Susceptibility of Culiseta longiareolata to insecticides in West Azerbaijan province, northwestern Iran.

Mosquitoes are involved in the transmission of a wide range of diseases and among them, Culiseta longiareolata, acts as a vector of avian malaria, tularemia and several arboviruses like West Nile fever. The current study was conducted to determine the susceptibility of C. longiareolata against deltamethrin 0.05% (pyrethroids), fenitrothion 1.00% (organophosphate) and bendiocarb 0.10% (carbamate). Mosquitoes were collected from different parts of Urmia county, the capital of West Azerbaijan. In the current study, 443 empty 3 - 5 days old adult female C. longiareolata were used for susceptibility tests. The susceptibility status of C. longiareolata was determined using WHO's recommended procedure. The results indicated higher mortality rates of Culiseta longiareolata against studied insecticides. Culiseta longiareolata, was resistant to all studied insecticides (deltamethrin, mortality rate: 62.50%, fenitrothion, mortality rate: 35.96% and bendiocarb, mortality rate: 20.16%). The current study represents the first report of susceptibility status of C. longiareolata to fenitrothion and bendicarb in Iran. Considering the climatic diversity and geographical conditions of northwestern Iran, reports of resistance of other mosquitoes' of this region, the susceptibility status of medically and veterinary important mosquitoes should be given serious attention.

Sindbis virus infection of mosquito species in the wetlands of northwestern Iran and modeling the probable ecological niches of SINV vectors in the country.

Sindbis virus (SINV) and Chikungunya virus (CHIKV) are among the most widely spread mosquito-borne viruses worldwide. Due to the key role of mosquitoes in the transmission cycle of vector-borne diseases, models such as Maximum Entropy (MaxEnt) have been used in recent years to predict the environmental suitability and ecological niches of mosquito vectors. Infection of three mosquito species (Anopheles maculipennis s.l., Culex tritaeniorhynchus, and Culiseta longiareolata) with CHIKV has recently been reported in Iran. However, given the importance of vector-borne diseases in the country, there is a need for extensive studies on the infection of mosquitoes with CHIKV and SINV in different areas of the country. Accordingly, the current research was conducted to investigate the infection of mosquitoes with the two aforementioned viruses in the northwestern part of Iran and also to model the ecological niches of the vectors of these mosquito-borne viruses in the country. In the current study, 4639 mosquito specimens, consisting of 2515 adults and 2124 larvae, were collected from the wetlands of West Azerbaijan Province and identified. Ten species belonging to four genera were identified in this study. The specimens were allocated to 149 pools for the determination of infection with CHIKV and SINV. The amplification pattern of five pools comprising two mosquito species (Culex pipiens complex and Cx. Theileri) corresponded to the reference strain of SINV, and the isolates were sequenced to confirm the presence of SINV genome. No cases of CHIKV infection were found among the 149 examined mosquito pools. Data on the distribution of Cx. Pipiens complex and Cx. Theileri were mapped using ArcMap 10.5. Prediction maps of the presence probability for these species revealed that they are most likely to be found in and spread to the north, northwest, south, and southeastern areas of the country and in areas with abundant water resources. For the first time in Iran, our study investigated the presence probability of SINV vectors using ecological niche modeling. Ecological niche profiling showed that the most suitable habitats for Cx. pipiens are mainly concentrated in the north and northwestern parts of the country, whereas Cx. theileri is mostly located in the northwest and western regions. However, there were some other areas of low suitability for these two species in the country. Further studies in a broader geographical area with more species of mosquitos and the determination of infection with other mosquito-borne viruses can provide a clear understanding of the potential spread of mosquito-borne diseases in various regions of Iran.

Molecular analysis of the mitochondrial markers COI, 12S rDNA and 16S rDNA for six species of Iranian scorpions.

