Experimental infection of Anopheles sinensis with Korean isolates of Plasmodium vivax.

The objectives of the present study were to (1) determine the susceptibility of Anopheles sinensis to Korean isolates of Plasmodium vivax, (2) establish a method to collect large quantities of P. vivax sporozoites for use as antigen in seroepidemiological studies, and (3) investigate the characteristics of Korean isolates of P. vivax sporozoites. Females of Anopheles sinensis were collected at non-epidemic area, Seokwha-ri, Cheongwon-gun and Chungcheongbuk-do using tent-trap methods coupled with dry ice. The females were artificially infected with gametocytes of P. vivax using blood obtained from P. vivax malaria patients. Individual mosquitoes were infected using either a parafilm-covered glass feeding apparatus or were allowed to feed on naturally infected volunteers. Mosquitoes were sacrificed between 16 and 18 days post-feeding and an enzyme-linked immunosorbent assay (ELISA) was used to detect sporozoites. Four (33.4%) of 12 mosquitoes, which were fed on naturally infected volunteers directly, were positive for sporozoites. In cases, the mosquitoes allowed to feed on whole blood which were extract from three different patients with heparin treated vacuutainers using a parafilm-covered glass apparatus. Two of 55 (3.6%) were positive which blood sample was maintained at room temperature for 8 hours, 1 of 68 (1.5%) was positive which blood was maintained at 4 degrees C for 24 hours and 1 of 47 (2.3%) was positive at 4 degrees C for 48 hours. The mean number of sporozoites was estimated about 818 (n = 8; range of 648-1,056) based on optical density values of ELISA.     

The Identification of Plasmodium vivax Infected Anopheles sinensis by Enzyme-Linked Immunosorbent Assay, Republic of Korea, 1998

ABSTRACT To figure out how far spread the malaria parasite infected mosquitoes from Demilitarized Zone (DMZ). We investigated the sporozoite-infected mosquitoes in Chosan-ri and Hajiseok-ri of Paju-shi, Tonae-dong of Koyang-shi, Mudeung-ri of Yonchon-gun, Soyo-dong of Tongduchon-shi, and Tukjeong-ri of Yangju-gun, Kyonggi-do province in 1998. They were known as highly epidemic areas in the Republic of Korea. Anopheline mosquitoes were collected by human biting collection method from the 2nd week of June to the 5th week of August. A total of 3,256 anopheles mosquitoes were captured in these areas. A sandwich enzyme-linked immununosorbent assay (ELISA) for identifying Plasmodium vivax sporozoites in mosquitoes was used in this survey. Only one mosquito which was captured in Chosan-ri, on 2nd week of July showed positive reaction in this test. This mosquito was identified as Anopheles sinensis. This showed a 0.068% (1/1,465) positive rate in Chosan-ri on 2nd week of July. We could not detect positive mosquitoes the other areas. So we concluded the parasite infected mosquitoes may be highly limited near the DMZ.     

Mosquito species composition and Plasmodium vivax infection rates on Baengnyeong-do (island), Republic of Korea

Vivax malaria is a significant military and civilian health threat in the north of the Republic of Korea (ROK). The island of Baengnyeong-do is the westernmost point of the ROK and is located close to the southwestern coast of the Democratic People's Republic of Korea (DPRK). Mosquitoes were collected using a black light trap on Baengnyeong-do, and Anopheles spp. were assayed by PCR, to identify the species, and screened for sporozoites of Plasmodium vivax. Of a subsample of 257 mosquitoes, Anopheles lesteri was the most frequently collected (49.8%), followed by Anopheles sinensis (22.6%), Anopheles pullus (18.7%), Anopheles kleini (7.8%), and Anopheles belenrae (1.2%). The overall sporozoite rate was 3.1%, with the highest rates observed in An. kleini (15.0%), An. sinensis (5.2%), and An. lesteri (1.6%). No sporozoite positive An. pullus or An. belenrae were observed. The results extend our knowledge of the distribution and potential role in malaria transmission of An. kleini, An. lesteri, and An. sinensis, for an area previously considered to be at a low risk for contracting vivax malaria.     

