A reassessment of the artificial infection of three predominant mosquito species with Plasmodium vivax in Shandong Province, China

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1 J Vector Borne Dis 53, September 2016, pp Research Articles A reassessment of the artificial infection of three predominant mosquito species with Plasmodium vivax in Shandong Province, China Chongxing Zhang 1-2, Guihong Shi 1, Peng Cheng 1, Haifang Wang 1, Hongmei Liu 1, Lijuan Liu 1, Xiuxia Guo 1, Maoqing Gong 1, Yong Huang 1 1 Department of Medical Entomology, Vector Biology Key Laboratory of Medicine and Health Shandong Province, Shandong Institute of Parasitic Diseases, Shandong Academy of Medical Sciences, Jining; 2 Department of Collaborative Innovation Center for the Origin and Control of Emerging Infectious Diseases, Taishan Medical University, Taian, Shandong, China ABSTRACT Background & objectives: Under certain ecological circumstances, pathogens are able to rapidly adapt to new vectors. The great capacity of Plasmodium spp. to adapt to new anopheline mosquito vectors on different continents and the continuous ecological changes attributed to humans might promote their adaptation to culicine vectors, which are known to infect humans. Based on our current knowledge, it is difficult to predict whether such adaptations will occur. This study was aimed to determine the infection susceptibility of Anopheles sinensis, Culex tritaeniorhynchus and Cx. pipiens pallens to Plasmodium vivax in Shandong Province of China. Methods: The susceptibility of the three predominant species of mosquitoes An. sinensis, Cx. tritaeniorhynchus and Cx. pipiens pallens in Shandong Province was compared with a direct membrane feeding assay with 15 batches of Shandong strain mono-infected gametocyte-containing blood collected from Plasmodium vivax-infected patients. Infectivity was measured by dissecting the midguts and salivary glands of the mosquitoes. The presence of oocysts and sporozoites was determined by microscopy at 6 and 22 days post-blood feeding. Results: From the 15 batches of mosquitoes that were fed infected blood, oocysts and sporozoites were detected only in 7th, 13th and 15th batches of infection for An. sinensis, and no oocysts or sporozoites were detected in Cx. tritaeniorhynchus or Cx. pipiens pallens. The positive rate of An. sinensis infection was 21.2, 13 and 36.3% in the three batches of mosquitoes, with an average infection rate of 23.5%. Interpretation & conclusion: The susceptibility of An. sinensis to P. vivax was very high in Shandong Province. Cx. tritaeniorhynchus and Cx. pipiens pallens failed to exhibit susceptibility to P. vivax. Key words Anopheles sinensis; Culex pipiens pallens; Culex tritaeniorhynchus; membrane blood feeding; Plasmodium vivax INTRODUCTION Malaria, one of the most devastating human parasitic diseases, is prevalent in tropical developing regions, causing great morbidity and mortality. Despite all control efforts, 3.4 billion people are still at risk of malaria, and approximately 207 million cases of malaria occur globally each year 1. It is well known that human malaria is transmitted exclusively by anopheline mosquitoes. The susceptibility of Anopheles spp. to different human malarial parasites has been widely investigated. In 1929, Hindle and Feng 2 studied the artificial infection rate of An. sinensis with the local Plasmodium vivax. Subsequently, more studies were undertaken to observe the development of P. vivax in An. sinensis However, other culicine mosquito species, such as Aedes spp, Culex spp, and Mansonia spp, are also abundantly present in malaria-endemic areas. Similar to the anophelines that feed on humans, culicines are important vectors for human disease, transmitting arboviruses and filarial worms. Although culicine mosquitoes have never been shown to transmit human malaria, they can transmit avian Plasmodium species to birds; and several studies have investigated whether different humaninfectious Plasmodium spp. can complete their development in culicine mosquitoes. Depending on the mosquito and parasite species used, partial or complete development of Plasmodium has been observed in culicine mosquitoes In 1937, Williamson and Zain 21 infected Culex bitaeniorhyncus with P. falciparum, P. vivax and P. malariae successfully, representing the first observation of human malarial parasites in a culicine mosquito. Oocysts have been observed in the midgut epithelium in Mansonia uniformis infected with P. falciparum 25. Simi-