OBJECTIVES: Annually, 1.2 million humans are stung by scorpions and severely affected by their venom. Some of the scorpion species of medical importance have a similar morphology to species with low toxicity. To establish diagnostic tools for surveying scorpions, the current study was conducted to generate three mitochondrial markers, Cytochrome Oxidase I (COI gene), 12S rDNA and 16S rDNA for six species of medically important Iranian scorpions: Androctonus crassicauda, Hottentotta saulcyi, Mesobuthus caucasicus, M. eupeus, Odontobuthus doriae, and Scorpio maurus. RESULTS: Phylogenetic analyses of the obtained sequences corroborated the morphological identification. For the first time, 12S rDNA sequences are reported from Androctonus crassicauda, Hottentotta saulcyi, Mesobuthus caucasicus and M. eupeus and also the 16S rDNA sequence from Hottentotta saulcyi. We conclude that the mitochondrial markers are useful for species determination among these medically important species of scorpions.

Molecular Characterisation of Cytochrome Oxidase I and Internal Transcribed Spacer 2 Fragments of Culiseta longiareolata.

Objective: Among the mosquitoes, Culiseta longiareolata plays a notable role in the transmission of avian malaria, tularemia and arboviral diseases, including West Nile fever. We conducted this study to characterise the cytochrome oxidase I (COI) and internal transcribed spacer 2 (ITS2) fragments of Cs. longiareolata in northwestern Iran to determine the classification status of this species. Methods: The COI and ITS2 fragments from six populations of Cs. longiareolata were amplified, sequenced and analysed. For phylogenetic analysis, the evolutionary history was estimated using the Tamura-Nei-based Maximum Likelihood approach. Results: Thirteen sequences (six for ITS2 and seven for COI) from six populations of Cs. longiareolata were acquired and deposited into the GenBank. Phylogenetic analysis showed that the COI sequences from the current study cluster together with the same species from other parts of the world. Moreover, the ITS2 sequences of the current study and sequences retrieved from the GenBank, despite intraspecies variation, fall into a distinct clade with acceptable bootstrap values. Conclusion: Notable genetic variations were observed between various Cs. longiareolata populations based on the evaluations of ITS2 and COI fragments. By conducting such studies, the exact classification status of this species can be determined.

Larval habitats and species diversity of mosquitoes (Diptera: Culicidae) in West Azerbaijan Province, Northwestern Iran.

BACKGROUND: The characteristics of a larval habitat is an important factor which affects the breeding pattern and population growth of mosquitoes Information about the larval habitat characteristics and pupal productivity can be utilized for the surveillance of the level of population growth, species diversity, and preferred breeding sites of mosquitoes, which are important aspects of integrated vector control. In the present study, mosquito larvae were collected from 22 natural habitats in five counties of the West Azerbaijan Province in the Northwest of Iran during May-November 2018. Physicochemical characteristics of the habitats were investigated. These included alkalinity, chloride (Cl) content, water temperature (°C), turbidity (NTU), Total Dissolved Solids (TDS) (ppm), Electrical Conductivity (EC) (µS/cm), and acidity (pH). The index of affinity between the collected species was calculated using Fager & McGowan test. RESULTS: A total of 2715 specimens were collected and identified. Seven different species belonging to four genera were identified in our study sites. The species included, Culex pipiens Linnaeus 1758, Culex theileri Theobald 1903, Culex mimeticus Noé 1899, Culex modestus Ficalbi 1947, Culiseta longiareolata Macquart 1838, Anopheles maculipennis Meigen 1818complex, and Aedes caspius Pallas 1771. There was a significant difference in chloride content and water temperature preferences among the different species (P < 0.05). Also, there was no significant difference in pH, Alkalinity, Turbidity, TDS, and EC preferences among the different species (P > 0.05). The affinity between the pair of species Cx. mimeticus/Cs. longiareolata was 0.526. There was no affinity between other pairs of species or the affinity was very weak. CONCLUSIONS: The physicochemical and biological characteristics of mosquito larval habitats play an important role in zoning of areas suitable for breeding and distribution. Surveillance of these characteristics can provide valuable information for entomological monitoring of mosquito vectors and for designing targeted control programs. Also, further studies should be undertaken in a wider geographical area, taking into account the complex characteristics of the physicochemical and ecological factors of the study area and their interaction with various mosquito species.

