J Arthropod-Borne Dis, March 2017, 11(1): 27–35 K Yadav et al.: Disappearance of … 27 http://jad.tums.ac.ir Published Online: March 14, 2017 Original Article Disappearance of Anopheles minimus and Anopheles dirus from Certain Ma- laria Endemic Areas of Assam, India *Kavita Yadav 1, Sunil Dhiman 1, Bipul Rabha 1, Diganta Goswami 1, PK Saikia 2, Vijay Veer 1 1Medical Entomology, Defence Research Laboratory, Tezpur, Assam, India 2Zoology Department, Gauhati University, Guwahati, Assam, India (Received 2 July 2014; accepted 17 Nov 2015) Abstract Background: Orang Primary Health Centre (OPHC) and Balipara Primary Health Centre (BPHC) of Assam (India) report mosquito borne diseases annually. Current study was performed to ascertain the prevalence of known malaria and Japanese Encephalitis (JE) vectors and their possible role in disease transmission. Methods: Malaria epidemiological data for 2006–2010 and JE data for 2008–2013 of Assam, India were obtained from the health authority. Mosquitoes were collected using CDC light traps and identified morpho-taxonomically. Results: Plasmodium falciparum cases (81.5%, 95% CI= 72.0–91.1) were statistically higher in OPHC (P< 0.0001, t= 8.0) during the recent years. There was 4.4 folds rise in the confirmed acute encephalitis syndrome (AES) and 3.2 folds increase in the confirmed JE cases during 2013 as compared to 2008. Altogether 9,218 mosquito specimens (PTND= 153.6), comprising of 44.1% anophelines (PTND= 67.7), 42.3% culicines (PTND= 65.0) and 9.5% manso- nia (PTND= 14.6) were recorded. In BPHC, Anopheles vagus was recorded in high density (P< 0.0001), whereas Culex quinquefasciatus was the predominant JE vector (P= 0.04). In OPHC, among the known malaria vectors, the density of Anopheles annularis was significantly high (P< 0.0001). However Culex bitaeniorhynchus was the pre- dominant known JE vector (P< 0.0001) followed by Cx. quinquefasciatus. Conclusion: Even in the absence of known efficient vectors, many Anopheles species are still involved in malaria transmission. There was disappearance of Anopheles minimus and Anopheles dirus and establishment of An. annu- laris, An. vagus and An. philippinensis/nivipes mosquitoes in study area. Keywords: Mosquito vectors, Malaria, Japanese encephalitis, Ecology, India Introduction Mosquitoes spread pathogenic agents of malaria, Japanese Encephalitis (JE), dengue, lymphatic filariasis and chikungunya in many countries. The data on prevalence of known mosquito vectors constitutes vital and useful information to control the mosquito-borne diseases. Despite concerted vector borne dis- eases intervention efforts in India during 2014, approximately 1.07 million confirmed malar- ia cases and 535 deaths have been reported, while 1,661 confirmed cases and 293 deaths have been attributed to JE (NVBDCP 2014). Mosquitoes are remarkably adaptable in- sect group, which continue to successfully coexist with human being and survive by feed- ing on human host and his domestic animals in addition to plant nectar. Assam is the largest state (population wise) in northeast region of India, where malaria transmission is endemic. In India, An. minimus Theobald, 1901, An. dirus Peyton and Harrison, 1979, An. fluviatilis James, 1902 and An. culicifa- cies Giles, 1901 have been recognised as po- tential malaria vectors, while An. annularis Van der Wulp, 1884, An. philippinensis Lud- low, 1902 and An. varuna Iyengar, 1924 play limited role in malaria transmission (Dev et al. 2003, Prakash et al. 2004, Bhattacharyya et al. 2010, Dhiman et al. 2011, 2012). JE out- breaks are common in northeastern states *Corresponding author: Dr Kavita Yadav, E-mail: kavitanami@gmail.com J Arthropod-Borne Dis, March 2017, 11(1): 27–35 K Yadav et al.: Disappearance of … 28 http://jad.tums.ac.ir Published Online: March 14, 2017 including Assam and occur mainly during rainy season. Sixteen mosquito species have been incriminated as JE