J Arthropod-Borne Dis, December 2013, 7(2): 147–153 R Bueno-Marí and R Jiménez-Peydró: Anophelism in … http://jad.tums.ac.ir Published Online: August 31, 2013 Original Article Anophelism in a Former Malaria Area of Northeastern Spain *Rubén Bueno-Marí, Ricardo Jiménez-Peydró Laboratorio de Entomología y Control de Plagas, Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Universitat de València-Estudi General, Valencia, Spain (Received 6 Oct 2012; accepted 14 May 2013) Abstract Background: A field study on diversity and distribution of anophelines currently present in a past endemic malaria area of Spain was carried out in order to identify possible risk areas of local disease transmission. Methods: Multiple larval sites were sampled from June to October of 2011 in the Region of Somontano de Barbastro (Northeastern Spain). The sampling effort was fixed at 10 minutes which included the active search for larvae in each biotope visited. Results: A total of 237 larval specimens belonging to four Anopheles species (Anopheles atroparvus, An. claviger, An. maculipennis and An. petragnani) were collected and identified. Conclusions: Malaria receptivity in the study area is high, especially in the area of Cinca river valley, due to the abundance of breeding sites of An. atroparvus very close to human settlements. Although current socio-economic conditions in Spain reduce possibilities of re-emergence of malaria transmission, it is evident that certain entomo- logical and epidemiological vigilance must be maintained and even increased in the context of current processes of climate change and globalization. Keywords: Malaria, Mosquitoes, Entomological surveillance, Vector borne diseases, Spain Introduction Malaria was a widespread disease in Eu- rope until the second half of 20th century. The parasitosis was particularly devastating between XVI and XIX centuries in Southern Europe due to the boom of irrigation tech- niques based on long flooding periods, main- ly rice cultivation. Although the disease is cur- rently considered eradicated from Europe, it must be noted that cycles of malaria trans- mission still are relatively common in coun- tries like Georgia, Azerbaijan, Kyrgyzstan, Ta- jikistan, Uzbekistan and Turkey (WHO 2010). Moreover, the increasing of imported ma- laria cases in last decades together with the high presence of anophelines in many Southern Europe regions (Romi et al. 1997, Ponçon et al. 2007, Bueno Marí and Jiménez Peydró 2012) has enabled the appearance of few autoch- thonous or probable autochthonous malaria cases, as recently has occurred in countries like Italy (Baldari et al. 1998, Romi et al. 2012), Greece (Kampen et al. 2002), France (Doudier et al. 2007) or Spain (Santa-Olalla Peralta et al. 2010). From the fifteen Anopheles species report- ed in Spain (Bueno Marí et al. 2012a), only two were considered primary malaria vec- tors: An. atroparvus Van Thiel, 1927 and An. labranchiae Falleroni, 1926. An. labranchiae was found to be abundant in a restricted area of the contiguous Alicante and Murcia Prov- inces (Southeastern Spain) in 1946 (Clavero and Romeo Viamonte 1948), but had disap- peared by 1973 (Blázquez and de Zulueta 1980) probably due to abandonment of rice cultivation in this area (Eritja et al. 2000). On the other hand, An. atroparvus still is well distributed in different regions of Ibe- *Corresponding author: Dr Rubén Bueno-Marí, E- mail: ruben.bueno@uv.es 147 J Arthropod-Borne Dis, December 2013, 7(2): 147–153 R Bueno-Marí and R Jiménez-Peydró: Anophelism in … http://jad.tums.ac.ir Published Online: August 31, 2013 rian Peninsula (Encinas Grandes 1982, Bueno Marí 2011). Anopheles labranchiae and An. atroparvus are considered the most important eastern and western Mediterranean malaria vectors respectively. The aim of this study was to analyze the diversity and distribution of anophelines cur- rently present in a past endemic malaria area of Northeastern Spain in order to identify pos- sible risk