Malaria Vector Surveillance in Ganghwa-do, a Malaria-Endemic Area in the Republic of Korea

We investigated the seasonality of Anopheles mosquitoes, including its species composition, density, parity, and population densities of mosquitoes infected with the parasite in Ganghwa-do (Island), a vivax malaria endemic area in the Republic of Korea. Mosquitoes were collected periodically with a dry-ice-tent trap and a blacklight trap during the mosquito season (April-October) in 2008. Anopheles sinensis (94.9%) was the most abundant species collected, followed by Anopheles belenrae (3.8%), Anopheles pullus (1.2%), and Anopheles lesteri (0.1%). Hibernating Anopheles mosquitoes were also collected from December 2007 to March 2008. An. pullus (72.1%) was the most frequently collected, followed by An. sinensis (18.4%) and An. belenrae (9.5%). The composition of Anopheles species differed between the mosquito season and hibernation seasons. The parous rate fluctuated from 0% to 92.9%, and the highest rate was recorded on 10 September 2008. Sporozoite infections were detected by PCR in the head and thorax of female Anopheles mosquitoes. The annual sporozoite rate of mosquitoes was 0.11% (2 of 1,845 mosquitoes). The 2 mosquitoes that tested positive for sporozoites were An. sinensis. Malarial infections in anopheline mosquitoes from a population pool were also tried irrespective of the mosquito species. Nine of 2,331 pools of Anopheles mosquitoes were positive. From our study, it can be concluded that An. sinensis, which was the predominant vector species and confirmed as sporozoite-infected, plays an important role in malaria transmission in Ganghwa-do.     

Vector competence of Anopheles lesteri Baisas and Hu (Diptera: Culicidae) to Plasmodium vivax in Korea

Three anopheline mosquitoes in Korea were studied for their abilities as vectors for Plasmodium vivax. The female mosquitoes of Anopheles lesteri, An. pullus and An. sinensis were allowed to suck malaria patient blood until fully fed, and they were then bred for 2 weeks to develop from malaria parasites to sporozoites. The result from the above confirmed the sporozoites in one An. lesteri of one individual and five An. sinensis of six individuals. We also confirmed that An. sinensis was the main vector to transmit malaria and An. lesteri as well as An. sinensis were able to carry Korean malaria parasites. Therefore, we propose that diversified study is needed to manage malaria projects.     

Synchronous peaks in trap catches of malaria-infected mosquito species at Daeseongdong, a border village between North and South Korea

Malaria continues to be a major health threat near the Demilitarized Zone (DMZ) that separates North and South Korea. Adult mosquitoes were collected from 20 July through 21 October, 2010 at Daeseongdong, a small village within the DMZ. Molecular techniques were used to identify Anopheles to species and for detection of Plasmodium vivax sporozoites in their head and thorax. Trap catches showed concordant peaks of Anopheles belenrae and An. kleini early in the study period and concordant peaks of An. pullus and An. sinensis later in the season. Three well defined peaks of the 107 sporozoite positive mosquitoes were observed: 34.6% were An. kleini, 23.4% were An. belenrae, 21.5% were An. sinensis, 19.6% were An. pullus, and 0.9% were An. lesteri. Estimation of the extrinsic incubation period from daily temperatures did not help identify preceding biting peaks of An. pullus and An. sinensis, when infection should have been acquired. We explore possible reasons for the sudden appearance and disappearance of sporozoite-infected mosquitoes, including the influx of infected mosquitoes from adjoining areas, and weather patterns. Regular surveillance for infected mosquitoes near border areas of the Republic of Korea may provide advance warning of increased malaria risk potential.     

A mark-release-recapture experiment with Anopheles sinensis in the northern part of Gyeonggi-do,Korea

In order to study the range of flight and feeding activity of Anopheles sinensis, the dispersal experiment was conducted in Paju city, located in the northern part of Gyeonggi-do, Republic of Korea, during the period of 7th to 28th September 1998. Unfed females An. sinensis were collected in cowshed and released after being marked with fluorescent dye at 23:00 hours on the same day. Released female mosquitoes were recaptured everyday during 21 days using light traps, which were set at 10 sites in the cowsheds located 1, 3, 6, 9 and 12 km north-northwest and north-northeast and at 3 sites located 1, 6 and 9 km toward south-west from the release point. In addition, to study the longest flight distance in one night, we set the light traps at 16 and 20 km toward north-northeast from the release site. All the collected mosquitoes were placed on filter papers and observed on UV transilluminator after treatment with one drop of 100% ethanol. Out of 12,773 females of An. sinensis released, 194 marked females mosquitoes were recaptured, giving 1.52% recapture rate. Of 194, 72 mosquitoes (37.1%) were recaptured in light traps from three places set at 1 km from the release point, 57 mosquitoes (29.4%) from two places at 1-3 km, 41 mosquitoes (21.1%) from three places at 3-6 km, 20 mosquitoes (10.3%) from three places at 6-9 km, and 4 mosquitoes (2.1%) from two places at 9-12 km. Since 170 female mosquitoes (87.6%) out of 194 marked mosquitoes were captured within 6 km from the release point, this flight radius represents the main activity area. An sinensis was found to be able to fly at least 12 km during one night.     