Molecular characterization of Ribosomal DNA (ITS2) of hard ticks in Iran: understanding the conspecificity of Dermacentor marginatus and D. niveus.

OBJECTIVES: Hard ticks (Acari: Ixodidae) are ectoparasites of medical and veterinary importance. They are obligate blood-feeding vectors with the ability to transmit a wide variety of pathogens. Standard morphological keys are normally used for the identification of tick species. However, considering the importance of accurate species identification and the determination of bio-ecological characteristics of species, relying on morphological keys alone can be questionable. In this study, two DNA fragments (ITS2 and COI) were selected for phylogenetic evaluation of Iranian hard tick species belonging to the genera Dermacentor, Hyalomma, and Rhipicephalus. RESULTS: 1229 specimens of Dermacentor marginatus, D. niveus, Hyalomma anatolicum, Rhipicephalus bursa, and R. sanguineus s.l constituting 11 populations were collected from three different climatic and zoogeographical zones in Iran. Morphological studies revealed notable differences in important morphological characteristics between different populations of D. marginatus. The results of ITS2 sequence analysis provided additional evidence which supports the conspecificity of D. niveus and D. marginatus. Contrary to this finding, the sequence analysis of COI and phylogeny favored the separation of the two species. Given the greater importance of COI in identifying and discriminating species, a possibility heterospecificity between the two species should be considered.

Identification of symbiotic bacteria in the midgut of the medically important mosquito, Culiseta longiareolata (Diptera: Culicidae).

OBJECTIVE: The potential use of symbiotic bacteria for the control of mosquito-borne diseases has attracted the attention of scientists over the past few years. Culiseta longiareolata is among the medically important mosquitoes that transmit a wide range of vector-borne diseases worldwide. However, no extensive studies have been done on the identification of its symbiotic bacteria. Given the role of this species in the transmission of some important diseases and its widespread presence in different parts of the world, including northwestern parts and the West Azerbaijan Province in Iran, a knowledge about the symbiotic bacteria of this species may provide a valuable tool for the biological control of this mosquito. Accordingly, the present study was conducted to isolate and identify the cultivable isolates bacterial symbionts of Culiseta longiareolata using 16S rRNA fragment analysis. RESULTS: The midguts of 42 specimens of Cs. longiareolata were dissected, and the bacteria were cultured on agar plates. After the purification of the bacterial colonies, 16srRNA region amplification and gene sequence analysis were performed, and the sequences were confirmed by biochemical methods. In the present study, 21 isolates belonging to the genera Acinetobacter, Aerococcus, Aeromonas, Bacillus, Carnobacterium, Klebsiella, Morganella, Pseudomonas, Shewanella and Staphylococcus were identified.

The Potential of West Nile Virus Transmission Regarding the Environmental Factors Using Geographic Information System (GIS), West Azerbaijan Province, Iran.

BACKGROUND: West Nile fever, as an expanding zoonotic disease, has been reported from different creatures involved in the disease from Iran. In addition to biological mosquito-associated factors, various elements such as their activities, distribution, behavior and vectorial capacity could be affected by environmental factors. We determined the distribution of West Nile virus (WNV) vectors, the environmental factors affecting WNV transmission and the high-risk areas across West Azerbaijan Province (Northwestern Iran), regarding the potential of WNV transmission using Geographical Information System (GIS). METHODS: Mosquitoes' larvae and adults were collected from different habitats of the province in 2015 and identified using standard morphological keys. The data regarding the distribution of mosquitoes across the studied area were organized in ArcMap databases. Inverse Distance Weighted (IDW) interpolation analysis was conducted on the data of synoptic stations to find climatic variables in the collection sites of different mosquito species. Layers of transmission-related environmental factors were categorized and weighed based on their effects on disease transmission. RESULTS: Overall, 2813 samples of different mosquito species from different regions of the province were collected and identified. According to the GIS analysis, areas in the northeastern province, which have lower altitudes and slopes with higher temperatures and more water bodies, were found to have better condition for the activity of mosquitoes (as high-risk areas: hot spots). CONCLUSION: The precision of our results was proven to be in line with previous study results that identified high-risk areas, where WNV-infected vectors were captured from these same areas.