areas of local disease transmission. Materials and Methods Study area The study area selected was the Somontano de Barbastro Region (N 42º02’47’’/ E 0º07’23’’), which is situated in Huesca Prov- ince (Northeastern Spain) (Fig. 1). This re- gion is a transition zone between high mountain area (Pyrenees) and vast plain area (Monegros) that is characterized approximately by 15 ºC of average annual temperature and 400–700 mm of annual rainfall. In this region malaria was a common disease during XVIII century, even have been described several epidemic episodes as occurred between 1783 and 1785 with hundreds of deaths (Nieto Callén and Bosch Ferrer 1991). When malaria was pre- sent, two different areas could be distin- guished in terms of disease morbidity and mortality: for one side lowland territories ad- jacent to Cinca River where the anthroponosis was very intense and on the other side the rest of the region where malaria incidence was very low. The area of Cinca river valley has been traditionally associated with var- ious types of irrigated crops and the presence of An. atroparvus was supposed to be very frequent according to old registers of malaria incidence. The study area is also character- ized by a mountainous region at north with several small rivers and streams, all of them tributaries of Cinca River, and a dry region dedicated to the development of rainfed crops at south. Sampling methods and species identification Multiple larval sites were sampled using the standard dipping method (Service 1993) from June to October of 2011. The sampling effort was fixed at 10 minutes which includ- ed the active search for larvae in each bio- tope visited (Bueno Marí 2010). For small lar- val habitats such as tree holes or small con- tainers, the sampling was done by emptying or pipetting the contents for immature stages. Tree holes are particularly interesting since An. plumbeus Stephens, 1828 is a strictly dendrolimnic species frequent in the Pale- arctic Region and has been recognized as a minor vector for human malaria in Europe since the beginning of the 20th century (Shute 1954, Krüger et al. 2001, Bueno Marí and Jiménez Peydró 2011a, Schaffner et al. 2012). Data were recorded from all identifiable aquatic environments across the three differ- ent regions (eastern region of Cinca river Val- ley, northern mountainous region and south- ern region of rainfed crops) of the study area. The sampling effort was fixed at 10 minutes, which included the active search for larvae in each biotope visited (Bueno Marí 2010). Larval exemplars collected were identified at specific level according to the taxonomic criteria of Schaffner et al. (2001) and Bueno Marí (2010). Fig. 1. Situation of study area 148 J Arthropod-Borne Dis, December 2013, 7(2): 147–153 R Bueno-Marí and R Jiménez-Peydró: Anophelism in … http://jad.tums.ac.ir Published Online: August 31, 2013 Results A total of 237 Anopheles larval specimens were collected and identified (Table 1). Four anophelines were recorded: An. atroparvus, An. claviger (Meigen 1804), An. maculipennis Meigen, 1818 and An. petragnani Del Vecchio, 1939. Besides these anophelines collections, nine species of mosquitoes were also caught in the study area, namely Culex hortensis Ficalbi, 1889 (n= 23), Cu.impudicus Ficalbi, 1890 (n= 27), Cu. modestus Ficalbi, 1889 (n= 11), Cu. mimeticus Noè, 1899 (n= 67), Cu. pipiens Lin- naeus, 1758 (n= 481), Cu. territans Walker, 1856 (n= 85), Culiseta annulata (Schrank 1776) (n= 5), Cu. longiareolata (Macquart 1838) (n= 133) and Ochlerotatus caspius (Pallas 1771) (n= 48). None mosquito exem- plar was collected in tree holes. Table 1. Information about anopheline records Species Coordinates N Coordinates W/E Alti- tude (m) Biotope Region Nº exem- plars An. atroparvus 41°58' 12.6'' 0°10' 15.3''E 273 River margin Cinca valley 6 An. atroparvus 41°58' 11.1'' 0°10' 12.4''E 266 River margin Cinca valley 3 An. atroparvus 42°03' 54.3'' 0°12' 58.5''E 323 River margin Cinca valley 