mosquitoes , the most important group of nuisance pest insects , due to their diversity and abundance , demonstrated vector competence and frequent infection in nature , they are regarded as one of the most important vectors of diseases ( sardelis et al . 2008a , b ) . tracking the movement of mosquitoes in their natural habitat is critically important for understanding their basic biology , demography , ethology and vector - borne disease control as well as prevention . animal marking have been used since 218 bc when fisher and peterson used banding to distinguish ownership of birds ( fisher and peterson 1964 ) . unfortunately , for marking insects , most marking techniques for vertebrate , such as bands , brands , tattoos , tags , notches , paints are not practical because they are tedious , time - consuming , heavy and costly ( southwood 1978 , basavaraju et al . insect marking for scientific studies dates back to 1920 , since then , a variety of marking techniques , paints , dyes etc . were used to in studies of insect population dynamics ( geiger et al . 1919 , dudley and searles 1923 ) . 2007 ) , paints ( trpis and hausermann 1986 , niebylski and meek 1989 , service 1993 , bellini et al . 2007 ) , and radioactive isotopes ( jenkins 1949 , abdel - malek 1966 , lindquist et al . 1967 , hood - nowotny et al . however , for study on the behavioral characteristics of post bloodmeal , existing techniques tend to be labor intensive , as they require rearing mosquitoes , marking them in large quantities , and then inspecting large numbers of individuals to detect recaptures ( walker et al . furthermore , compared with natural populations , rearing mosquitoes , marking them in large quantities by using artificial methods , and releasing them may change their behavior ( reisen et al . 2003 , silver 2008 ) . for study the behavior characteristics after blood meal , these methods are not ideal however , the problem is how to mark breeding mosquitoes without inhibiting their normal biology , and with long - term retention after blood meal , it is still bothering most biological scientists . ( 2014 ) marked adult mosquitoes by feeding them on cow injected with isotope p and subsequent ecological investigations . in some studies , successfully labeled mosquitoes by feeding them on radioactive animal blood ( hassett and jenkins 1951 ) , use of large bait animals for marking wild population of adult anophels aquasalis injected with a dose of 1.7 curies of p ( bruce - chwatt 1956 ) . however , such high dosage would be dangerous to the animal ( winteringham london meeting , 1953 ) . the aim of the current study was to develop a marking technique as an internal marker to mark post blood meal mosquitoes by using stable phosphate isotope p and determine the optimal concentration of it . before experimenting , 6 healthy rabbits ( 2 kg ) were selected and physically examined by veterinarian . the injection method of normal saline solution to the rabbits was according to smith et al . ( 1951 ) , i e. an isotonic buffered saline solution , containing different concentration of na2hpo4 , was injected intravenously of rabbits . the dosage for no . 1 , 2 rabbits , no . 3 , 4 rabbits , no . 5 rabbit and no . 6 were 0.2 mci , 0.4 mci , 0.8 mci and 1.7 mci , respectively . for the negative control , 1 healthy rabbits was injected with isotonic buffered saline solution without na2hpo4 . then 20 to 50 emerged female cx . pipiens fed on these rabbits at 6 , 12 , 24 , 48 , 72 h and 120 h after injected with na2hpo4 . at the same time , 0.2 ml of blood was extracted from rabbits , as well as at 16 and 32 days for radioactivity level measure . pipiens radioactivity levels were measured at 2 h , 6 h , 12 h , 24 h , 48 h , 72 h and 120 h after blood meal . the measurement of radioactivity was conducted using a liquid scintillation counters ( model ysj-76 ) . before injection of p to rabbits , to normalize background radiation , radioactivity level of 30 emerged adult cx . mosquitoes tested were anesthesia with ether , placed in a  bell counter tube of the vitriol chambers for 1 minute , as for the rabbits blood test , 0.2 ml of blood was dipped on the paper in the tinfoil sample plates , counts that exceeding 50% of the background was as the standard that were labeled . at the 2 day after no . 6 rabbit was injected with na2hpo4 , up to 5 g feces was placed into a container with 300 ml of tap water and cx . pipiens larvae and pupae inside . as the negative control , placed 5 g feces of the rabbit without injected na2hpo4 into the water . the experimental project was reviewed and approved by the ethical committee of shandong academy of medical sciences ( jinan , shandong ) . urine and feces of the rabbits were collected and sent to the institute of radiation medicine , shandong academy of medical sciences for appropriate processing to prevent spread of the isotope . before experimenting , 6 healthy rabbits ( 2 kg ) were selected and physically examined by veterinarian . the injection method of normal saline solution to the rabbits was according to smith et al . ( 1951 ) , i e. an isotonic buffered saline solution , containing different concentration of na2hpo4 , was injected intravenously of rabbits . the dosage for no . 