2 Zhang et al: Artificial infection of mosquito with Plasmodium vivax 209 larly, ookinetes have been observed in the blood meals of mosquitoes infected with Cx. salinarius 23 and Cx. quinquefasciatus 26. Although, Alavi et al 24 observed P. berghei ookinetes in the blood meals of experimental Ae. aegypti, the transmission experiment was unsuccessful. Under certain ecological circumstances, pathogens are able to rapidly adapt to new vectors. For example, the chikungunya virus (CHIKV) was originally transmitted by Ae. aegypti. A single point mutation resulting in an amino acid replacement significantly increased the transmission efficiency of the virus in Ae. albopictus 27. Plasmodium vivax adaptation to different anophelines in Mexico is also a very good example; in coastal areas, the parasites are transmitted by An. albimanus, the most common anopheline in these areas, whereas at higher altitudes, the main vector is An. pseudopunctipennis, a foothill mosquito These examples show that pathogens, including Plasmodium spp, are able to adapt to new vectors rapidly following environmental changes 30. Due to the great capacity of Plasmodium spp. to adapt to new vectors on different continents, ongoing ecological changes caused by humans might promote the adaptation of human-infectious Plasmodium parasites to culicines. The transmission of avian Plasmodium spp. by anopheline and culicine mosquitoes clearly suggests that such transmission might be possible for mammalian-infectious Plasmodium parasites. Based on our current knowledge, it is difficult to predict whether such adaptations will occur, and more research is needed to provide a more reliable prediction 30. In China, An. sinensis Wiedemann plays a major role in the maintenance of P. vivax malaria transmission 31. Culex tritaeniorhynchus Giles is the primary vector of Japanese encephalitis (JE) virus, and Cx. pipiens pallens Coquillett is the primary vector of JE virus and filariasis in China Anopheles sinensis Wiedemann, Cx. pipiens pallens Coquillett and Cx. tritaeniorhynchus Giles (Diptera: Culicidae) are three dominant species in Shandong Province 34. However, data on the susceptibility of these three mosquitoes to P. vivax are rare in Shandong Province, and Cx. tritaeniorhynchus and Cx. pipiens pallens have never been observed to be infected with P. vivax in China. The aim of this study was to investigate the susceptibility and development of P. vivax in the three main species of mosquitoes in Shandong Province, China. MATERIAL & METHODS Colonization of mosquitoes This study was carried out in Jining, Shandong Province, China from July to October 2010 and three mosquito species An. sinensis, Cx. tritaeniorhynchus and Cx. pipiens pallens were maintained in the laboratory of the Shandong Institute of Parasitic Diseases, Jining. These mosquitoes were reared using the methods described by Park et al 35 in an insectary room at 27 ± 1 C and at 70-80% relative humidity in a 12 h light/12 h dark cycle, and adult mosquitoes were raised with 10% (w/v) glucose with a sponge. For two days prior to blood feeding, the 6- to 8- day-old mosquitoes were provided only water. Selection and screening of gametocyte patients The only malaria parasite reported in the study area is Plasmodium vivax 36. The patients who sought clinical treatment in Shandong Institute of Parasitic Diseases were screened for malarial parasites by a standard finger prick and Giemsa-stained blood smear. The patients who presented with a high density of P. vivax in the blood ( P. vivax/100 leucocytes) and with an appropriate gametocyte count (0.5 30/100 P. vivax), as observed in a thin blood smear under a microscope, were selected. The screening took place the same day as the experimental infection of mosquitoes. A total of ml of venous blood from the selected patients