The role of midgut symbiotic bacteria in resistance of Anopheles stephensi (Diptera: Culicidae) to organophosphate insecticides.

In the current study, the effects of the presence of symbiotic bacteria on the activity of the enzymes involved in An. stephensi resistance to temephos are evaluated for the first time. Four different strains (I. susceptible strain, II. resistant strain, III. resistant strain + antibiotic, and IV. resistant strain + bacteria) were considered in order to determine the possible effects of the symbiotic bacteria on their hosts' resistance to temephos. The median values of all enzymes of susceptible strain were compared with those of other resistant strains. The results of this study indicated a direct relationship between the presence of bacteria in the symbiotic organs of An. stephensi and resistance to temephos. The profile of enzymatic activities in the resistant strain changed to a susceptible status after adding antibiotic. The resistance of An. stephensi to temephos could be completely broken artificially by removing their bacterial symbionts in a resistant population.

Molecular Characterization of Mosquitoes (Diptera: Culicidae) in Northwestern Iran by Using rDNA-ITS2.

Several mosquito species are vectors of disease; however, to understand their role in disease transmission, accurate species identification is of particular importance. Morphological identification is the main method used, but molecular techniques have emerged as a tool for the identification of closely related species. In this study, mosquitoes from the West Azerbaijan Province in northwestern Iran were characterized on the basis of their rDNA-ITS2 sequences. Nine populations of 6 species of mosquitoes belonging to the genera Anopheles, Culex, Culiseta, and Ochlerotatus were studied. To the best of our knowledge, ITS2 sequences of Culiseta longiareolata and Culex hortensis have been reported for the first time. In addition, ITS2 sequences of Culex theileri and Ochlerotatus caspius have been reported for the first time in Iran. Phylogenetic analysis based on ITS2 showed that subfamilies Anophelinae and Culicinae of the family Culicidae could be differentiated successfully and subgenera Anopheles and Cellia of the genus Anopheles were separated. The analysis showed that the genera Culex, Culiseta, and Ochlerotatus have diverged separately.

West Nile Virus in Mosquitoes of Iranian Wetlands.

The West Nile virus (WNV) transmission cycle includes a wide range of migratory wetland birds as reservoirs, mosquitoes as biological vectors, and equines and humans as dead-end hosts. Despite the presence of potential vector species, there is no information about the existence of WNV in mosquito vectors in Iran. The Iranian West Azerbaijan Province is located in the northwestern part of Iran and has borders with Turkey, Iraq, Armenia, and the Republic of Azerbaijan. The current study was conducted to identify the wetland mosquitoes of the West Azerbaijan Province and their infection with WNV. In this study, 2143 specimens were collected, comprising 1541 adults and 602 larvae. Six species belonging to four genera were collected and identified: Anopheles maculipennis sensu lato (s.l.), Culex (Cx.) hortensis, Cx. pipiens s.l., Cx. theileri, Culiseta longiareolata, and Aedes (Ae.) (Ochlerotatus) caspius. In total, 45 pools of mosquitoes were examined. Two of the adult pools collected from the same location showed the presence of WNV in Ae. (Och.) caspius, from Sangar, Makoo County, as confirmed by PCR and sequencing. Due to the discovery of WNV in the mosquito population of the region, and the presence of wetlands and significant populations of migratory birds, the health sector should carefully monitor the factors involved in the cycle of this disease.