vector in India, of which Culex tritaeniorhynchus Giles, 1901 and Culex vishnui Theobald, 1901 subgroup has been reported as prominent JE vectors in the endemic areas (Kanojia 2007, Saxena et al. 2008, Dhiman et al. 2013). Many culicine and mansonia species, namely, Cx. vishnui, Cx. bitaeniorhynchus Giles, 1901, Cx. gelidus Theobald, 1901, Ma. uniformis Theobald, 1901, Ma. annulifera Theobald, 1901 and Ma. indiana Edwards, 1930 are well known vec- tors of JE and reported in many parts of As- sam and other north-eastern states of India (Saxena and Dhole 2008, Dhiman et al. 2009, 2013). Udalguri and Sonitpur districts of Assam are highly endemic for malaria and contrib- ute considerably to the malaria cases in the state. A recent study conducted in Udalguri district has indicated that, the number of health centres having annual parasitic index (API) of > 5 and more than 30% of malaria cases due to P. falciparum Welch, 1897 were increased in the recent years. The study ex- hibited that 11 health centres were malaria hot spots, of which 9 were part of Orang Primary Health Centre (OPHC) (Yadav et al. 2012). Similarly, in Sonitpur District 10 health centres including Balipara have been identified as malaria hot spots, which have an extremely high malaria risk (Nath et al. 2013). The forestlands of both the districts have remained the areas of intense malaria parasite reservoir providing foci for re-infec- tion in the other neighbouring areas. Moreo- ver, ecological changes due to deforestation have brought some changes in geo-climate that has significantly influenced the mosquito vector ecology and diseases transmission (Nath et al. 2012). The dynamic distribution and transmis- sion of malaria and JE in Assam poses a se- rious epidemiological challenge due to vari- ous socio-economic, geo-political and envi- ronmental factors (Dev et al. 2003, 2010, Dhiman et al. 2011, Rabha et al. 2012, Yadav et al. 2014). Information on vector entomol- ogy is an essential component in disease man- agement, which depends upon the knowledge of vector species density and composition. Many studies have been conducted in vari- ous parts of the state, but anthropogenic eco- system modifications in the past few years might have influenced the known malaria and JE vectors composition. Therefore, it is inevitable to update data on prevalence of vector mosquitoes for reviewing vector con- trol strategies. The present study was undertaken during April 2012 to August 2013 in malaria endemic Primary Health Centres of Udalguri and Sonit- pur districts of Assam to generate information on known mosquito vector prevalence. Materials and Methods Study area Current study was conducted in randomly selected four sentinel survey sites each (Table 1) in OPHC of Udalguri District and Balip- ara Primary Health Centre area (BPHC) of Sonitpur District. OPHC (92 ̊ 07’–92 ̊ 22’ E longitude and 26 ̊ 33’–26 ̊ 56’ N latitude) sit- uated at 105.2 meters, is dominated by var- ious ethnic tribes primarily engaged in agri- culture and tea cultivation. The climate is sub-tropical humid and experience an aver- age annual rainfall of about 2,000mm, while the temperature and relative humidity varies between 34.5 °C to 13.5 °C and 65 to 90% respectively. Study area has many small riv- ers, scattered tea gardens and vast paddy fields, which create suitable breeding ecol- ogy for mosquito vectors. BPHC (92 ̊ 38’–92 ̊ 59’ E longitudes and 26 ̊ 41’–27 ̊ 02’ N lati- tude, 74.7 meters) is dominated by different ethnic groups, including Bodo, Nepali, Aa- divasi and Assamese, with agriculture based very low socio-economic status. The average temperature ranging from 15 °C to 35 °C, about J Arthropod-Borne Dis, March 2017, 11(1): 27–35 K Yadav et al.: Disappearance of … 29 http://jad.tums.ac.ir Published Online: March 14, 2017 1,900mm average rainfall and 55–90% rel- ative humidity plays a major role in deter- mining the climate of the