mosquitoes are of major health concern not only because of their irrititating bites but also because of the capability to transmit life - threatening diseases to human being and his livestock ( tolle 2009 ) . while this is so , the effective control of mosquitoes is still a far - reaching dream ( ranson et al . apart from the resistance problem , one of the main obstacles is the scarcity of control measures , which are specific and also safe to use ( kamareddine 2012 ) . in this context , the discovery of a new event on the natural enemies of mosquitoes may be a promising occurence in the field of finding environmental - friendly control measures to overcome these troublesome insects . microsporidia are single celled intracellular eukaryotic fungi parasitizing all groups of animals ( corradi et al . more than 1300 species of these microorganisms have been described in the literature ( vvra et al . while 14 species are known to infect humans , at least 150 more species have been recognized to parasitize 14 genera of mosquitoes ( canning 2001 , andreadis 2007 ) . based on these evidences , it has been stated that all of the mosquito species could be possibly the host of at least one microsporidium agent ( andreadis 2007 ) . microsporidia are transmitted horizontally and vertically between and within hosts by means of a specific form called spore . the spores are very small and characterized by their unique invading behavior and the exclusive ultrastructure ( wittner et al . while the monomorphic forms ( eg anncaliia and vavraia ) produce only one spore type , the polymorphic ones ( eg amblyospora and parathelohania ) have a complex life cycle and produce different types of spores . the latter are generally more host specific and commonly there is an obligatory intermediate copepod host in their life cycle ( becnel et al . microsporidia can be recognized by light microscopy techniques such as geimsa and different versions of trichrome staining ( garcia 2002 ) . the molecular information on the ss rrna sequence is an asset and facilitates phylogenetic analysis ( andreadis 2007 ) . anopheles superpictus grassi ( diptera : culicidae ) is a major malaria vector in asia ( zahar 1974 ) . it has been incriminated in the stable malaria as its longevity frequently fits for the completion of the parasite sporogonic cycle ( macdonald 1957 ) . this mosquito is also known to transmit malaria in iran with a widespread distribution ( saebi 1987 , edrissian 2006 ) . recently , in a study on the mosquito fauna of chahar mahal and bakhtiari province , at the central western part of iran , a few lactophenol - preserved larvae of an . superpictus were found disfigured by the swelling of their thorax and/or first abdominal segments ( fig . close observation showed that the multiple packets of a tiny microorganism have been infiltrated in the affected areas . thus , a preliminary study was conducted to identify the responsible microorganism at the genus level . apparent infection the thorax and first 3 abdominal segments in a lactophenol - preserved larva of anopheles superpictus the locality was the same area in kiar district of chahar mahal and bakhtiari province where the infected larvae of an . anopheline larvae were collected with the aid of a dipper and an eyedropper based on their surface swimming behavior . close observation of the larvae in a small black pan was used to detect the whitish discoloration of body segments as the primary sign of microsporidium infection . in laboratory , larval identities were checked with the aid of a valid local taxonomic key ( azari - hamidian and harbach 2009 ) . the infected larvae were observed under a low power stereomicroscope and dissected to remove their infective contents . wet preparations were prepared by adding a small sample from the extracted tissues to a drop of tap water . simple pressure with the tip of a pencil was applied on the coverslip to test if it is possible to extrude polar tube . other preparations were prepared by the gentle spreading of the infective material on microscope slides . the air - dried specimens were fixed in absolute methanol for 10 minutes . for geimsa staining 1:14 v / v concentrate solution all of the preparations were viewed under the 1000-x power of a conventional compound microscope . spore measurements were performed at random for 50 spores in both wet and fixed preparations . the morphological characters proposed by andreadis ( 2007 ) and hazard and anthony ( 1974 ) were used to identify the microsporidium genus . the infected larvae were recollected from the same locality previously discovered in chahar mahal and bakhtiari in 2012 ( fig . the first 3 abdominal segments was the primary site of infection . at times , the thorax was also affected . the infected larvae were sluggish and tended to remain more silent at the bottom of the pan once they were physically stimulated by an eyedropper . one of the typical breeding places in which the infected anopheles superpictus larvae were collected close observation of the involved segments under a stereomicroscope showed that they were filled with the globular masses of a fine opaque material indicative of a microsporidium infection ( fig . 