13 An. atroparvus 42°07' 09.5'' 0°13' 47.1''E 355 River margin Cinca valley 9 An. atroparvus 42°07' 10.8'' 0°13' 49.5''E 372 River margin Cinca valley 7 An. atroparvus 42°11' 28.9'' 0°09' 14.6''E 568 River margin Cinca valley 10 An. atroparvus 42°11' 11.4'' 0°09' 15.1''E 557 River margin Cinca valley 11 An. atroparvus 42°08' 23.3'' 0°01' 31.3''E 447 River margin Mountainous 5 An. atroparvus 42°08' 59.1'' 0°01' 40.7''E 463 River margin Mountainous 14 An. atroparvus 42°15' 01.2'' 0°05' 13.2''W 637 River margin Mountainous 8 An. atroparvus 42°10' 26.2'' 0°04' 26.5''W 628 Container Mountainous 2 An. atroparvus 41°56' 49.8'' 0°02' 05.2''W 409 Container Crops 5 An. atroparvus 41°55' 22.7'' 0°05' 37.8''W 348 Irrigation channel Crops 9 An. atroparvus 41°57' 42.2'' 0°04' 03.6''E 315 Temporal puddle Crops 7 An. atroparvus 41°56' 35.6'' 0°03' 36.6''E 301 Rice field Crops 45 An. atroparvus 41°56' 56.9'' 0°03' 42.8''E 306 Temporal puddle Crops 12 An. claviger 42°14' 09.9'' 0°02' 37.5''O 672 Temporal puddle Mountainous 2 An. maculipennis 42°03' 54.3'' 0°12' 58.5''E 323 River margin Cinca valley 4 An. maculipennis 42°07' 10.8'' 0°13' 49.5''E 372 River margin Cinca valley 2 An. maculipennis 42°04' 39.1'' 0°02' 14.6''E 375 River margin Crops 9 An. maculipennis 42°08' 23.3'' 0°01' 31.3''E 447 River margin Mountainous 6 An. maculipennis 42°08' 59.1'' 0°01' 40.7''E 463 River margin Mountainous 5 An. maculipennis 42°09' 54.4'' 0°01' 36.3''E 479 River margin Mountainous 7 An. maculipennis 42°15' 01.2'' 0°05' 13.2''O 637 River margin Mountainous 3 An. maculipennis 42°08' 47.9'' 0°03' 34.2''O 565 River margin Mountainous 6 An. petragnani 42°10' 28.1'' 0°03' 25.1''E 574 Container Mountainous 12 An. petragnani 42°09' 54.0'' 0°1' 59.1''O 372 Container Mountainous 6 An. petragnani 42°14' 09.9'' 0°02' 37.5''O 672 Temporal puddle Mountainous 9 149 J Arthropod-Borne Dis, December 2013, 7(2): 147–153 R Bueno-Marí and R Jiménez-Peydró: Anophelism in … http://jad.tums.ac.ir Published Online: August 31, 2013 Discussion The presence of An. atroparvus in the Cinca river valley has been confirmed decades af- ter. The occurrence of An. atroparvus is par- ticularly interesting not only all along Cinca River, but also in irrigation channels and flooding areas such as small plots trans- formed in rice fields where high densities of An. atroparvus were found. It is important to note that in the neighboring Region of Monegros, where large territories are flood- ed artificially and used for rice cultivation, a probable autochthonous case of malaria was diagnosed in 2010 presumably transmitted by An. atroparvus (Bueno Mari et al. 2012b). This is probably the first indigenous case of the disease in Spain since 1961. It is very difficult to compare the ento- mological data obtained in the study area with other neighboring regions, since current researchers about mosquito fauna in these regions are practically nonexistent. However, if we compare the epidemiological situation of the Region of Somontano de Barbastro with the available information of other bor- dering territories such as the Region of Monegros, we can conclude that An. atroparvus densities are clearly higher in the second one, mainly because rice fields are strongly colonized by the species. Despite both re- gions are characterized by high extensions of dry environments, the abundance of rice fields in Monegros Region makes this area more suitable the development of An. atroparvus. At phenological level, it is important to note that larval exemplars of An. atroparvus were collected during all the months of the study (June-October) which means that the species has a multivoltine cycle and overlap- ping of generations is quite frequent as has been suggested by other authors for Northern Spain areas (Encinas Grandes 1982). More- over, also following the criteria of Encinas Grandes (1982) it is likely than our last collec- tions of October corresponds to future over wintering females, since the activity of the species