Malpighian tubules are important determinants of Pseudomonas transstadial transmission and longtime persistence in Anopheles stephensi.

BACKGROUND: Pseudomonas is a genus of bacteria commonly found in investigations of gut microbes in malaria mosquitoes. Among those mosquitoes is the dominating malaria vector in Asia, Anopheles stephensi, where Pseudomonas is a prevailing bacterium and natural inhabitant of its breeding places. In order to explore the reason for finding Pseudomonas so frequently, an investigation of its localization and transstadial properties was undertaken. METHODS: A Pseudomonas isolate from An. stephensi was transformed successfully with an endogenous plasmid modified to express green fluorescent protein (GFP). Subsequently, the Pseudomonas-GFP was added to the laboratory larval breeding place of An. stephensi and taken up by the larvae. After 24 hours, the larvae were cleaned and moved to a bath with double-distilled water. Also, female adults were fed sugar solution containing Pseudomonas-GFP. The Pseudomonas-GFP was traced in the alimentary canal of larvae, pupae and adults. RESULTS: Fluorescent microscopy and PCR assays showed that the Pseudomonas bacteria underwent transstadial transmission from larvae to pupae and then to adults. In blood-fed female mosquitoes, the bacteria increased in numbers and remained in the mosquito body for at least three weeks after eclosion. In addition to the midgut, the Malpighian tubules of both larvae and adult mosquitoes were colonized by the bacteria. Also Pseudomonas-GFP that was distributed through sugar solution was able to colonize the Malpighian tubules of adult females. CONCLUSIONS: Colonization of the Malpighian tubules by Pseudomonas bacteria seems to be important for the transstadial passage from larvae to adult and presumably for the longevity of the bacteria in the adult mosquito. The existence of an entry point in the larval stage, and the long duration in the female gut, opens up for a possible use of Pseudomonas in mosquito paratransgenesis.

Phylogenetic analysis of the oriental-Palearctic-Afrotropical members of Anopheles (Culicidae: Diptera) based on nuclear rDNA and mitochondrial DNA characteristics.

The phylogenetic relationships of Anopheles spp. at the junction of Oriental, Palearctic, and Afrotropical regions in the Iranian plateau were investigated using molecular markers. A 711-bp mitochondrial DNA cytochrome oxidase C subunit I (COI) fragment and the entire second internal transcribed spacer (ITS2) region (286-576 bp) of the nuclear ribosomal DNA (rDNA-ITS2) were sequenced from 14 and 28 taxa, respectively. The analyses included 12 species within Anopheles and 4 within the Myzorhynchus Series of the subgenus Anopheles, 8 within Neocellia, 6 within Myzomyia, 3 within Paramyzomyia, and 1 within the Pyretophorus Series of the subgenus Cellia. The congruent tree topologies of both molecular markers strongly supported monophyly of subgenera Anopheles and Cellia. Phylogenetic trees constructed on the basis of ITS2 sequences could accurately categorize all of the series according to the classical taxonomy but could not distinguish Pyretophorus (Anopheles subpictus) from Paramyzomyia Series. Although sequence data of the COI region were available for only 14 species, the inferred trees revealed good classification among the series but could not show the monophyletic relationship of Cellia spp. Except for a few cases, the tree inferred from ITS2 sequences revealed the best classification for the species studied. The molecular data could significantly improve our understanding of the phylogenetic position of the taxa.

Isolation and identification of culturable bacteria from wild Anopheles culicifacies, a first step in a paratransgenesis approach.