3 ) . in wet preparations , the in situ stirring spores ranged 4.330.192.670.12 micron . the anterior , smaller and opaque half was seemed to be a bit blunt and composed of heterogenous components with a small projection into the aforementioned section . the refractive surrounding border was in favor of the presence of a tick wall around the body . heavy infection of first 3 abdominal segments ( above ) multiple globular masses of the infective material in thorax ( below ) microsporidia in wet preparations from infected anopheles superpictus larva in the stained specimens , spores were ranged 4.180.432.450.33 micron . the posterior and anterior light spots could be attributed to the posterior vacuole and anterior sporoplast , respectively . geimsa ( above ) and ryan - blue modified trchrome staining ( below ) of the microsporidia in anopheles superpictus larva typical clusters of parathelohania sp spores in anopheles superpictus larva . ( above ) geimsa and ( below ) ryan - blue modified trichrome stained specimens simple pressure applied on wet preparations was not able to extrude the polar filaments of spores . overall , these features suggested the presence of a microsporidium infection from the genus parathelohania . it is also the first report of natural microsporidium infection in a mosquito species from of iran . parathelohania , with at least 22 species , is the second largest polytypic genus next to amblyospora in the family amblyosporidae ( andreadis 2007 ) . this genus which was firstly described by codreanu in 1966 , redescribed later by anthony and hazard in 1974 ( hazard and anthony 1974 ) . the type species is parathelohania legeri ( synonym : thelohania legeri ) which was discovered in anopheles maculipennis meigen sl about 110 years ago ( andreadis 2007 ) . the latest described species , p. tomski , p. divulgata , p. sibirika , p. formosa and p. teguldeti have been reported by simakova and pankova from russia in 2004 ( simakova and pankova 2004 ) . all of these microsporidia were isolated from anopheline mosquitoes . in reality , anopheline mosquitoes constitute the principal hosts for the genus parathelohania ( hazard and anthony 1974 ) . parathelohania chagrasensis and p. barra are the only species described from aedomyia and ochlerotatus mosquitoes ( pillai 1968 , hazard and oldacre 1976 ) . vertical and horizontal transmission is a common feature in parathelohania and normally there is an intervening obligatory copepod host in the life cycle ( becnel et al . 2005 ) . in the infected larvae , the first 3 abdominal segments were primarily involved ( fig . 3 ) . this region corresponds with the middle part of the midgut , which is highly alkaline . a series of digestive enzymes are maximally active in this portion of the gut ( clements 1992 ) . perhaps , from here the germinated spores access to their target cells in the fat tissues and progressively extend to invade other areas . while the severe over - growth of spores kills male larvae , the infection in females is less pronounced . these larvae transmit their infection to the adulthood during which the vertical transmission will occur ( andreadis 2007 ) . the extrusion of the polar tube is a reliable sign of a microsporidium spore ( wittner and weiss 1999 ) . in this study , this might be regarded as an exception to the general recommendation proposed by andreadis ( andreadis 2007 ) . geimsa staining is a routine technique in the diagnosis of microsporidia infection in mosquitoes ( andreadis 2007 ) . in the present study , with this method however , a better shadow of the internal structures was obtained by ryan - blue modified trichrome technique ( fig . , it was possible to detect the position of posterior vacuole and/or anterior poloroplast as one or two light spots in the stained spores . since the introduction of the weber - green trichrome staining technique , while a few have addressed the quality of the visualization of the morphological characters , the others have tried to improve the practical items , or both . this study suggests that the trichrome staining might be considered as a preferred technique in those situations in which more details of the spores are aimed at . in the present study , the size of