has been traditionally circumscribed between April and September months for Northern Spain, although local climatology could obviously alter this situation. Although it has been shown that adults of An. atroparvus can migrate distances close to 12 km in search of optimal hosts for feeding (Kaufmann and Briegel 2004), we must high- light that breeding sites of the species de- tected in our study have been mainly situated near from human settlements. From an epide- miological point of view, of course the prox- imity of vectors and humans is a very im- portant factor in order to evaluate the pos- sibilities of a disease outbreak. Infectivity tests carried out on European populations of An. atroparvus showed that this species can transmit Asian strains of Plasmodium vivax (Grassi and Feletti 1890), but is refractory to African strains of Pl. falciparum (Welch 1897) (Ramsdale and Coluzzi 1975). However, more recent studies have shown the ability of An. atroparvus to generate oocysts of P. faliciparum (Marchant et al. 1998), but not to complete the sporogony. Despite An. atroparvus was the main responsible in the maintenance of malaria endemicity in much of Europe during centuries, the vectorial role of the species was particularly interesting in northern countries due to its endophagic be- havior and semiactive overwintering females (Huldén et al. 2005). Endemic northern malaria reached to 68°N latitude in Europe during the 19th century, where the summer mean temperature only irregularly exceeded the lower limit of 16 °C needed for sporogony of P. vivax (Garnham 1988). This temperature conditions should have caused that malaria transmission have main- ly occurred in indoor conditions due to trans- mission of sporozoites throughout the winter by semiactive hibernating mosquitoes (Huldén et al. 2005), since it is well known that in 150 J Arthropod-Borne Dis, December 2013, 7(2): 147–153 R Bueno-Marí and R Jiménez-Peydró: Anophelism in … http://jad.tums.ac.ir Published Online: August 31, 2013 warm conditions the overwintering females of An. atroparvus can take several blood meals (Encinas Grandes 1982). Therefore northern malaria existed in a cold climate by means of summer dormancy of P. vivax hypnozoites in addition to the indoor feeding activity of over- wintering females of An. atroparvus and also probably other species of maculipennis com- plex (Huldén et al. 2005, Bueno Marí and Jiménez Peydró 2011b). With regards to the collections of An. claviger and An. maculipennis, their aquatic stages were mainly found in fresh or slightly brackish water in the mountainous area away from anthropised environments. Although these bioecological aspects, together with their zoo- philic tendency, indicate a minor role in malaria transmission, it must be noted that both species have been related with several vivax malaria outbreaks in some Eastern Med- iterranean countries (Gramiccia 1956, Coluzzi et al. 1964, Schaffner et al. 2001). Respect to An. petragnani, the species was only found in small biotopes distributed in the mountainous area. An. petragnani is a strictly zoophilic spe- cies very frequent in low anthropised envi- ronments of southern Europe (Bueno Marí and Jiménez Peydró 2011c). Consequently, An. petragnani is not considered as an im- portant malaria vector. This information co- incides with an absence of data about the vectorial status of the species in scientific literature. Ultimately we can conclude that malaria receptivity is high, especially in the area of Cinca river valley, due to the abundance of breeding sites of An. atroparvus very close to human settlements. Although An. atroparvus is also present in the other areas, the short- age of suitable larval biotopes for the species in the dry area of crops and the low percent- age of humans that lives and can serve con- sequently as a regular hosts for An. atroparvus in the mountainous area, provokes that the epidemiological interest of these areas would be less in comparison