Quantitation of malaria sporozoites in the salivary glands of wild Afrotropical Anopheles

The number of malaria sporozoites in the salivary glands was determined microscopically for 1137 wild, naturally infected Anopheles from western Kenya. Infective Anopheles gambiae Giles sensu lato (n = 874) contained a geometric mean (GM) of 962 sporozoites and An.funestus Giles (n = 263) contained 812. No significant differences were detected in geometric mean numbers of sporozoites between species, collection techniques or sites. Of the infective An.gambiae, 1.7% (15/874) contained more than 41,830 sporozoites, the maximum observed for An.funestus. Microscopic techniques were found to be more sensitive than enzyme-linked immunosorbent assays (ELISA) for detecting low-grade sporozoite infections in salivary glands. Salivary gland sporozoites from 83.6% of the 1137 gland infections were identified by ELISA as either Plasmodium falciparum Welch (n = 910), P.ovale Stephens (n = 7), P.malariae Grassi & Feletti (n = 3) or mixed (n = 30). The 187 gland infections which could not be identified by ELISA contained significantly fewer sporozoites (GM = 242) than those which could be identified (GM = 1200).     

Population dynamics of five anopheles species of the Hyrcanus group in northern Gyeonggi-do, Korea

To investigate the population densities of potential malaria vectors, Anopheles species were collected by light traps in malaria endemic areas, Paju and Gimpo, Gyeonggi-do of Korea. Five Anopheles Hyrcanus sibling species (An. sinensis, An. pullus, An. lesteri, An. kleini, and An. belenrae) were identified by PCR. The predominant species, An. pullus was collected during the late spring and mid-summer, while higher population consists of An. sinensis were collected from late summer to early autumn. These 2 species accounted for 92.1% of all Anopheles mosquitoes collected, while the other 3 species accounted for 7.9%. Taking into account of these population densities, late seasonal prevalence, and long-term incubation period (9-13 months) of the Korean Plasmodium vivax strain, An. sinensis s.s is thought to play an important role in the transmission of vivax malaria in the study areas.     

Studies on Anopheles sinensis, the vector species of vivax malaria in Korea

Extensive previous studies on taxonomy, behavior/bionomics and control of Anopheles sinensis are reviewed and summarized. Recent molecular identification revealed that the population of An. sinensis complex includes An. sinensis, An. pullus, An. lesteri and at least two new species, and An. yatsushiroensis is synonmy of An. pullus. An. sinensis is the main vector species of vivax malaria in Korea. Larvae of An. sinensis breed in wide range of habitats which are naturally-made clean water, stagnant or flowing; main habitats include rice fields, ditches, streams, irrigation canals, marshes, ponds, ground pools, etc. Their host preferences are highly zoophilic. Human blood rate is very low (0.7-1.7%); nevertheless An. sinensis readily feeds on man when domestic animals are not found near by. They feed on hosts throughout the night from dusk to dawn with a peak period of 02:00-04:00 hours; they are slightly more exophagic (biting outdoors); much larger numbers come into the room when light is on. Main resting places are outdoors such as grasses, vegetable fields and rice fields. A mark-release-recapture study resulted that 37.1% was recaptured within 1 km, 29.4% at 1-3 km, 21.1% at 3-6 km, 10.3% at 6-9 km and 2.1% at 9-12 km distance. An. sinensis hibernate outdoors (mostly under part of dense grasses) during October-March. At the end of the hibernation period (March-April) they feed on cows at daytime. Until today any single measure to effectively control An. sinensis population has not been found. Indoor residual spray with a long-lasting insecticide can not reduce vector population densities, but shorten their life spans in some degree, so contributes to malaria control.     