1 , 2 rabbits , no . 3 , 4 rabbits , no . 6 were 0.2 mci , 0.4 mci , 0.8 mci and 1.7 mci , respectively . for the negative control , 1 healthy rabbits was injected with isotonic buffered saline solution without na2hpo4 . then 20 to 50 emerged female cx . pipiens fed on these rabbits at 6 , 12 , 24 , 48 , 72 h and 120 h after injected with na2hpo4 . at the same time , 0.2 ml of blood was extracted from rabbits , as well as at 16 and 32 days for radioactivity level measure . pipiens radioactivity levels were measured at 2 h , 6 h , 12 h , 24 h , 48 h , 72 h and 120 h after blood meal . the measurement of radioactivity was conducted using a liquid scintillation counters ( model ysj-76 ) . before injection of p to rabbits , to normalize background radiation , radioactivity level of 30 emerged adult cx . mosquitoes tested were anesthesia with ether , placed in a  bell counter tube of the vitriol chambers for 1 minute , as for the rabbits blood test , 0.2 ml of blood was dipped on the paper in the tinfoil sample plates , counts that exceeding 50% of the background was as the standard that were labeled . at the 2 day after no . 6 rabbit was injected with na2hpo4 , up to 5 g feces was placed into a container with 300 ml of tap water and cx . pipiens larvae and pupae inside . as the negative control , placed 5 g feces of the rabbit without injected na2hpo4 into the water . the experimental project was reviewed and approved by the ethical committee of shandong academy of medical sciences ( jinan , shandong ) . urine and feces of the rabbits were collected and sent to the institute of radiation medicine , shandong academy of medical sciences for appropriate processing to prevent spread of the isotope . pipiens and rabbits blood was measured , the background was determined as was 1213 counts per minute ( cpm ) , i e counts that exceeded 50% of background ( > 20 cpm ) were considered positive . within 5 days after no pipiens fed on the rabbits , among the 222 mosquitoes blood feeding , except one mosquito was fewer than 20 cpm , the others 221 mosquitoes were labeled no matter how much dosage of na2hpo4 was injected . the more dosage the rabbits injected with na2hpo4 , the higher radioactive levels of cx . the radioactive levels started to decrease from the 48 hours ( 2 day ) after injection . compared with radioactive level at 48 h , though the radioactive level of labeled mosquitoes was higher than background at 72 h , 96h and 120 h , radioactive level decreased to a lower level ( figs . 1 , 2 , 3 ) . levels of radioactivity of culex pipiens after fed on no . 1 , 2 rabbits after injection of p ( 0.2 mci ) legend 6 , 12 , 24 , 48 , 72 , 120 : mosquito fed time after injection p into rabbit ( hours ) levels of radioactivity of culex pipiens after fed on no . 3 , 4 rabbits after injection of p ( 0.4 mci ) legend 6 , 12 , 24 , 48 , 72 , 120 : mosquito fed time after injection p into rabbit ( hours ) levels of radioactivity of culex pipiens after fed on no . 5 rabbits after injection of p ( 0.8 mci ) legend 6 , 24 , 48 , 72 , 120 : mosquito fed time after injection p into rabbit ( hours ) the radioactive level decreased very fast in the blood of rabbits . at 6 hour the radioactive level was 100% , the radioactive level decreased 2648% , 6075% and 9597% at the 2 , the 5 and the 32 day , respectively ( table 1 ) . radioactivity levels of rabbit blood after injection of p ( 1.7 mci ) at different time cpm = pulse per minute , - do not measured no . 1 to 4 rabbits showed good health , have no change on body temperature and body weight . on the 2 day , no . 5 rabbit abortion , and gave birth to 2 died bunnies , the radioactive level was 15 mr / h at 5 cm away from these died bunnies . the no . 5 rabbit presented no abnormalities and was dissected at the 143 day after injected with na2hpo4 , no pathological changes were found in the internal organs . the radioactivity level of liver was measured , which decreased to similar level of background . 6 rabbit showed diarrhea , appetite loss , weight loss and other symptoms . at the 31 days , and no special lesions it still presented a higher radioactivity level in liver , spleen , intestine , heart , lung , kidney , muscle etc ( table 2 ) . 6 rabbit tissue after injection of p ( 1.7 mci ) 31 days background radiation= 22 cpm at the 2 day after no . 