with the area of Cinca river Valley. All this information fit perfectly with old descriptions of malaria outbreaks in the Region (Nieto Callén and Bosch Ferrer 1991). In any case we must highlight that malariogenic potential of the study area is low due to the low vulnerabity of the coun- try. Vulnerability is a key factor to assess about possible re-emergence of disease and can be defined by the number of gametocyte car- riers (malaria patients) during the suitable period for malaria transmission. In Spain the imported malaria cases (mainly tourists and immigrants arrived form endemic regions) re- ported yearly by National Ministry of Health are around 400 cases. Moreover, malaria is a Notifiable Disease in Spain, so these patients are quickly identified and treated in order to minimize the possible dispersion of parasite by anophelines. Despite current socio-economic conditions in Spain which reduce possibilities of re-emer- gence of malaria transmission (Bueno Marí and Jiménez Peydró 2008), it is evident that certain entomological and epidemiological vig- ilance must be maintained and even increased in the context of current processes of climate change and globalization. Acknowledgements Dr Rubén Bueno Marí wishes to thank to Centro de Estudios del Somontano de Barbastro (CESB) and Culture Commission of Barbastro Council for the grant of the XXII Research Award López Novoa to him because this was a key factor to carry out this study. Moreover authors want to acknowledge to Instituto Aragonés de Gestión Ambiental (INAGA) for the grant of insects capture permissions in natural enclaves of the study area. Finally we want to recognize the important task of Alberto Bernués-Bañeres and Francisco Al- berto Chordá-Olmos (PhD Students) in the field part of the study. The authors declare that there is no conflict of interest. 151 J Arthropod-Borne Dis, December 2013, 7(2): 147–153 R Bueno-Marí and R Jiménez-Peydró: Anophelism in … http://jad.tums.ac.ir Published Online: August 31, 2013 References Baldari M, Tamburro A, Sabatinelli G, Romi R, Severini C, Cuccagna P, Fiorilli G, Allegri MP, Buriani C, Toti M (1998) Introduced malaria in Maremma, Italy, decades after eradication. The Lancet. 351(9111): 1246–1248. Blázquez J, Zulueta J (1980) The disappearance of Anopheles labranchiae from Spain. Parassitol. 22(1–2): 161–163. Bueno Marí R (2010) Bioecología, diversidad e interés epidemiológico de los culícidos mediterráneos (Diptera: Culicidae). Servei de Publicacions de la Universitat de València, Valencia. Bueno Marí R (2011) El anofelismo de la Comunidad Valenciana: un ejemplo de estudio del potencial malariogénico de España. Bol Asoc esp Ent. 35(1–2): 47–83. Bueno Marí R, Jiménez Peydró R (2008) Ma- laria en España: aspectos entomológicos y perspectivas de futuro. Rev Esp Salud Pública. 82(5): 467–489. Bueno Marí R, Jiménez Peydró R (2011a) Anopheles plumbeus Stephens, 1828: a neglected malaria vector in Europe. Ma- laria Reports. 1. Bueno Marí R, Jiménez Peydró R (2011b) Re-emergence of malaria and dengue in Europe. In: Rodríguez Morales AJ (Ed) Current Topics in Tropical Medicine. In Tech, Rijeka, pp. 483–512. Bueno Marí R, Jiménez Peydró R (2011c) Dif- ferences in mosquito (Diptera: Culicidae) biodiversity across varying climates and land-use categories in Eastern Spain. Entomol Fennica. 22(3): 190–198. Bueno Marí R, Jiménez Peydró R (2012) Study of the Malariogenic Potential of Eastern Spain. Trop Biomed. 29(1): 39–50. Bueno Marí R, Bernués Bañeres A, Jiménez Peydró R (2012a) Updated checklist and distribution maps of mosquitoes (Dip- tera: Culicidae) of Spain. Eur Mosq Bull. 30: 91–127. Bueno Marí R, Bernués Bañeres A, Chordá Olmos FA, Jiménez Peydró R (2012b) Entomological surveillance in a recent autochthonous malaria area of Spain. J Vector Borne Dis. 49(1): 45–47. Clavero G, Romeo Viamonte JM (1948) EI paludismo en las huertas de Murcia y Orihuela. Ensayos de aplicación de los insecticidas modernos, DDT y 666, en la lucha antipalúdica. Rev San Hig Públ. 22: 199–228. Coluzzi M, Saccà G, Feliciangeli D (1964) Sulla identita’ delle popolazioni di Anopheles claviger nel Medio Oriente. Riv Parassitol. 