in 2-3 years. HighlightsO_LIThe seasonal biology of Australpavlovskyella gurneyi, found in sandy depressions wallows formed by the activity of the red kangaroo, under sparse semi-arid desert shade trees was examined for the first time. C_LIO_LIEngorged ticks placed in kangaroo wallows survived for at least one year without food. C_LIO_LIIn this environment, the entire life cycle could be completed in 2-3 years. C_LI

BackgroundTemperature choice is a vector trait that influences microhabitat selection and can have important implications for vector species, as it may affect how often vectors encounter hosts. Aedes aegypti and Ae. albopictus are disease vectors whose geographic ranges continue to expand each year. One aspect that remains largely understudied is the altitudinal range of these species and the extent of differences in thermal behavior between lowland and highland populations. MethodsI collected Ae. aegypti and Ae. albopictus on the islands of Bioko and Sao Tome. I compared the distribution of the two species along an altitudinal cline spanning 2,000 m of elevation. I then used live specimens to test temperature preference for both species in a laboratory thermocline. ResultsI report the distribution of these two species on the island of Bioko and show that the abundance of immature stages of both species follows a negative exponential decay with altitude. I compare this distribution with that observed on the neighboring island of Sao Tome, also in the Gulf of Guinea. Overall, the distribution patterns of the two species are similar, but models indicate a higher abundance at sea level in Sao Tome than in Bioko. I used specimens from this survey to study temperature preference under controlled conditions. I found no significant differences between species or between sexes; however, I detected an altitudinal cline in temperature preference, with high-elevation populations preferring cooler temperatures on both islands. ConclusionsThese results indicate the presence of phenotypic variation in a key trait--temperature choice--that may alter the likelihood of contact between these vectors and humans.

Tropilaelaps spp. are a parasitic mite that feed and reproduce within honey bee brood (Apis spp.) and cause significant damage and mortality to Apis mellifera colonies. T. mercedesae is found outside the range of its natural host A. dorsata and was recently detected in Europe for the first time in 2024. It is widely believed that Tropilaelaps spp. are unable to survive without brood. However, studies have demonstrated that T. mercedesae can survive during broodless periods while parasitising A. dorsata and without brood in overwintering colonies of A. mellifera in temperate climates. This study sought to examine the survival of T. mercedesae on a range of matrices and found that mites could survive for more than 96 hours on live adult A. mellifera and more than 144 hours and 168 hours on decomposing A. mellifera pupae and adults respectively. These findings could indicate one possible mechanism which allows T. mercedesae to survive without brood. These findings also have implications towards better understanding possible transmission routes for Tropilaelaps. Previously bee sales in the form of queens and packages and used beekeeping equipment were considered a relatively low risk for transmitting Tropilaelaps spp. due to the absence of live brood. This study demonstrates Tropilaelaps ability to survive in these scenarios and the increased potential for it to spread globally.

Identifying the current geographic range of disease vectors is a critical first step towards determining effective mechanisms for controlling and potentially eradicating them. This is particularly true given that historical vector ranges may expand due to changing climates and human activity. The Aedes subgenus Stegomyia contains over 100 species, and among them, Ae. aegypti and Ae. albopictus mosquitoes represent the largest concern for public health, spreading dengue, chikungunya, and Zika viruses. While Ae. aegypti has been observed in the country of Zambia for decades, Ae. albopictus has not. In 2015 we sampled four urban and two rural areas in Zambia for Aedes species. Using DNA barcoding, we confirmed the presence of immature and adult Ae. albopictus at two rural sites: Siavonga and Livingstone. These genotypes seem most closely related to specimens previously collected in Mozambique based on CO1 sequence from mtDNA. We resampled Siavonga and Livingstone sites in 2019, again observing immature and adult Ae. albopictus at both sites. Relative Ae. albopictus frequencies were similar between sites, with the exception of immature life stages, which were higher in Siavonga than in Livingstone in 2019. While Ae. albopictus frequencies did not vary through time in Livingstone, both immature and adult frequencies increased through time in Siavonga. This report serves to document the presence of Ae. albopictus in Zambia, which will contribute to the process of determining the potential public health implications of this disease vector in Central Africa.