Does moonlight influence the biting behaviour of Anopheles funestus?

The possible effect of moonlight on the biting behaviour of mosquitoes in southern Mozambique, in particular that of Anopheles funestus (Diptera: Culicidae), a primary vector of malaria, was investigated by comparing catches indoors and outdoors using CDC light traps and 'Furvela' tent traps, respectively, for 35 consecutive nights, from 9 September to 15 October 2008. Collections were separated into three 4-hourly samples each night. A total of 17 591 mosquitoes belonging to nine species were collected, 6747 in light traps and 10 844 in tent traps. Anopheles funestus (n = 7634) and Mansonia africana (n = 4859) were the most abundant species. Fluctuations in temperature and humidity were the two environmental variables associated with changes in relative abundance of mosquitoes. Most An. funestus were collected indoors, with the majority collected in the first 4 h of the night. This was most evident on nights when moonlight was present in the early part of the night. A total of 3488 An. funestus were dissected for gonotrophic age determination. Parous rates did not change with lunar phase, but estimated oviposition cycle length was significantly shorter on nights when moonlight was present at the time of oviposition. Moonlight at dusk did not, however, affect the proportion of newly emerged insects with mating plugs collected. Outdoor transmission of malaria, especially on moonlit nights, remains a problem for control programmes.     

Late season commercial mosquito trap and host seeking activity evaluation against mosquitoes in a malarious area of the Republic of Korea

Field trials evaluating selected commercially available mosquito traps variously baited with light, carbon dioxide, and/or octenol were conducted from 18-27 September 2000 in a malarious area near Paekyeon-ri (Tongil-Chon) and Camp Greaves in Paju County, Kyonggi Province, Republic of Korea. The host-seeking activity for common mosquito species, including the primary vector of Japanese encephalitis, Culex tritaeniorhynchus Giles, was determined using hourly aspirator collections from a human and propane lantern-baited Shannon trap during hours when temperatures exceeded 15 degrees C. The total number of mosquitoes and number of each species captured during the test was compared using a block design. Significant differences were observed for the total number of mosquitoes collected, such that, the Mosquito MagnetTM with octenol > Shannon trap > ABC light trap with light and dry ice > Miniature Black Light trap (manufactured by John W. Hock) > or = New Jersey Trap > ABC light trap with light only. Significant differences in numbers collected among traps were noted for several species including: Aedes vexans (Meigen), Anopheles lesteri Baisas and Hu. An. sinensis Weidemann. An. sineroides Yamada, An. yatsushiroensis Miyazaki, Culex pipiens pallens Coquillett L., Cx. orientalis Edwards and Cx. tritaeniorhynchus. Host-seeking activity for most common species showed a similar bimodal pattern. Results from these field trap evaluations can significantly enhance current vector and disease surveillance efforts especially for the primary vector of Japanese encephalitis, Cx. tritaeniorhynchus.     

Seasonal prevalence of mosquitoes collected from light traps in the Republic of Korea, 2005

Adult mosquito surveillance was conducted during 2005 at 22 US military installations and two training sites located in six provinces in the Republic of Korea. Adult mosquitoes were collected in New Jersey light traps from 1 May to 15 October to determine threshold levels to initiate pesticide applications based on trap indices. A total of 27 363 adult mosquitoes (22 368 [81.7%] females and 4995 [18.3%] males) comprising 15 species (including five members of the Anopheles sinensis group) belonging to seven genera were collected. The most common species collected were members of the Anopheles sinensis Wiedemann group (50.2%), followed by Culex tritaeniorhynchus Giles (30.6%), Aedes vexans nipponii (Theobald) (11.8%), and Culex pipiens Coquillett (6.3%) females. Trap indices varied widely for species over their range, due in part to geographical distribution and degree of association with urban or rural communities.     

Seasonal prevalence of mosquitoes collected from light traps in the Republic of Korea, 2006

Adult mosquito surveillance was conducted during 2006 at 22 US Army and Air Force installations and two training sites located in six provinces in the Republic of Korea. Adult mosquitoes were collected in New Jersey light traps from 1 May through 15 October 2006 to determine threshold levels to initiate pesticide applications based on trap indices. In total 16 217 adult mosquitoes [13 612 (83.9%) females and 2605 (16.1%) males] comprising 16 species (including five members of the Anopheles sinensis Group) belonging to eight genera were collected. Females of the most commonly collected species were members of the Anopheles sinensis Wiedemann Group (58.9%), followed by Aedes vexans nipponii (Theobald) (21.3%), Culex tritaeniorhynchus Giles (11.8%) and Culex pipiens Coquillett (5.2%). Trap indices varied widely for species over their range, due in part, to their geographical distribution and degree of association with urban or rural communities.     