25(2): 123–128. Doudier B, Bogreau H, De Vries A, Ponçon N, Stauffer WM, Fontenille D (2007) Possible autochthonous malaria from Marseille to Minneapolis. Emerg Infect Dis. 13(8): 1236–1238. Encinas Grandes A (1982) Taxonomía y biología de los mosquitos del área salamantina (Diptera: Culicidae). Uni- versidad de Salamanca, Salamanca. Eritja R, Aranda C, Padrós J, Goula M, Lucientes J, Escosa R, Marqués E, Cáceres S (2000) An annotated check- list and bibliography of the mosquitoes of Spain (Diptera: Culicidae). Eur Mosq Bull. 8: 10–18. Garnham PCC (1988) Malaria parasites of man: life-cycles and morphology (ex- cluding ultrastructure). In: Wernsdorfer WH, McGregor I (Eds) Malaria: Princi- ples and Practice of Malariology, Vol. I. Churchill Livingstone, Edinburgh, pp. 61–96. Gramiccia G (1956) Anopheles claviger in the Middle East. Bull WHO. 15(3–5): 816–821. Huldén L, Huldén L, Heliövaara K (2005) Endemic malaria: an 'indoor' disease in 152 J Arthropod-Borne Dis, December 2013, 7(2): 147–153 R Bueno-Marí and R Jiménez-Peydró: Anophelism in … http://jad.tums.ac.ir Published Online: August 31, 2013 northern Europe. Historical data ana- lysed. Malaria J. 4(19). Kampen H, Maltezos E, Pagonaki M, Hunfeld KP, Maier WA, Seitz HM (2002) Indi- vidual cases of autochthonous malaria in Evros Province, northern Greece: se- rological aspects. Parasitol Res. 88(3): 261–266. Kaufmann C, Briegel H (2004) Flight perfor- mance of the malaria vectors Anopheles gambiae and Anopheles atroparvus. J Vector Ecol. 29(1): 140–153. Krüger A, Rech A, Su XZ, Tannich E (2001) Two cases of autochthonous Plasmo- dium falciparum malaria in Germany with evidence for local transmission by indigenous Anopheles plumbeus. Trop Med Int Health. 6: 983–985. Marchant P, Rling W, Van Gemert GJ, Leake CJ, Curtis CF (1998) Could british mos- quitoes transmit falciparum malaria? Parassitol Today. 14(9): 344–345. Nieto Callén JJ, Bosch Ferrer JR (1991) La epidemia de tercianas de 1783-85 en Barbastro y su corregimiento: miseria, mortandad y asistencia a finales del antiguo régimen. Somontano. 2: 63–83. Ponçon N, Toty C, L' Ambert G, Le Goff G, Brengues C, Schaffner F, Fontenille D (2007) Biology and dynamics of poten- tial malaria vectors in Southern France. Malaria J. 6(18). Ramsdale CD, Coluzzi M (1975) Studies on the infectivity of tropical African strains of Plasmodium falciparum to some southern European vectors of malaria. Parassitol. 17(1–3): 39–48. Romi R, Pierdominici G, Severini C, Tamburro A, Cocchi M, Menichetti D, Pili E, Marchi A (1997) Status of malaria vec- tors in Italy. J Med Entomol. 34(3): 263– 271. Romi R, Boccolini D, Menegon M, Rezza G. Probable autochthonous introduced ma- laria cases in Italy in 2009–2011 and the risk of local vector-borne transmis- sion (2012). Euro Surveill. 17(48). Santa-Olalla Peralta P, Vázquez-Torres MC, Latorre-Fandós E, Mairal-Claver P, Cortina-Solano P, Puy-Azón A, Adiego-Sancho B, Leitmeyer K, Lucientes-Curdi J, Sierra-Moros MJ (2010) First autochthonous malaria case due to Plasmodium vivax since erad- ication, Spain, October 2010. Euro Surveill. 15(41). Schaffner F, Angel G, Geoffroy B, Hervy JO, Rhaeim A (2001) The mosquitoes of Europe/ Les moustiques d’Europe. IRD Éditions and EID Méditerranée. Schaffner F, Thiéry I, Kaufmann C, Zettor A, Lengeler C, Mathis A, Bourgouin C (2012) Anopheles plumbeus (Diptera: Culicidae) in Europe: a mere nuisance mosquito or potential malaria vector? Malar J. 26(11). Service MW (1993) Mosquito Ecology. Field Sampling Methods. Elsevier Science Publishers, London. Shute PG (1954) Indigenous P. vivax malaria in London believed to have been trans- mitted by Anopheles plumbeus. Mon Bull Minist Health Publ Health Lab Serv. 13: 48–51. WHO (2010) Centralized information system for infectious diseases, Malaria, Den- mark. 153