The green peach aphid, Myzus persicae (Sulzer) (Homoptera: Aphididae), is a major pest of Brassica L. species in Australia, where it can transmit >100 viruses. Globally, this species has evolved resistance to 74 insecticides from numerous chemical groups. Although Integrated Pest Management (IPM) strategies are being implemented, chemical treatment remains the predominant method used to control aphids. Insecticide seed treatments are viewed as a softer alternative to chemical sprays and are widely used in Australian canola fields. The effects of imidacloprid, thiamethoxam, and a mixture of thiamethoxam & lambda-cyhalothrin canola seed treatments were investigated on the parasitoid, Aphidius colemani Viereck (Hymenoptera: Aphidiidae) and the predator, the green lacewing Mallada signatus (Schneider) (Neuroptera: Chrysopidae); both important natural enemies of M. persicae. The number of mummies formed by A. colemani on the untreated plants was lower than those formed on the thiamethoxam & lambda-cyhalothrin and imidacloprid treated plants. The number of A. colemani reared from mummies on thiamethoxam & lambda-cyhalothrin plants was higher than those reared from thiamethoxam and untreated plants. Significant effects of insecticide seed treatments were only noted for mummies produced while the parent parasitoids were on the plants, not for those mummies produced after their removal. This suggests seed treatment effects were immediate but not long lasting. Based on cumulative parasitoid survival days for two generations, A. colemani exposed to thiamethoxam & lambda-cyhalothrin and imidacloprid treatments had a greater fitness than those exposed to the thiamethoxam and untreated controls, possibly due to the phenomenon of insecticide hormoligosis. Despite the treatment effects observed, we did not detect any behavioural differences in M. persicae or A. colemani. Mallada signatus were not negatively affected by feeding on M. persicae on insecticide seed treated plants, suggesting they are more tolerant of seed treatments than A. colemani. The findings from this study provide a useful platform for further experimentation on the effects of seed treatments on natural enemies of M. persicae.

The introduction of the western honey bee Apis mellifera to Asia has seen the parasitic mites Varroa destructor and Tropilaelaps spp. transfer from their native Asian honey bee hosts (Apis cerana and Apis dorsata respectively) to infest the brood of A. mellifera causing significant damage to colonies and colony losses. T. mercedesae was recently detected in Europe for the first time in A. mellifera colonies and is considered a more damaging parasite of A. mellifera than Varroa. Beekeepers rely heavily on the use of synthetic miticides and organic chemicals to control Varroa and Tropilaelaps which has resulted in Varroa developing resistance to many synthetic miticides and these treatments becoming less effective. Less is known about chemical resistance in Tropilaelaps as no study has been undertaken that specifically looks at this issue, but there is evidence to suggest that Tropilaelaps do have resistance to chemicals such as Amitraz, Coumaphos, Flumethrin and Fluvalinate. The use of synthetic miticides is widely recommended for surveillance and detection of Tropilaelaps and this recommendation forms a part of the contingency response of several government agencies. The study developed a novel chemical resistance test for Tropilaelaps and sought to test the efficacy of commercially available synthetic miticides and found that mites were resistant to all the synthetic chemical treatments tested apart from Amitraz which was shown to be 64% effective. Understanding and managing miticide resistance in this species is critical to prevent its further spread and colony losses.

BackgroundKnockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) gene are a key mechanism of insecticide resistance in mosquitoes. In Asian Aedes aegypti populations two main VGSC haplogroups with kdr mutations have been identified: one carrying the F1534C mutation and another with V1016G and/or S989P mutations. Functional studies have demonstrated that these three mutations on a single haplotype confer up to a 1100-fold increase in pyrethroid resistance, underscoring the need to monitor these triple mutations within distinct populations. This study investigates the prevalence of kdr mutations in Indian populations and explores the linkage association between these mutations and two distinct conserved types of introns located between exons 20 and 21. MethodsAe. aegypti specimens collected from eight different locations were genotyped for kdr alleles and intron (between exons 20 and 21) haplotypes using PCR-based assays. Representative samples underwent DNA sequencing of VGSC regions. ResultsFive kdr mutations namely S989P, V1016G, T1520I, F1534C, and F1534L, were identified, each exhibiting varying distribution and frequencies across different geographical regions. Two distinct and stably-diverged intron haplotypes, designated as intron-A and intron-B, were identified between exons 20 and 21. Seven haplotypes, including two wild-type variants, were observed among Indian populations. The kdr-bearing haplotypes can be classified into three distinct haplogroups: haplogroup G (V1016G with/or without S989P and with intron-A), haplogroup L (F1534L and intron-A), and haplogroup C (F1534C with/or without T1520I and with intron-B). Importantly, no evidence of recombination within Indian populations was detected among these three haplogroups. ConclusionsFive kdr mutations were identified in the VGSC of Indian Ae. aegypti populations, each showing a definitive linkage with one of the two types of intron haplotypes. The lack of recombination among haplogroups bearing 1016G with 989P, 1534C and 1534L mutations suggests that the most potent insecticide resistance haplotype, bearing the triple kdr mutation, is currently absent. This finding has significant operational implications, as it may indicate that current vector control measures remain effective against these populations, potentially delaying the emergence of highly resistant phenotypes