area. There are many rivers and large spreads of tea meadows and paddy fields. The prevailing climatic condi- tions are conducive for the breeding and pro- liferation of different vector mosquitoes. A recent study conducted in Sonitpur district using normalized difference vegetation index (NDVI) has shown that the forest-covered area was 1.2 folds decreased in two years (Nath et al. 2012). Malaria and Japanese Encephalitis situa- tion in the area Malaria epidemiological data for the years 2006–2010 were collected from the District Malaria Office of the concerned district and analysed to understand the malaria situation during preceding years. Japanese Encephali- tis data of Assam for the years 2008–2013 was obtained from the NVBDCP and de- picted in Table 2. Collection and identification of known mos- quito vectors Mosquitoes were collected from dusk to dawn (1800–0600 h) with 6-volt battery op- erated miniature light traps (Centres for Dis- ease Control, Atlanta, USA) and indoor rest- ing using WHO aspirator tubes. The mud plastered kuchha houses and bamboo houses having a thatched roof and adjacent to the cattle sheds were selected for the study. The study area had numerous small ponds and ir- rigation channels, while number of domestic animals such as pig, fowl and duck were also available. Smoking and burning was prevent- ed during the operation of the traps. The traps were installed near unscreened win- dows in the rooms at about 2 m above the ground and kept on throughout the night un- til removed in the early morning hour. The collected mosquitoes were etherised (if alive) and identified to species complex level with the help of standard keys (Barraud 1934, Wattal and Kalra 1961). Densities of known vector mosquitoes were calculated in terms of mean numbers of mosquito of a species caught per trap night and expressed as per trap night density (PTND) of that particular species. Data analysis The collected mosquitoes were expressed in per trap night density (PTND), whereas known vector density for each species was presented as mean±SEM. Comparison of mosquitoes was performed using ANOVA followed by Tukey Krammer test of multiple comparison. PTND of known malaria and JE vectors was compared using unpaired students’ “t” test. Results Past malaria and Japanese Encephalitis sit- uation Malaria situation during the past years (2006–2010) in BPHC and OPHC is shown in Fig. 1. The slide positivity rate (SPR) in both the locations was similar in the recent years (P= 0.8, t= 0.3), however cases attributed to P. falciparum malaria in OPHC were 81.5% (95% CI= 72.0–91.1) and found to be statis- tically higher than in BPHC (P< 0.0001, t= 8.0). Further, the average annual parasitic index (API) during the years 2006–2010 was 6.9± 12 (95% CI= 3.6–10.3) in OPHC as com- pared to 2.4±0.5 (5% CI= 1.1–3.7) in BPHC (P= 0.01, t= 3.4). Past encephalitis data sug- gests that as compared to 2008, there has been 4.4 folds increase in the confirmed acute encephalitis syndrome (AES) cases and 3.2 folds increase in the confirmed JE cases in 2013. However, confirmed deaths due to AES showed 2.7 folds while due to JE showed 4.1 increases during the last six years. Known malaria and Japanese Encephali- tis vectors abundance A total of 9,218 mosquito specimens were collected in the current study using CDC light J Arthropod-Borne Dis, March 2017, 11(1): 27–35 K Yadav et al.: Disappearance of … 30 http://jad.tums.ac.ir Published Online: March 14, 2017 traps and aspirators in 60 trap nights (PTND= 153.6). Of the total mosquitoes, anophelines were 44.1% (PTND= 67.7), culicines were 42.3% (PTND= 65.0), mansonia were 9.5% (PTND= 14.6) while remaining 4.1% (PTND= 6.3) corresponded to other mosquito species belonged to Stegomyia, Neomelaniconion, Coquillettidia and Armigeres. In BPHC, total 7,201 (PTND= 180.0) mosquitoes constituting 37.9% anophelines (PTND= 68.2), 45.3% culicines (PTND= 81.6) and 12.0% mansonia (PTND= 21.7) were re- ported during the study. The density of anophe- lines and culicines mosquitoes was statistical- ly higher than the other mosquitoes, however no significant difference was found among both of them (P= 0.8, t=0.3). Among the re- ported malaria vectors in India and neigh- bouring Bangladesh, present study found An. annularis, An. culicifacies, An. philippinensis and An. vagus Doenitz, 1902 mosquitoes. Anopheles vagus (58.1%, PTND= 37.7 of the total known malaria vectors) was recorded in significantly high density (F= 18.4, P< 0.0001), but did not differ statistically from An. annu- laris (P= 0.2, t= 1.6). Among the known JE vectors, Cx. quinquefasciatus Say, 1823 was predominant (F= 2.6, P= 0.04) and account- ed for 49.5% of the total JE vectors (PTND= 44.6). Malaria and JE vectors density ob- tained in BPHC is depicted in Table 3. There was no statistically difference between ma- laria and JE vectors density (P≥ 0.3, t≤ 0.6) in BPHC throughout the study period. In OPHC, of the total 2,017 (21.9%) mos- quitoes in 20 trap nights (PTND= 100.9), 66.2 % were anophelines, 47.6% were culicines, while 1.8% corresponded to mansonia spe- cies. The PTND was highest for anophelines (66.8) followed by culicines (31.8). Density of anophelines was statistically higher than the other mosquitoes (F= 748.6, P< 0.0001). Among the known malaria vectors, An. cu- licifacies, An. vagus, An. fluviatilis and An. an- nularis were prevalent, however the density of An. annularis was significantly high (F= 180.3, P< 0.0001). Culex bitaeniorhynchus was the predominant known JE vector (F= 92.1, P< 0.0001) followed by Cx. quinque- fasciatus. Malaria and JE vector density in OPHC is shown in Table 4. Malaria vector density was statistically higher than the JE vectors in OPHC (F= 15.1, P< 0.0001). Between BPHC and OPHC, the PTND of known JE and malaria vectors (Fig. 2) be- tween both the study areas were similar (P≥ 0.4, t≤ 1.0). (BSR- blood smear rate, SPR- slide positivity rate, % Pf- percent Plasmodium falciparum, API- annual par- asitic index) Fig. 1. Past malaria situation depicting blood smear rate (BSR), slide positivity rate (SPR), % Plasmo- dium falciparum and annual parasitic index (API): (A) In BPHC, (B) In OPHC B A J Arthropod-Borne Dis, March 2017, 11(1): 27–35 K Yadav et al.: Disappearance of … 31 http://jad.tums.ac.ir Published Online: March 14, 2017 (Mal- Malaria, BPHC/OPHC- Balipara/Orang Primary Health Centre) Fig. 2. Per trap night density of known malaria and Japanese Encephalitis vectors in four sentinel collection sites (S1–S4) in BPHC and OPHC areas Table 1. Global Positioning System coordinates of the sentinel survey locations (S1-S4) in both study areas of Orang Primary Health Centre (OPHC) and Balipara Primary Health Centre (BPHC) of Assam (India) Study area Survey site GPS location Balipara Primary Health Centre (BPHC) S1 92°46'43.0" E 26°41'18.6" N S2 92°47'36.2" E 26°42'02.3" N S3 92°47'39.6" E 26°40'35.3" N S4 92°48'09.7" E 26°41'31.8" N Orang Primary Health Centre (OPHC) S1 92°16'15.8"E 26°38'23.8" N S2 92°15'49.9"E 26°41'45.4" N S3 92°17'32.4"E 26°41'52.0" N S4 92°19'38.3"E 26°41'55.3"N Table 2. Past acute encephalitis syndrome and Japanese Encephalitis situation in Assam Year Acute encephalitis syndrome (AES) Japanese Encephalitis (JE) Reported cases Deaths (%) Reported cases Deaths (%) 2008 319 99 (31.0) 157 33 (21.0) 2009 462 92 (19.9) 218 46 (21.1) 2010 469 117 (24.9) 142 40 (28.2) 2011 1319 250 (19.0) 489 113 (23.1) 2012 1343 229 (17.1) 463 100 (21.6) 2013 1388 272 (19.6) 495 134 (27.1) J Arthropod-Borne Dis, March 2017, 11(1): 27–35 K Yadav et al.: Disappearance of … 32 http://jad.tums.ac.ir Published Online: March 14, 2017 Table 3. Known malaria and Japanese Encephalitis vectors in Balipara Primary Health Centre area Species Density (mean±SEM) F (P) Malaria vectors An. vagus 377.3±81.5 18.4 (< 0.0001)An. annularis 241.5±15.9 An. culicifacies 