6 rabbit was placed into a container , at the 2 and 7 day , there were total 39 adult cx . pipiens mosquitoes emerged , among them , 22 ( 56.5% ) were marked , their cpm was 21875 , the average was 323 . pipiens and rabbits blood was measured , the background was determined as was 1213 counts per minute ( cpm ) , i e counts that exceeded 50% of background ( > 20 cpm ) were considered positive . within 5 days after no pipiens fed on the rabbits , among the 222 mosquitoes blood feeding , except one mosquito was fewer than 20 cpm , the others 221 mosquitoes were labeled no matter how much dosage of na2hpo4 was injected . the more dosage the rabbits injected with na2hpo4 , the higher radioactive levels of cx . the radioactive levels started to decrease from the 48 hours ( 2 day ) after injection . compared with radioactive level at 48 h , though the radioactive level of labeled mosquitoes was higher than background at 72 h , 96h and 120 h , radioactive level decreased to a lower level ( figs . 1 , 2 , 3 ) . levels of radioactivity of culex pipiens after fed on no . 1 , 2 rabbits after injection of p ( 0.2 mci ) legend 6 , 12 , 24 , 48 , 72 , 120 : mosquito fed time after injection p into rabbit ( hours ) levels of radioactivity of culex pipiens after fed on no . 3 , 4 rabbits after injection of p ( 0.4 mci ) legend 6 , 12 , 24 , 48 , 72 , 120 : mosquito fed time after injection p into rabbit ( hours ) levels of radioactivity of culex pipiens after fed on no . 5 rabbits after injection of p ( 0.8 mci ) legend 6 , 24 , 48 , 72 , 120 : mosquito fed time after injection p into rabbit ( hours ) the radioactive level was 100% , the radioactive level decreased 2648% , 6075% and 9597% at the 2 , the 5 and the 32 day , respectively ( table 1 ) . radioactivity levels of rabbit blood after injection of p ( 1.7 mci ) at different time cpm = pulse per minute , - do not measured 1 to 4 rabbits showed good health , have no change on body temperature and body weight . on the 2 day , 5 rabbit abortion , and gave birth to 2 died bunnies , the radioactive level was 15 mr / h at 5 cm away from these died bunnies . the no . 5 rabbit presented no abnormalities and was dissected at the 143 day after injected with na2hpo4 , no pathological changes were found in the internal organs . the radioactivity level of liver was measured , which decreased to similar level of background . however , no . 6 rabbit showed diarrhea , appetite loss , weight loss and other symptoms . at the 31 days , and no special lesions were found in internal organs after dissection . it still presented a higher radioactivity level in liver , spleen , intestine , heart , lung , kidney , muscle etc ( table 2 ) . 6 rabbit tissue after injection of p ( 1.7 mci ) 31 days background radiation= 22 cpm at the 2 day after no . 6 rabbit was injected with na2hpo4 . after 5 g feces of no . 6 rabbit was placed into a container , at the 2 and 7 day , there were total 39 adult cx . pipiens mosquitoes emerged , among them , 22 ( 56.5% ) were marked , their cpm was 21875 , the average was 323 . we could see that the more dosage the rabbits injected with na2hpo4 , the higher radioactive levels of cx . the radioactive levels started to decrease from the 48 hours ( 2 day ) after injection . compared with radioactive level at 48 h , though the radioactive level of labeled mosquitoes was higher than background at 72 h , 96 h and 120 h , radioactive level decreased to a lower level . the more injection of na2hpo4 into rabbits , the higher radioactivity level in the blood and the slower decrease , was also consistent with the labeling condition of cx . 1 , 2 , 3 ) . no . 1 to 4 rabbits showed good health , but as for the no . 5 rabbit injected with 0.8 mci of na2hpo4 , on the 2 day , abortion , and gave birth to 2 died bunnies . at the 143 day , the no . 5 rabbit presented no abnormalities , no pathological changes were found in the internal organs . 6 rabbit injected with 1.7 mci of na2hpo4 showed diarrhea , appetite loss , weight loss and other symptoms . at the 31 days , it still presented a higher radioactivity level in liver , spleen , intestine , heart , lung , kidney , muscle etc . therefore , the appropriate concentration for not only marking mosquitoes but also no harm to rabbits was not more than 0.4 mci . stable isotopes occur naturally in the environment , are safe and non - invasive , pose no health or environmental risks ( hood - nowotny and knols 2007 ) . in medical research , most stable isotopes are non - toxic and are routinely used for mosquito feeding trials , in which human adults are labelled up through supplementary feeding with stable isotopes , may be useful for host seeking behavior and repellent testing , etc . several tracers were studied , such as co , sr , zn , i , ca and p , whereas p was the most applied radioisotope for tagging due to its short half - life , safety , activity and easy of detection ( obrien and wolfe 1964 ) . hasset and jenkins ( 1951 ) also performed a detailed study of the conditions affecting mosquitoes labelled with p and compared stages , p concentrations and age . the filarial larvae , setaria digitata linstow was marked after adult mosquitoes ( armigerea obturbans walker ) fed on cows or men infected with microfilaria , that larvae of mosquitoes were reared in water containing 1 c of p / ml ( dissanaike et al . 