BACKGROUND: Due to the effect of midgut bacteria on proliferation of parasites and their potential as paratransgenesis tools, their identification in malaria vector mosquitoes is important. Anopheles culicifacies s.l. is one of the main malaria vectors in Asia; however, its midgut microbiota remains un-studied. This work was primarily designed to isolate potential candidates for use in a paratransgenesis approach, but also to give a picture of the midgut microbiota of wild-caught An. culicifacies larvae and adults from the southeast corner of Iran, which has the highest malaria endemicity in the country. METHODS: A total of 68 larvae and 34 adult females (newly eclosed and older) from three different biotopes in Iran were analyzed for their midgut microflora. The mosquitoes had their midgut bacterial contents plated on three different culture media (brain heart agar, nutrient agar and blood agar) yielding 57 bacterial isolates. The 16S rRNA genes of the isolates were sequence analyzed for species designation, which then was confirmed by biochemical analysis. RESULTS: A total of twelve bacterial genera were identified: Acinetobacter, Aeromonas, Bacillus, Chryseobacterium, Delftia, Exiguobacterium, Kurthia, Microbacterium, Pseudomonas, Staphylococcus, Thorsellia and Variovorax. In older females, only Gram-negative bacteria were found, whereas larvae and newly-eclosed adults also harbored Gram-positive bacteria. The diversity of isolates also varied between sampling sites and mosquito stages, with the largest number of genera found in the Anguri district and in larvae, respectively. Pseudomonas was the most common genus retrieved from all sampling sites, and in both larvae and adults, suggesting a potential transstadial passage of these bacteria. Interestingly, identical 16S sequences of Pseudomonas were found in mosquitoes originating from different habitats at least 45 km apart, which could suggest that these bacteria have been adapted to the mosquitoes. CONCLUSIONS: The study of vector mosquito microbiota has recently gathered increased interest because of the potential influence on vector competence. By adding data from a hitherto uncharacterized malaria mosquito, a better picture of gut flora in vector mosquitoes was obtained. Furthermore, some species of the predominant genus Pseudomonas will be evaluated for the selection of a paratransgenesis candidate.

Molecular characterization, biological forms and sporozoite rate of Anopheles stephensi in southern Iran.

OBJECTIVE: To identify the biological forms, sporozoite rate and molecular characterization of the Anopheles stephensi (An. stephensi) in Hormozgan and Sistan-Baluchistan provinces, the most important malarious areas in Iran. METHODS: Wild live An. stephensi samples were collected from different malarious areas in southern Iran. The biological forms were identified based on number of egg-ridges. Molecular characterization of biological forms was verified by analysis of the mitochondrial cytochrome oxidase subunit I and II (mtDNA-COI/COII). The Plasmodium infection was examined in the wild female specimens by species-specific nested-PCR method. RESULTS: Results showed that all three biological forms including mysorensis, intermediate and type are present in the study areas. Molecular investigations revealed no genetic variation between mtDNA COI/COII sequences of the biological forms and no Plasmodium parasites was detected in the collected mosquito samples. CONCLUSIONS: Presence of three biological forms with identical sequences showed that the known biological forms belong to a single taxon and the various vectorial capacities reported for these forms are more likely corresponded to other epidemiological factors than to the morphotype of the populations. Lack of malaria parasite infection in An. stephensi, the most important vector of malaria, may be partly due to the success and achievement of ongoing active malaria control program in the region.

Fauna and Larval Habitats of Mosquitoes (Diptera: Culicidae) of West Azerbaijan Province, Northwestern Iran.

BACKGROUND: Several important diseases are transmitted by mosquitoes. Despite of the potential of the occurrence of some mosquito-borne diseases such as West Nile, dirofilariasis and malaria in the region, there is no recent study of mosquitoes in West Azerbaijan Province. The aim of this investigation was to study the fauna, composition and distribution of mosquitoes and the characteristics of their larval habitats in this province. METHODS: Larvae and adult collections were carried out from different habitats using the standard methods in twenty five localities of seven counties across West Azerbaijan Province. RESULTS: Overall, 1569 mosquitoes including 1336 larvae and 233 adults were collected from 25 localities. The details of geographical properties were recorded. Five genera along with 12 species were collected and identified including: Anopheles claviger, An. maculipennis s.l., An. superpictus, Culex pipiens, Cx. theileri, Cx. modestus, Cx. hortensis, Cx. mimeticus, Culiseta Longiareolata, Ochlerotatus caspius s.l., Oc. geniculatus and Uranotaenia unguiculata. This is the first record of Oc. geniculatus in the province. CONCLUSION: Due to the geographical location of the West Azerbaijan Province, it comprises different climatic condition which provides suitable environment for the establishment of various species of mosquitoes. The solidarity geographical, cultural and territorial exchanges complicate the situation of the province and its vectors as a threat for future and probable epidemics of mosquito-borne diseases.