15.3±6.0 An. philippinensis 15.5±2.5 JE vectors Cx. quinquefasciatus 446.3±270.3 F= 2.6 (P= 0.04) Cx. vishnui 175.3±89.9 Cx. bitaeniorhynchus 120.8±100.2 Cx. whitmorei 25.5±4.2 Cx. gelidus 8.5±2.9 Ma. uniformis 131.8±115.7 Ma. indiana 30.3±19.2 Ma. annulifera 44.8±33.6 Table 4. Known malaria and Japanese Encephalitis vectors in Orang Primary Health Centre area Species Density (mean±SEM) F (P) Malaria vectors An. vagus 156.0±11.7 180.3 (< 0.0001) An. annularis 162.8±6.7 An. culicifacies 1.8±0.5 An. fluviatilis 3.5±0.6 JE vectors Cx. quinquefasciatus 63.8±7.7 92.0 (< 0.0001) Cx. bitaeniorhynchus 90.5±6.5 Cx. whitmorei 0.6±0.6 Cx. gelidus 3.3±0.8 Ma. indiana 0.8±0.4 Ma. annulifera 1.8±0.8 Discussion Vector borne diseases control programmes have always stressed that up-to-date knowledge of spatial distribution and diversity of mos- quito vectors across the endemic areas is in- evitable for planning and implementing the effective intervention measures. In the cur- rent study, known malaria and JE vectors were collected in two ecologically distinct en- demic primary health centres of Assam, where malaria and JE transmission is supported by many efficient mosquito vectors. Anopheles dirus, An. fluviatilis and An. minimus mosquito species have been considered as important ma- laria vectors, however in north-eastern states and neighbouring country Bangladesh the role of An. annularis, An. culicifacies, An. philip- pinensis and An. vagus in malaria transmis- sion is determined (Prakash et al. 2004, Alam et al. 2010, Bhattacharyya et al. 2010, Dhiman et al. 2012). The present study has indicated that many known malaria vectors were abundant in both the study areas, however could not report even a single specimen of established vectors An. minimus and An. dirus (Dev et al. 2003, Das et al. 2004, Sarma et al. 2012). Both An. minimus and An. dirus are known for unin- terrupted spread of malaria in the region, but the surprising results of current study indi- cate that the other malaria vectors of com- paratively lesser epidemiological importance might have taken over malaria transmission J Arthropod-Borne Dis, March 2017, 11(1): 27–35 K Yadav et al.: Disappearance of … 33 http://jad.tums.ac.ir Published Online: March 14, 2017 in absence of the well-established vectors. In BPHC of Assam An. annularis was found harbouring both P. falciparum and P. vivax Grassi and Feletti 1890 and its density out- numbered the density of An. minimus during malaria season (Dhiman et al. 2012). Anophe- les culicifacies, although comes in lower counts but have strong anthropophilic character and malaria transmission potential in the suburbs of the region (Dhiman et al. 2012). Further, An. nivipes Theobald, 1903 and An. vagus have also been found positive for sporozoite in different malaria endemic areas of northeast- ern India (Prakash et al. 2004, Bhattacharyya et al. 2010). Presently, An. annularis and An. vagus were recorded in large number in ar- eas where malaria is still endemic and many cases are reported annually (Rabha et al. 2012, Yadav et al. 2012, Nath et al. 2013). Although both these species are primarily considered zoophilic and exophilic in nature, but they have been considered to be oppor- tunistic in the host selection for blood meal and have been thought to maintain malaria transmission in the region (Prakash et al. 2004, Dhiman et al. 2012). Large number of specimens corresponding to these two spe- cies was collected indoor resting which indi- cates that both of these might be shifting ex- ophilic and exophagic behaviour to endophilic and endophagic. In Assam-Meghalaya bor- der, An. annularis prefers resting indoors and a considerable proportion feed on human blood (Dhiman et al. 2014). In the recent years, there has been tremen- dous deforestation, and new resettlements are coming up rapidly in the study area. The forestlands, which provide favourable breed- ing habitats for