was drawn into heparinized tubes for the experimental feedings. Experimental infections Prior to the infectious blood meal, mosquitoes were starved for 1 2 days in the mosquito cages. Freshly drawn blood was immediately transferred to artificial membrane feeders that had been prewarmed to 37 C, as described previously 16, Finally, a fresh, washed amniotic membrane was tightly placed on the bowl and allowed to dry in the shade. Before the experiment, the dry amniotic membranes were prepared by moistening them with physiological saline and stretching them across the bottom of a 250 ml wide-mouth bottle, ensuring a proper gap between the amnion and the bottom, and using a rubber stopper with two glass tubes to plug the bottle mouth. During blood feeding, a constant-temperature (37 C) circulatingwater system was used to prevent exflagellation of the microgametocytes 39. Mosquitoes were allowed to feed for 20 min to 2 h each time. After being blood fed, all the engorged mosquitoes were reared for 10 days, and the stomach and the salivary glands of each mosquito were dissected under the microscope and checked for the presence of oocysts and sporozoites. Temperature and humidity indoors The temperature and humidity were measured and recorded twice daily (800 and 1700 hrs) in the insectary

3 210 J Vector Borne Dis 53, September 2016 during the study period. The average temperature was C, and the relative humidity was 70 98%. From blood feed to dissection, the average temperature was C in the insectary for 15 batches of mosquitoes. Ethical approval All human-subject research conducted in this study was reviewed and approved by the Institutional Ethics Committee of the Shandong Institute of Parasitic Diseases (SIPD), Shandong Academy of Medical Sciences. All the patients involved in this study read and signed informed consent forms. RESULTS Patient data More than 400 symptomatic malaria patients visited the Shandong Institute of Parasitic Diseases in Shandong during the study period. After screening, 185 volunteers were enrolled for the present study. Percentage of positive mosquitoes For An. sinensis, 195 mosquitoes were dissected among which 154 showed ovarian development and 41 were undeveloped (Tables 1 and 2), wheraeas 26 (13.3%) mosquitoes were positive for P. vivax; 11 (5.6%) were positive for oocysts; and 18 (9.2%) were positive for sporozoites. In the 154 mosquitoes with developed ovaries, 10 (6.5%) were positive for stomach oocysts, 18 (11.6%) were positive for salivary gland sporozoites, three were positive for oocytes in the stomach and sporozoites in the salivary glands, and a total of 25 (16.2%) mosquitoes were positive for stomach oocysts and salivary gland sporozoites. Only 1 of 41 (2.4%) of the ovarianimmature An. sinensis mosquitoes had an oocyst in the stomach, but sporozoites were not found in the salivary glands. For Cx. tritaeniorhynchus, 203 mosquitoes were dissected (149 showed ovarian development, 54 were undeveloped), and 19 Cx. pipiens pallens were dissected (15 showed ovarian development, four were undeveloped). The oocysts and sporozoites were not found in the stomachs or salivary glands in either of these two species of mosquitoes. In the 11th batch, one blood-fed Cx. bitaeniorhyncus mosquito with an undeveloped ovary, and no oocysts or sporozoites were detected. Cx bitaeniorhyncus, however, is not a predominant mosquito species and was not included in Tables 1 and 2. Infection condition (i) Of the 15 batches of infectious blood that were Table 1. Artificial infection in three predominant mosquito species with Plasmodium vivax (mosquitoes with full ovarian development). Batch Clinical P. vivax/100 Gametocytes/ Feeding Days Temperature An. sinensis Cx. tritaeniorhynchus Cx. pipiens attack leucocytes 100 P. vivax date after ( C) No. No. positive No. No. positive No. No. positive times blood dissected Salivary Stomach dissected Salivary Stomach Dissected Salivary Stomach feeding gland gland gland 1 2/Tertian Jul /Tertian Jul /Day Jul /Day Aug /Day Aug /Tertian Aug /Day Aug /Tertian Aug /Tertian Aug /Tertian Aug /Tertian Sep /Tertian Sep /Tertian Sep /Tertian Sep /Day Sep Total ( ) Not detected.