Seasonal Prevalence of Mosquitoes Collected from Light Traps in the Republic of Korea, 2001

ABSTRACT Adult mosquito surveillance was conducted during 2001 at 28 US military installations and training sites located in five provinces in the Republic of Korea (ROK). Adult mosquitoes were collected in New Jersey and dry ice-baited CDC-type light traps from 1 May through 15 October. Mosquito surveillance was conducted to determine threshold levels to initiate pesticide applications and identify malaria infection rates at selected Army installations and training sites. A total of 61,584 adults [45,814 (74.4%) females and 15,770 (25.6%) males] comprising 17 species and 7 genera were collected during 2001. The most common species collected were Anopheles sinensis Wiedemann (36.3%), Aedes vexans nipponii (Theobald) (23.1%), Culex tritaeniorhynchus Giles (22.3%) and Culex pipiens Coquillett (17.6%). Trap indices (TIs) varied widely for species over their range, due in part, to geographical distribution and degree of association with urban communities. During 2001, the TI for An. sinensis was 19.6 females/trap night compared to years 1999 and 2000, when the TI for the Munsan area was 1.4 and 2.9 times (27.7 and 57.4 TI) greater, respectively. The weekly population densities for some species were variable for each of the years, apparently as a result of variable annual weather conditions. None of the 16,302 Anopheles sinensis assayed for plasmodium vivax (Pv) malaria infections by Enzyme Linked Immunosorbent Assay (ELISA) were positive.     

Transmission of malaria in the Tesseney area of Eritrea: parasite prevalence in children, and vector density, host preferences, and sporozoite rate.

Malaria transmission was studied from July to September, 2002 in three villages of the Tesseney sub-zone, in the western lowlands of Eritrea. The three methods used for mosquito collection were light traps, pyrethrum spray catches, and pit shelter collections. All anopheline mosquitoes that were collected belonged to the Anopheles gambiae complex and they were identified by PCR as the sibling species Anopheles arabiensis (Patton). Apart from An. arabiensis, the only other mosquitoes caught were culicines. The vector population increased greatly for about a month after the start of the rains. The anthropophilic indices obtained from the blood-fed An. arabiensis resting indoors and outdoors were only 20% and 25%, respectively, with most of the other meals on goats. ELISA for P. falciparum circumsporozoite protein revealed only one positive out of 1,026 tested. The malaria prevalence among children <10 years was only 3.3% (all P.falciparum) from 300 slides examined. These low rates seem to reflect recent success in malaria control in Eritrea.     

Moderate transmission but high prevalence of malaria in Madagascar

Malaria transmission remains poorly documented in areas of low transmission. A study has been carried out over two consecutive years in Analamiranga, a village located at an altitude of 885m on the western edge of the Malagasy highlands, with the aim of generating and updating malariometric indexes for both mosquitoes and schoolchildren. In this village, no vector control measures were performed during the study period nor during previous decades. Mosquitoes were collected monthly when landing on human volunteers and in various resting-places. Blood samples were taken every 3 months from schoolchildren aged 6-12 years and microscopically examined. Of 7,480 mosquitoes collected on human subjects, 5,790 were anophelines. Ten anopheline species were represented and three of these, Anopheles funestus, Anopheles arabiensis and Anopheles mascarensis, accounted for 59.2% of the collection. Of these three species 4,640 were also collected in resting places. The proportion of mosquitoes fed on bovids was high; conversely, the anthropophilic rate (mosquitoes fed on human beings) was especially low: 31%, 7% and 1%, respectively, for A. funestus, A. arabiensis and A. mascarensis. The only confirmed malaria vector was A. funestus with a low sporozoite index (of 6,830 A. funestus, five were positive for Plasmodium falciparum and four for Plasmodium vivax). The annual entomological inoculation rate (number of bites of infected anophelines per adult person) was estimated at 2.49 with low variation over the 2 years. Overall, 909 thick blood smears were tested from blood samples taken from schoolchildren with 30.3% being malaria-positive. The four Plasmodium species infecting human subjects were detected in the following proportions: P. falciparum 78.9%, P. vivax 19.4%, Plasmodium malariae 1.0% and Plasmodium ovale 0.7%. The proportions of children who were infected with any Plasmodium ranged from 10.7% in February to 51.0% in September. Parasitemic children with fever (axillary temperature >37.5 degrees C) accounted for 16.4% of the children sampled. This study demonstrates that there are substantial parasitological consequences of even a relatively low entomological transmission and also recommends including exterior resting-places of mosquitoes in future spraying campaigns in the highlands of Madagascar.     