malaria vectors An. dirus and An. minimus, have been reduced significantly during last few years (Nath et al. 2012). There- fore, disruption in the ecology of these two vectors might have persuaded other anopheline species such as, An. annularis, An. vagus and An. philippinensis/nivipes to establish them- selves as major species owing to the vast paddy cultivation in the area. Among the JE vectors Cx. bitaeniorhynchus, Cx. quinquefasciatus and Cx. vishnui were re- corded in large density, whereas Cx. gelidus was recorded in very low number. These vec- tors have been reported from other areas of Assam and found associated with the JE trans- mission in the region (Dhiman et al. 2013). In the past few years, the JE is emerging as serious vector borne disease in the entire north-east region, where reported cases and deaths during recent years has increased to many folds (NVBDCP 2013). However, the vector abundance of potential JE vectors has not been monitored regularly (Dhiman et al. 2013). JE outbreaks are common during the rainy season and occur at regular intervals in different parts of northeast region including study area. Therefore, data on population dynamics of JE vectors is important for fo- cused control measures implementation. Mos- quito density builds up during the epidemics, however abundance, survival and longevity of vector mosquitoes directly influence the dynamics of disease transmission annually. The study area reports high incidence of malaria throughout the year. Various ethnic tribes having poor economic condition and their socio-cultural customs and beliefs make malaria vector control difficult (Dhiman et al. 2011, Rabha et al. 2012, Yadav et al. 2014). Malaria vector density was high in OPHC, which corroborates the high number of reported malaria cases. The API reported in OPHC is about 2.8 folds, while the per- cent P. falciparum is about 2.3 folds higher than in BPHC. Higher density of known vec- tors has been found associated with increase in disease incidence across many endemic settings (Alam et al. 2012, Dhiman et al. 2012). Although not much JE cases are re- ported in OPHC as compared to BPHC, but the tribal villages in the OPHC area have high pig density, which could serve as reser- voir of JE virus, and also the high density of known JE vectors may sprout the disease J Arthropod-Borne Dis, March 2017, 11(1): 27–35 K Yadav et al.: Disappearance of … 34 http://jad.tums.ac.ir Published Online: March 14, 2017 during favourable transmission conditions. Ecological changes might have involved in the replacement of well-established mosquito species by other species. Deforestation and irrigation projects resulted in change of mosquito species involved in malaria trans- mission in Sri Lanka and Thailand (Amer- asinghe et al. 1991, Prothero 1999). Conclusion The present study is limited in its scope and reveals that variety of little known ma- laria and JE vectors are maintained in the study area, while some well-known vectors were disappeared or maintained at very low density. We have not attempted to incrimi- nate any known malaria and JE vectors, but suggest that even in the presence of compre- hensive vector control measures some little known vectors might have been playing a leading role in disease transmission. High density of known vectors may increase the risk of increasing insecticide resistance there- by circumventing the protection from insec- ticides. Further investigation on breeding ecol- ogy and insecticide susceptibility status of commonly used insecticides is important to provide information for adopting suitable control measures. Acknowledgements Authors are thankful to the local health staff for assisting in the collection of malaria and JE epidemiological data. The help ren- dered by the villagers during the study is al- so solicited. The authors declare no conflict of interests. 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