1957 ) . toxicity to the insect was also a serious problem to be considered in many studies ( quarterman et al . the radioisotopes ca and i were very toxic when fed to adult houseflies at 1 c / ml of milk , whereas p was satisfactory ( quarterman et al . 1954 ) . by using n - labeled potassium nitrate and c - labeled glucose to mark larval mosquitoes , there were no consistent effects of isotopic enrichment on immature mosquito survival or adult mosquito body size ( hamer et al . although fluorescent dyes or powders are also suitable for marking mosquitoes ( takken et al . 1998 , mccall et al . 2001 , pates 2002 , lapointe 2008 , baber et al . 2010 ) , and no effect of these dyes and powders on performance of ant ( pogonomyrmex owyheei ) , mountain pine beetle ( dendroctonus ponderosae ) , grasshopper ( melanoplus spp . ) , ae . aegypti , anopheles sinensis in some studies ( porter and jorgenson 1980 , linton et al . others have found a reduced longevity , behavioural response or survival of opine parasitoids ( diachasmimorpha spp . ) , weevils , ae . aegypti , asian citrus psyllid ( diaphorina citri ) and codling moths ( laspeyresia pomonella ) ( sheppard et al . be caused of the restrictions of the retention , most studies researches primarily focus on dispersal of nulliparous female mosquitoes during the initial host - seeking event and sometimes a second host - seeking event ( hamer et al . , mosquitoes were successfully marked after feeding on hosts injected with trace element rb and cs ( anderson et al . they are not radioactive , safe for workers and for the environment . as for insects marked with an element , no tags , paints , dyes , dusts , or visible marks were found left to alter insects behavior or interactions with other insects ( hagler and jackson 2001 ) . however , a limitation to use of trace elements as insect markers in large fields is that the detection of elements can be difficult , expensive , and time - consuming , requires technical expertise and expensive detection equipment ( akey and burns 1991 ) . some trace elements are not retained very well in certain insect species , for example , rb could be detected for only 26 days after marking aphids ( guillebeau et al . high concentrations of trace elements can adversely affect development , survival , increased adult deformity , reduced pupation , eclosion , egg production and fecundity of certain insects ( stimmann et al . naturally , occurring stable isotope markers are useful , as they do not require the pre - marking of individuals ( hood - nowotny and knols 2007 ) . labelling a distinct portion of an ecosystem with stable isotopes is a useful , minimally invasive method to study insect dispersal from an ecophysiological perspective ( macneale et al . stable isotopes occur naturally in the environment , unlike painting , dusting , etc . , stable isotope methods are non - invasive and samples require only minimal preparation following collection , which makes the cost of the process as a completely comparable to methods such as polymerase chain reaction ( hood - nowotny and knols 2007 ) . other advantages are the analysis costs ( depending on the isotope and the matrix , the cost per sample may range from us$ 5100.00 ) , shipping stable isotope samples is simple , safe and inexpensive ( iaea 2009 ) . it would cost between $ 150250 to label 1 000 000 anopheles mosquitoes with c - labelled glucose in the larval stages ( hood - nowotny et al . it is possible to trace the fate of labelled sperm into female spermatheca in studies of male mosquitoes labelled with c ( helinski et al . these stable isotope markers meet the usual criteria for use in insect studies : retention , no effect on behavior , durability , easily applied , clearly identifiable , and not expensive ( hagler and jackson 2001 ) . a labelled blood source also provides an easily identifiable point source for post feeding dispersal studies . tracing of labelled blood to determine resource allocation to the eggs or other tissues could also provide useful physiological information . it was possible to mark large numbers of mosquitoes by providing blood meals from a host fed injected with a stable isotope substance . na2hpo4 was injected via the jugular of rabbit vein ; these rabbits were bitten by emerged female cx . pipiens , so mosquitoes were successfully marked . the appropriate dosage ( not only can label mosquito but also have no ill effects on rabbits ) of na2hpo4 was 0.1 mci / kg . pipiens mosquitoes were also can be marked by placing rabbit feces or na2hpo4 into container . the technique was less time - consuming , more sensitive , and safer , can be used to study post - blood meal dispersal of mosquitoes and other blood - sucking insects . the short half - life of 14 days of p , however , was a disadvantage in studies where prolonged observations were necessary .