spores overlaps with the spores of p. anophelis , p. evansae , p. periculosa , p. indica , p. tomski , p. divulgata and p. formosa ( hazard and anthony 1974 , garcia and becnel 1994 , simakova and pankova 2004 ) . while the first 3 species have been discovered in the nearctic region , the remainder has been described from the palearctic region . it is clear that the spore size can not be used as a reliable method for species differentiation . instead , with the aid of electron microscopy techniques , comparisons that are more accurate would be possible . in some occasions , the definitive identification of a microsporidium species needs a good knowledge on its ecological attributes and the ultrastructural detail of developmental stages in the hosts ( andreadis 2007 ) . on the other hand , molecular information on the ss rrna sequence is a valuable tool in the phylogenetic analysis of the microsporidium species ( vossbrinck et al . the checklist of the culicidae of iran contains the name of 64 species ( azari - hamidian 2007 ) . a number of these mosquitoes might be considered as the potential host for microsporidia infection . this is because there is at least one report of natural microsporidium infection in these species from elsewhere ( castillo 1980 ) . messeae falleroni , ochlerotatus caspius ( pallas ) s.l , aedes vexans ( meigen ) , culex modestus ficalbi , cx . the vital consequences of artificial infection with anncaliia algerae ( synonym : nosema algerae , synonym : brachiola algerae ) on an . stephensi liston has been explored in laboratory , as well ( haq et al . 1981 ) . in the past , at least two extensive countrywide mosquito larval surveys have been performed in iran ( saebi 1987 , zaim 1987 ) . although there is no report of microsporidium infection in these studies , it is still possible that new cases of microsporidium infection could be found with more intensive larval surveys . there are few reports , which declare that some microsporidia species are connected with human infections ( cheney et al . however , the research on the application of these microorganisms in the biological control of mosquitoes is going on ( solter and becnel 2007 ) . mathematical modeling is also in favor of their effectiveness in the control of mosquitoes ( koella et al . recent efforts have been focused on species with polymorphic development and maximum phylogenetic distance to human parasitizing microsporidia ( andreadis 2007 ) . although parathelohania is a genus with average characters , the possibility of the utilization of these pathogens in the control of specific mosquitoes can not be completely excluded . the first report of an . superpictus larval infection with a microsporidium species from the genus parathelohania although the definitive diagnosis of the species needs specific ultrastructural and molecular data , current ecological attributes predicts that it is likely a new microsporidium species .
background : microsporidia are known to infect a wide variety of animals including mosquitoes ( diptera : culicidae ) . in a recent study on the mosquito fauna of chahar mahal and bakhtiari province , at the central western part of iran , 
 a few larvae of anopheles superpictus were infected with a microsporidium - resembled microorganism . 
 current investigation deals with the identification of the responsible microorganism at the genus level.methods:fresh infected larvae were collected from the field . after determining the species identity 
 they were dissected to extract their infective contents . 
 wet preparations were checked for general appearance and the size of the pathogenic microorganism . 
 fixed preparations were stained with geimsa and ryan - blue modified trichrome techniques to visualize further morphological characters . 
 the obtained light microscopy data were used in the identification process.results:the infected larvae were bulged by a whitish material filling the involved segments corresponding to a microsporidium infection . 
 bottle - shaped semioval spores ranged 4.330.192.670.12 and 4.180.432.450.33 micron in wet and fixed preparations , respectively . 
 they were mostly arranged in globular structures comprised of 8 spores . 
 these data was in favor of a species from the genus parathelohania in the family ambliosporidae.conclusion:this is the first report of a microsporidium infection in an . 
 superpictus . 
 the causative agent is diagnosed as a member of the genus parathelohania . further identification down to the species level needs to determine its ultrastructural characteristics and the comparative analysis of ss rrna sequence data . 
 it is also necessary to understand the detail of the components of the transmission cycle .