Upwind responses of Anopheles stephensi to carbon dioxide and L-lactic acid: an olfactometer study.

Excretion of carbon dioxide and L-lactic acid through exhalation and perspiration provides olfactory signals to mosquitoes which allow them to find and bite humans; however, mosquito species differ in this regard. This study investigated upwind responses of Anopheles stephensi, mysorensis form, an important malaria vector in Asia, to carbon dioxide and L-lactic acid under laboratory conditions. While a minimal dose of carbon dioxide (90 ppm) activated the mosquitoes, 10 times this amount suppressed them. L-lactic acid alone did not produce a significant effect by itself, but addition of 6 microg/min of L-lactic acid to a range of 90 to 410 ppm carbon dioxide resulted in attraction. The results provide further support for the hypothesis that CO2 plays an important role in the host-seeking behaviour of zoophilic mosquitoes, and suggests that L-lactic acid might play a more critical role than CO2 in the attraction of An. stephensi

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 Anopheles 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 Anopheles 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

Differential Responses of Anopheles stephensi (Diptera: Culicidae) to Skin Emanations of a Man, a Cow, and a Guinea Pig in the Olfactometer

Background: Biting habit of mosquitoes plays an important role in the epidemiology of mosquito-borne diseases. Mosquitoes use a set of elaborate sensory modalities to find their preferred hosts by exploiting cues emanating from a nearby host. It has been suggested that the chemical profile of skin can provide further support for anthropophilic mosquito species to find their suitable hosts. This study aimed at revealing the value of skin emanation for a zoophilic species like Anopheles stephensi as a model. Methods: Skin emanations of a man, a cow and a Guinea pig were collected by ethanol soaked cottons. Upwind responses of mosquitoes to 100 and 200 mu l of filtered skin materials were non-competitively explored in a dual-choice olfactometer. L-lactic acid and other chemical content of the skin samples were identified by an enzymatic kit and GC-MS, respectively. Results: Unexpectedly, only human skin emanation was resulted in the statistically significant activation and attraction responses of An. stephensi in the wind tunnel. L-lactic acid content of this skin sample was 10 and 29 times more than the cow and the Guinea pig, respectively. The possible role of lactic acid and a few other identified compounds have been discussed here. Conclusion: Anopheles stephensi showed higher and more specific upwind responses to human skin emanation in the olfactometer. Undoubtedly, the thorough explanation of this unexpected finding needs further investigation. But, if new data verify this result, then, it may be necessary to reconsider the role of skin emanation and thence the human blood index and vectorial capacity of this zoophilic mosquito

Fabrication of an olfactometer for mosquito behavioural studies

Background & objectives: Olfaction is the major sensory modality involved in the resource searching behaviour of insects including vector mosquitoes (Diptera: Culicidae). To date, our current country-wide knowledge on the host-seeking behaviour of Iranian mosquitoes is mainly confined to host preference which has exclusively come from field studies. Olfactometer is a scientific tool by which more naive aspects of man-vector contact can be clarified under controlled and less biased conditions. Methods: The wind tunnel and stimulus delivery system was constructed from acrylic materials based on previously introduced models with some modifications. Air supply and required light were ensured by a powerful compressor and incandescent bulbs, respectively. Desired level of temperature was maintained by controllable heating radiators. For humidity production a unique in-built piezo system was devised in the course of the air flow. Fine regulators facilitated the continuous generatation of the humidity at a preset level. Results: Titanium tetrachloride smoke plus monitoring of the wind speed revealed that the flow of air was proper and invariable. A desired level of humidity and temperature could be set up in just 10 and 15-45 min, respectively. These physical parameters varied only +/-2% (humidity) and +/-0.15 degrees C (temperature) in a typical 20 min duration. Conclusion: The first sophisticated olfactometer in the field of medical entomology in Iran is reported here. Fast set up and stability of physical parameters are its salient features. It is expected that with the aid of this olfactometer further information on the physiological principles of the host-seeking behaviour of mosquitoes become available soon

Molecular Characterization of Paederus Spp (Coleoptera: Staphylinidae, Paederinae) the Agent of Human Linear Dermatitis in the Caspian Sea Coast, North of Iran

