Background: Despite recent advances in our understanding of the basic biology behind transmission of zoonotic infectious diseases harbored by arthropod vectors these diseases remain threatening public health concerns. For effective control of vector and treatment, precise sampling indicating the prevalence of such diseases is essential. With an aim to develop a quick and simple method to survey zoonotic pathogen-transmitting vectors, LAMP (loop-mediated isothermal amplification) was applied to the detection of filarial parasites using a filarial parasite-transmitting experimental model that included one of the mosquito vectors, Aedes aegypti, and the canine heartworm, Dirofilaria immitis. Results: LAMP reactions amplifying the cytochrome oxidase subunit I gene demonstrated high sensitivity when a single purified D. immitis microfilaria was detected. Importantly, the robustness of the LAMP reaction was revealed upon identification of an infected mosquito carrying just a single parasite, a level easily overlooked using conventional microscopic analysis. Furthermore, successful detection of D. immitis in wild-caught mosquitoes demonstrated its applicability to field surveys. Conclusion: Due to its simplicity, sensitivity, and reliability, LAMP is suggested as an appropriate diagnostic method for routine diagnosis of mosquito vectors carrying filarial parasites. This method can be applied to the survey of not only canine filariasis but also lymphatic filariasis, another major public health problem. Therefore, this method offers great promise as a useful diagnostic method for filarial parasite detection in endemic filariasis regions.

Malaria control relies heavily on treated bed nets and indoor residual spraying with pyrethroid insecticides. Unfortunately, the resistance to pyrethroid insecticides, mainly due to the kdr mutation, is spreading in the main malaria vector Anopheles gambiae s.l., decreasing the insecticides’ efficacy. To manage the insecticide resistance rapidly and flexibly, simple and effective tools for the early detection of resistant mosquitoes are needed. This study aimed to develop an allele-specific, loop-mediated, isothermal amplification (AS-LAMP) method to detect the West African-type kdr mutation (kdr-w; L1014F) in field-collected mosquitoes.

Background: The proboscis is an essential head appendage in insects that processes gustatory code during food intake, particularly useful considering that blood-sucking arthropods routinely reach vessels under the host skin using this proboscis as a probe. Results: Here, using an automated device able to quantify CO₂-activated thermo (35°C)-sensing behavior of the malaria vector Anopheles stephensi, we uncovered that the protruding proboscis of mosquitoes contributes unexpectedly to host identification from a distance. Ablation experiments indicated that not only antennae and maxillary palps, but also proboscis were required for the identification of pseudo-thermo targets. Furthermore, the function of the proboscis during this behavior can be segregated from CO₂ detection required to evoke mosquito activation, suggesting that the proboscis of mosquitoes divide the proboscis into a "thermo-antenna" in addition to a "thermo-probe". Conclusions: Our findings support an emerging view with a possible role of proboscis as important equipment during host-seeking, and give us an insight into how these appendages likely evolved from a common origin in order to function as antenna organs.

Background: There is a need for improving the method for counting oocysts of Plasmodium berghei in the midgut of Anopheles mosquitoes. The two methods currently used, the formalin fixation method and the mercurochrome staining method, have contradicting advantages and disadvantages. In the formalin fixation method, the specimen can be preserved but unstained oocysts were often indistinct from the insect tissue. While in the mercurochrome staining method, stained oocysts can be clearly distinguished from insect tissue but the specimen are not well preserved. These two methods were combined in this study to develop a new improved technique in counting the oocysts, in which the specimen can be both stained and preserved well. This technique was evaluated for its accuracy and suitability in observing the oocyst development. Findings: In the improved technique, the parasite-infected midgut was first stained with mercurochrome, and then fixed with formalin. The specimens were finally observed using light microscopy. To evaluate the accuracy in the oocyst counting with the improved technique, mosquitoes were infected with the green fluorescent protein (GFP)-expressing parasite. Then, the midgut oocysts were counted using both the GFP marker and the improved technique. Results were then compared and showed that the improved technique retrieved 78%-123% (arithmetic mean = 97%) of the oocysts counted using the GFP marker. Furthermore, it was also possible to evaluate the oocyst development with a green filter using the light microscopy. Conclusions: The improved technique for oocyst counting will be a useful tool for evaluating midgut oocyst numbers and determining the developmental stage of oocysts in parasite-infected mosquitoes.