Studies on the resting behaviour and host choice of Anopheles gambiae and An. arabiensis from Muleba,Tanzania

The relative efficacy of a mechanical (Prokopack) collection method vs. manual aspiration in the collection of resting mosquitoes was evaluated in northern Tanzania before and after an intervention using indoor residual spraying and longlasting insecticide‐treated nets. In smoke‐free houses mosquitoes were collected from the roof and walls, but in smoky houses mosquitoes were found predominantly on the walls. Anopheles gambiae (Diptera: Culicidae) constituted 97.7% of the 312 An. gambiae complex specimens identified before but only 19.3% of the 183 identified after the intervention. A single sampling with the Prokopack collected a third of the available insects. Anopheles gambiae completed its gonotrophic development indoors, whereas Anopheles arabiensis did so outdoors. In both species gonotrophic development took 2 days. Most unfed resting An. arabiensis collected outdoors were virgins, whereas the majority of engorged insects were parous (with well‐contracted sacs). Daily survival was estimated to be 80.0%. Only 9.4% of the engorged An. arabiensis collected outdoors and 47.1% of those collected indoors had fed on humans. Using the Prokopack sampler is more efficient than manual methods for the collection of resting mosquitoes. Malaria transmission may have been affected by a change in vector composition resulting from a change in feeding, rather than reduced survival. Monitoring the proportions of members of the An. gambiae complex may provide signals of an impending breakdown in control.     

The effect of Plasmodium yoelii nigeriensis infection on the feeding persistence of Anopheles stephensi Liston throughout the sporogonic cycle.

Vector-borne parasites such as malaria have been shown to modify the feeding behaviour of their invertebrate hosts so as to increase the probability of transmission. However, evolutionary consideration of developmental changes in malaria within Anopheles mosquitoes suggests that the nature of altered feeding by mosquitoes should differ depending on the developmental stage of the parasite. We present laboratory evidence that the feeding persistence of female Anopheles stephensi towards a human host is decreased in the presence of Plasmodium yoelii nigeriensis oocysts (which cannot be transmitted), but increased when the malaria has developed into transmissible sporozoites in the salivary glands. In ten-minute trials, 33% of uninfected mosquitoes gave up their feeding attempt before the test period had ended, 53% of those harbouring oocysts had given up, but only 20% of those infected with sporozoites gave up by this time. We conclude that changes in feeding behaviour of mosquitoes mediated by parasite infection are sensitive to the developmental stage of the parasite and that these changes have important implications for malaria epidemiology.     

Adaptation of the AMRU-1 strain of Plasmodium vivax to Aotus and Saimiri monkeys and to four species of anopheline mosquitoes.

A chloroquine-resistant strain of Plasmodium vivax (AMRU-1) from Papua New Guinea has been adapted to grow in 4 species of Aotus monkeys (Aotus lemurinus griseimembra, Aotus vaciferans, Aotus nancymai, and Aotus azarae boliviensis), hybrid Aotus monkeys, and Saimiri boliviensis monkeys. Whereas it was possible to infect Saimiri monkeys with this parasite by inoculation of parasitized erythrocytes, only 42% of Saimiri monkeys became infected, compared to 92% of Aotus monkeys attempted. Comparative mosquito feedings showed that only A. vociferans, A. l. griseimembra, and Saimiri boliviensis monkeys produced infections in mosquitoes. Oocysts were observed on the guts of the 4 species of mosquitoes used (Anopheles gambiae, Anopheles stephensi, Anopheles freeborni, and Anopheles dirus), but sporozoite transmission was effected only with the intravenous inoculation of sporozoites from An. dirus into an A. l. griseimembra monkey.