4 Zhang et al: Artificial infection of mosquito with Plasmodium vivax 211 Table 2. Number of mosquitoes with undeveloped ovaries after blood feeding Batches An. sinensis Cx. tritaeniorhynchus Cx. pipiens No. dissected No. positive No. dissected No. positive No. dissected No. positive Stomach Salivary Stomach Salivary Stomach Salivary gland gland gland Total ( ) Not detected. fed to the An. sinensis, Cx. tritaeniorhynchus, Cx. pipiens pallens and Cx. bitaeniorhyncus species of mosquitoes, only the 7th, 13th, and 15th batches yielded positive results. In the three batches, 125 An. sinensis (103 with ovarian development, 22 undeveloped), 60 Cx. tritaeniorhynchus (21 with ovarian development, 39 undeveloped); and five Cx. pipiens pallens (2 with ovarian development, three undeveloped) were fed to engorgement. The positive rates of infection with P. vivax in An. sinensis mosquitoes were 21.2, 13 and 36.3% in the three batches of mosquitoes, respectively, with an average of 23.5%; (ii) For the three batches of blood that yielded positive mosquitoes, the experimental blood was taken from patients who experienced malarial attacks 4 5 times per day. Among the three blood batches, two had >15 gametocytes per 100 leukocytes and the remaining batch had six gametocytes. The gametocyte rates were 19, 4 and 3.9% for the 7th, 13th and 15th batches, respectively; and (iii) The days and temperature required for the appearance of sporozoites in the mosquito salivary gland were nine days at an average temperature of 28.2 C in mid- to late August, 12 days at an average temperature of 24 C in mid- to late September, and 17 days at an average temperature of 20.5 C in late September to early October. In this experimental study, the sporozoites and oocysts were observed for last time on the 11 October 2010 (Tables 1 and 2). DISCUSSION From mid-july to early October 2010, individuals from the three predominant species of mosquitoes in Shandong Province, China, i.e. 195 An. sinensis, 203 Cx. tritaeniorhynchus and 19 Cx. pipiens pallens, were subjected to artificial infection with the human malarial parasite P. vivax via membrane feedings. For Cx. tritaeniorhynchus and Cx. pipiens pallens mosquitoes, this is the first study to evaluate the potential for infection with P. vivax in China. Total 15 batches of 417 mosquitoes were dissected in three months during Of these, 26 mosquitoes from three batches totaling 190 mosquitoes (125 An. sinensis, 60 Cx. tritaeniorhynchus and 5 Cx. pipiens pallens) were vivax positive. The results of susceptibility of An. sinensis to P. vivax were also consistent with earlier experiments 2 4, 7, 9. The results proved that the susceptibility of An. sinensis to P. vivax in Shandong Province is still very high. The three batches of blood that produced P. vivax-positive mosquitoes were obtained from patients who had been attacked by clinical malaria 4 5 times per day, with two batches having >15 gametocytes/100 leukocytes and one batch with 6.4 gametocytes/100 leukocytes. The patient who donated blood for the 15th batch was treated with an eight-day regimen of chloroquine and primaquine twice daily. This finding suggests that, regular treatment of patients infected with malaria parasite is very important to control the spread and prevalence of malaria effectively. The mosquitoes with complete ovary development were engorged fully. Those that did not fully engorge, failed to do so because the temperature was too low, which affected body fat accumulation, or because the ovaries were not fully developed. For the 15th batch (blood feeding studied in late September, dissection in early October), mosquitoes with undeveloped ovaries accounted for more than half of the number of blood-fed mosquitoes due to the lower room temperature (average 20.3 C). Although, P. falciparum and other human-infectious Plasmodium spp. come closest to completing their developmental cycle in culicine mosquitoes 18, oocysts, as well as sporozoites, have been also reported in Mansonia uniformis 19. In infections of Ma. uniformis with P.