Malaria vectors have developed resistance to the four families of insecticides available for public health purposes. For example, the kdr mutation is associated with organochlorines and pyrethroids resistance. It is of particular concern that organophosphate and carbamate resistance associated with the G119S ace-1 R mutation has recently increased in West Africa in extent and frequency, and is now spreading through the Anopheles gambiae malaria vector population. There is an urgent need to improve resistance management using existing insecticides and new tools to quickly assess resistance level for rapid decision-making. DNA extracted from field-collected mosquitoes was used to develop the method. Specific primers were designed manually to match the mutation region and an additional mismatched nucleotide in the penultimate position to increase specificity. Other primers used are common to both wild and mutant types. The allele specific (AS)-LAMP method was compared to the PCR restriction fragment length polymorphism (PCR-RFLP) and real-time PCR (RT-PCR) methods using the genomic DNA of 104 field-collected mosquitoes. The primers designed for LAMP were able to distinguish between the wild type (ace-1 S ) and mutated type allele (ace-1 R ). Detection time was 50 min for the wild type homozygous and 64 min for the heterozygous. No amplification of the resistant allele took place within the 75-min test period when using the wild type primers. For the ace-1 R resistant type, detection time was 51 min for the resistant homozygous and 55 min for the heterozygous. No amplification of the wild type allele took place within the 75-min test period when using the resistant type primers. Gel electrophoresis of LAMP products confirmed that amplification was primer-DNA specific, i.e., primers could only amplify their target specific DNA. AS-LAMP, PCR-RFLP, and RT-PCR showed no significant difference in the sensitivity and specificity of their ace-1 R detection ability. The AS-LAMP method could detect the ace-1 R mutation within 60 min, which is faster than conventional PCR-RFLP. This method may be used to quickly detect the ace-1 R mutation for rapid decision-making, even in less well-equipped laboratories.

Filariasis is emerging as a public health concern in tropical and subtropical areas. Filariasis is an endemic problem commonly found in southeast Asian countries. Using the PCR-restriction fragment length polymorphism (PCR-RFLP) of the ITS1 region with Vsp I, the overall prevalence rates of Dirofilaria immitis (12.2% (41/337); 95% confidence interval: 9.1–16.1%) and Brugia pahangi (8.3% (28/337); 95% confidence interval: 5.8–11.8%) were determined based on 337 free-roaming community dogs from 20 districts in Northern Thailand. Microfilaremia was found in only 6.2% of dogs (21/337). Co-infection with D. immitis and B. pahangi was observed in two dogs. Of the 215 blood samples examined using a Canine Heartworm Ag Kit, only 3.72% (eight dogs) were D. immitis antigen positive. Among these eight, six dogs had occult D. immitis infections. In terms of geographic distribution, we found the abundance of D. immitis and B. pahangi in the central areas at altitudes less than 400 m to be 12.1% and 10.3%, respectively. In contrast, at higher altitudes between 400 and 800 m, a significantly higher number of B. pahangi compared with D. immitis infected individuals were observed at 14.29% and 4.1%, respectively. In conclusion, D. immitis and B. pahangi were the most common filarial infections found in community dogs in Northern Thailand. Dogs might be an important reservoir of B. pahangi in that region. Increasing awareness and concern and including proper deworming programs for community dogs should be endorsed to reduce the transmission risk. Additionally, the population dynamics of the mosquito vector of B. pahangi across altitudinal gradients deserved further investigation.