Identification of bacterial microflora in the midgut of the larvae and adult of wild caught Anopheles stephensi: a step toward finding suitable paratransgenesis candidates.

To describe the midgut microbial diversity and to find the candidate bacteria for the genetic manipulation for the generation of paratransgenic Anopheline mosquitoes refractory to transmission of malaria, the microbiota of wild larvae and adult Anopheles stephensi mosquito midgut from southern Iran was studied using a conventional cell-free culture technique and analysis of a 16S ribosomal RNA (rRNA) gene sequence library. Forty species in 12 genera including seven Gram-negative Myroides, Chryseobacterium, Aeromonas, Pseudomonas, Klebsiella, Enterobacter and Shewanella and five Gram-positive Exiguobacterium, Enterococcus, Kocuria, Microbacterium and Rhodococcus bacteria were identified in the microbiota of the larvae midgut. Analysis of the adult midgut microbiota revealed presence of 25 Gram-negative species in five genera including Pseudomonas, Alcaligenes, Bordetella, Myroides and Aeromonas. Pseudomonas and Exiguobacterium with a frequency of 51% and 14% at the larval stage and Pseudomonas and Aeromonas with a frequency of 54% and 20% at the adult stage were the most common midgut symbionts. Pseudomonas, Aeromonas and Myroides genera have been isolated from both larvae and adult stages indicating possible trans-stadial transmission from larva to adult stage. Fast growth in cheap media, Gram negative, and being dominantly found in both larvae and adult stages, and presence in other malaria vectors makes Pseudomonas as a proper candidate for paratransgenesis of An. stephensi and other malaria vectors.

Analysis of mosquito bloodmeals using RFLP markers.

An important variable in the amplification of arthropod vector-borne diseases is the degree of contact between human hosts and mosquito vectors. To analyze this interaction, a DNA based method was developed to differentiate human bloodmeals from other sources in the mosquito Anopheles stephensi (Diptera: Culicidae) Liston. A portion of the host mitochondrial DNA cytochrome B genes were PCR amplified and classified to the species level based on their restriction fragment length polymorphism (RFLP). The cytochrome B sequences showed sufficient interspecific polymorphism to distinguish between human, cow, sheep, chicken, and guinea pig hosts. XhoI could distinguish human from other vertebrates whereas TaqI alone could separate the others. The importance of these results in epidemiological studies of malaria and other vector borne diseases is discussed.

Effects of post-ingestion and physical conditions on PCR amplification of host blood meal DNA in mosquitoes.

The effects of post-ingestion and physical conditions under which killed mosquitoes are stored on the success of detecting blood meal DNA of Anopheles stephensi and Culex quinquefasiatus were investigated by polymerase chain reaction (PCR) amplification at the human mitochondrial DNA cytochrome b (cytB) gene. Host DNA extracted from the blood meal up to 33 h post-ingestion in both species acts as an efficient template for PCR amplification. However, more DNA concentration is needed for meals digested for a longer time, and successful PCR amplification from meals digested for 36 h,dropped to a faint band. There were no differences between PCR success rate for samples stored at +4 or -20 degrees C, but less successful products were observed in samples kept at 4 degrees C for the periods longer than 30 h digestion. The results of this study are important for conducting malaria epidemiological studies that provide information about the degree of contact between human hosts and mosquito vectors, impact of vector controls such as bed nets and repellents, and the transmission dynamics of human malaria and other vector-borne diseases.