5 212 J Vector Borne Dis 53, September 2016 falciparum, only oocysts were found in the midgut epithelium 25. The ookinetes have been observed in the blood meal of mosquitoes in studies with Cx. salinarius 23, Cx. quinquefasciatus 26 and Ae. aegypti 24. The ookinetes number of P. falciparum in the blood meals of Cx. quinquefasciatus were nearly the same as in their natural vector, An. gambiae 26. It remains unclear, however, whether the results were comparable because of the use of different genotypes for the parasite and the vector In this study, the locally predominant Cx. tritaeniorhynchus and Cx. pipiens pallens were not infected with P. vivax. The results are consistent with an earlier study on Cx. bitaeniorhyncus and P. falciparum, in which no oocysts were observed in the midgut epithelium 43. The transmission experiment with P. berghei in Ae. aegypti was also unsuccessful 24. In infections of Ma. uniformis with P. falciparum, no mosquitoes were maintained long enough to determine whether sporozoites would develop in the oocysts and invade the salivary glands 25. It is still difficult to determine whether P. vivax infects Cx. tritaeniorhynchus and Cx. pipiens pallens in nature. In nature, avian plasmodia develop in and are typically transmitted by culicine mosquitoes Culex and Aedes. However, some avian malarial parasites, for example, P. gallinaceum, have been observed of being transmitted by Anopheles quadrimaculatus 44 46, An. stephensi, and An. gambiae 24, under laboratory conditions. In a study also, Plasmodium-refractory and a Plasmodium-susceptible line of An. gambiae were genetically selected, conferring either refractoriness or susceptibility to P. cynomolgi and P. gallinaceum and to some P. falciparum (human malaria parasite) lines 47. Similarly, a highly Plasmodium-susceptible line of An. stephensi was selected, and within a few generations, this line showed over 85% infection prevalence in P. gallinaceum 48. Another species of avian malarial parasite, P. relictum, has been successfully transmitted between birds by An. quadrimaculatus, An. albimanus 46, 49, An. crucians 46, and An. freeborni 46, 50. These results demonstrate that the establishment of the parasite in new regions almost always depends on vector switches Nevertheless, human-infectious Plasmodium spp. have only adapted so far to different anopheline species and not to culicine mosquitoes. The ability of mammalian-infectious Plasmodium parasites to develop in culicine mosquitoes is highly dependent on the combination of the mosquito and parasite species used 30. CONCLUSION The susceptibility of An. sinensis in Shandong Province to P. vivax appeared very high when evaluated with membrane feeding assay under laboratory conditions. 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7 214 J Vector Borne Dis 53, September 2016 bitaeniorhynchus Giles, 1901, to Plasmodium relictum but not to Plasmodium gallinaceum and Plasmodium falciparum. Indian J Malariol 1955; 9(2): Eyles DE. Studies on Plasmodium gallinaceum: IV. A comparison of the susceptibility of Aedes aegypti, Anopheles quadrimaculatus and Anopheles freeborni. Am J Epidemiol 952; 56(1): Haas VH, Akins H. Transmission of Plasmodium gallinaceum by Anopheles quadrimaculatus. J Natl Malar Soc 1948; 6(4): Hunninen AV. Comparative susceptibility of four anopheline mosquitoes to Plasmodium relictum. J Natl Malar Soc 1951; 10(3): Collins FH, Sakai RK, Vernick KD, Paskewitz S, Seeley DC, Miller LH, et al. Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae. Science 1986; 234(4776): Hume JCC, Hamilton H, Lee KL, Lehmann T. Susceptibility of Anopheles stephensi to Plasmodium gallinaceum: A trait of the mosquito, the parasite, and the environment. PLoS One 2011; 6(6): e Hunninen AV. Comparative development of Plasmodium relictum oocysts in Anopheles quadrimaculatus, An. albimanus, and Culex pipiens. J Parasitol 1953; 39(1): Mok FF. Transmission of Plasmodium relictum Grassi and Feletti by Anopheles freeborni Aitken. Science 1951; 113(2939): Ricklefs RE, Fallon SM. Diversification and host switching in avian malaria parasites. Proc R Soc Sci 2002; 269(1494): Ricklefs RE, Fallon SM, Bermingham E. Evolutionary relationships, cospeciation, and host switching in avian malaria parasites. Syst Biol 2004; 53(1): Correspondence to: Mr. Yong Huang/Mr. Maoqing Gong, Department of Medical Entomology, Vector Biology Key Laboratory of Medicine and Health Shandong Province, Shandong Institute of Parasitic Diseases, Shandong Academy of Medical Sciences, Jining, Shandong , People s Republic of China @163.com; gmq2005@163.com Received: 13 May 2016 Accepted in revised form: 21 July 2016