Background: A combined morphological and molecular survey was performed to determine the agent of human linear dermatitis Paederus Fabricius, 1775 (Coleoptera: Staphylinidae, Paederinae) species composition in Mazandaran Prov­ince in the Caspian Sea coast in northern Iran, where most of linear dermatitis cases of the country occurred. Methods: Altogether, 397 Paederus specimens were collected from May to August 2021 and classified using morphological characters and ITS2-rDNA sequence analysis. Results: Morphological investigation revealed that all the specimens were Paederus fuscipes. ITS2 polymerase chain reaction (PCR) direct-sequences and the profiles of restriction fragment length polymorphism (RFLP) derived from digestion of PCR products by HinfI, HpaII, and SalI enzymes were identical confirming the morphological results, implying that all specimens belonged to a single taxon. Conclusion: Paederus fuscipes (Fabricius, 1775) is considered the dominant taxon and responsible for linear dermatitis in Ma­zandaran Province. To our knowledge, we have provided the first molecular typing of Paederus beetles at the species level, suggesting that ITS2-rDNA characterization is an alternative tool for species discrimination of Paederus spp

Experimental Study on Plasmodium berghei, Anopheles Stephensi, and BALB/c Mouse System: Implications for Malaria Transmission Blocking Assays

Background:Plasmodium berghei is a rodent malaria parasiteand has been very valuable means in the progress of our understanding of the essential molecular and cellular biology of the malaria parasites. Availability of hosts such as mice and vectors such as Anopheles stephensi has made this parasite a suitable system to study the parasite-host and vector-parasite relationships. Numerous studies have described life cycle and parameters influencing maintenance of the parasite within the mice or the mosquito. In this paper we revealed more details and have addressed some parameters and points influence maintenance of various life stages of the parasite (merozoites, macrogametocytes, ookinetes, oocysts and sporozoites) in the laboratory model P.berghei–A.stephensi-BALB/c mouse. This study helps understanding the biology of vertebrate-parasite and mosquito-malaria interactions that may aid in the development of a new generation of drug/vaccine and vector-based measures for malaria control

Biochemical Basis of Cyfluthrin and DDT Resistance in Anopheles stephensi (Diptera: Culicidae) in Malarious Area of Iran

Background: Anopheles stephensi is a key urban malaria vector in the Indian subcontinent and Middle East includ­ing south and southeast of Iran. Wide application of insecticides resulted in resistance of this species to various insec­ticides in these regions. This study was conducted to reveal the role of metabolic mechanisms in the development of resistance in An. stephensi to DDT and cyfluthrin. Methods: Field mosquito specimens were collected from Chabahar Seaport, southeast corner of Iran, in 2015. Insec­ticide susceptibility and enzyme assays were conducted as recommended by WHO. Results: Mean enzyme ratios were 3.95 and 3.04 for α- esterases and 2.40 and 1.97 for β- esterases in the DDT and cyfluthrin- resistant populations correspondingly compared with the susceptible strain. The GSTs enzyme mean ac­tivity ratios were 5.07 and 2.55 in the DDT and cyfluthrin- resistant populations compared with the susceptible beech strain. The cytochrome p450s enzyme ratios were 1.11 and 1.28 in the DDT and cyfluthrin- resistant populations respectively compared with the susceptible beech strain. Conclusion: Metabolic mechanisms play a crucial role in the development of DDT and cyfluthrin resistance in An. stephensi, therefore, further evaluation of the mechanisms involved as well as implementation of proper insecticide resistance management strategies are recommended

Toxicological Analysis of Insects on the Corpse: a Valuable Source of Information in Forensic Investigations

Background: Entomotoxicology as a subset of forensic entomology can be used by analysis of carcass feeding in­sects to detecting of drugs or toxins, as well as the cause and manner of death in cases of ante-mortem drugs intoxi­cation. Morphine is one of the deacetylate metabolites of heroin. The aim of this study was to determine the presence and quantity of morphine in insects on the carcass and compare them with decomposing carcass. Methods: Field of this study was in Chalabeh District and toxicological tests were carried out at the Department of Forensic Toxicology, Legal Medicine Center, Kermanshah, Iran in 2017. Morphine was inoculated into live rabbit as experimental model at concentrations of 12.5, 25, 50mg/ml, similar to those normally encountered in human over­doses, then quality and quantity of morphine were determined in insects such as Chrysomya albiceps (as the first wave of insect succession on human cadavers) fed on carcass. Results: Quantitative assessment at larvae showed that morphine was detected in all larvae (feeding and post feeding stage) fed on tissues from carcasses administered morphine, except for post-feeding larvae from R1 which received 12.5mg/ml dosage of morphine. Conclusions: Necrophagous insects are an indicator on the scene of crime and a potential source of information about the antemortem situation. Detection of drug in insects which is actually a reflection of the cause of death is possible