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Investigations of the Anopheline (Diptera: Culicidae) fauna from three areas belonging to the Danube Delta Biosphere Reserve in order to evaluate the risk of malaria re-emergence

FALCUTA Elena, PRIOTEASA Florian-Liviu, NICOLESCU Gabriela

Address: Cantacuzino National Institute of Research and Development for Microbiology and Immunology, 103 Splaiul Independentei, Bucharest 050096, Romania. Address of author responsible for correspondence: Elena Falcuta - Cantacuzino National Institute of Research and Development for Microbiology and Immunology, 103 Splaiul Independentei, Bucharest 050096, Romania.; email: efalcuta@yahoo.com, efalcuta@cantacuzino.ro

BSTRACT. The survey focused on the comparative analyses of the anopheline fauna belonging to the maculipennis group between three areas of the Danube Delta Biosphere Reserve, two of them situated near the Razim-Sinoe lagoonal complex and one belonging to the fluvial delta. The study that was carried out during 2006 and 2007 intended to establish the composition of the anopheline fauna as well as the longevity of the various species in order to evaluate the risk of malaria re-emergence. A number of 2437 mosquitoes, belonging to Anopheles maculipennis group were collected. The presence of the former vector species was pointed up: Anopheles atroparvus, Anopheles messeae and Anopheles maculipennis sensu stricto. The investigations of the number of egg batches laid by a female have shown the physiological age of the respective female and namely if the female could infect or not the humans.
Key words: Malaria, Danube Delta, Anopheles maculipennis group, Physiological age

INTRODUCTION The climate of the planet follows a warming-up period. Different models suggest that this phenomenon will continue and will have more prominent effects in temperate regions. Both local and global changes may, independently or combined, have a negative impact on humans health, by emergence of some diseases in Europe, or by re-emerging long-time sleeping diseases from this area. Malaria is one of the most important vector-diseases on the globe and kills almost 1.5 3 million people every year. During last century, malaria was eradicated from almost all European countries, but it has already reappeared in regions in which it had previously been eradicated. The main vectors of malaria in Palaearctic region including Europe belong to Anopheles maculipennis group. Romania is a malaria-free country since 1962. Mosquitoes of the genus Anopheles are widely spread in Romania [16], but few is known about their distribution and vector potential in current conditions of environmental changing. Four species of Anopheles were involved in malaria transmission in the former endemic areas in Romania: Anopheles atroparvus, Anopheles sacharovi, Anopheles maculipennis sensu stricto and Anopheles messeae [25]. During the first half of the last century the localities in the neighbourhood of the lagoonal complex RazimSinoe represented areas with high incidence of malaria. These areas were the only ones in the country where two important and strong vectors transmitted malaria, Anopheles atroparvus and Anopheles sacharovi, the latter beeing the main vector in this area [24, 26]. The fluvial delta was described by Zotta [23, 24] as an area of anophelism without malaria. The survey is intended to monitor the Anopheles fauna belonging to the maculipennis group in order to evaluate the risk of re-emergence of malaria under the present environmental condition. MATERIALS AND METHODS The survey was carried out during the years 2006 (between April and October) and 2007 (between March and September) in three areas of the Danube Delta Biosphere Reserve, two of them belonging to the Razim

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- Sinoe lagoonary complex and one being situated in the fluvial delta at Mila 26. From these study areas very few recent data concerning Anopheles were available. Slcioara is a vilage situated in the western part of the Razim-Sinoe lacustine complex. Here we collected mosquitoes in two investigational sites with adjoining locations: Site A is represented by the vilage. Here we investigated some animal shelters (2 pigsties and one chicken coops), a house and the outdoor. Site B is a fishpond located near Slcioara village. Here are some buildings used by fishermans. Grindul Lupilor is a marine sand bank situated in the central part of the Razim-Sinoe lacustrine complex. Is a large area were the vertebrate fauna is very well represented and anthropic settlements are very few. Here mosquitoes were collected inside of a pigsty, in a house and outdoor. Mila 26 is located in Maliuc area on the Sulina branch of the Danube. The mosquitoes were collected in two investigational sites: Site A is represented by an anthropic habitat, limited to a courtyard situated on the bank of Sulina branch. Site B is represented by a large area with many reed-beds chanels which are populated by many water-birds. A large number of domestic animals (pigs, cattle, horses) are living in a semi-wild state here. In this area there is a stable in which some of these animals find shelter during the night. Capturing methods: Indoor capturing Inside of the houses and animal shelters the anophelines were captured using entomological aspirators [17] (Fig 4.). The anophelines species can be differentiated from other species during capturing by their oblique position on the substrate with the proboscis, the head and the body on the same line. Indoor capturing was the main method because in the resting sites the anophelines are fed and they are able to lay egg batches. This fact is very important because the species of maculipennis complex can be differentiated among themselves by the appearance of the eggs. If the anophelines females dont lay egg batches it is impossible to differentiate morphologicaly the species belonging to maculipennis group. Outdoor capturing Outside of the human or animal shelters the anophelines were caught along other species using CDC light traps (Fig 5.), entomological handnet and entomological aspirators [17]. Species identification: Fully fed females belonging to the maculipennis complex were kept in cloth cages at a temperature of approximately 27 2 and a high relative humidity of 7080% for optimal ovarian C development. After having laid egg batches, the Anopheles individuals were determined on a species level by the morphological characters of their eggs (Fig. 6. and Fig. 7.) using a Nikon dissecting microscope. Estimation of the physiological age of females: Simultaneously with the monitoring of the anopheline fauna, we estimated the physiological age of the females via dissections and observation of the stages of their ovaries [21]. RESULTS AND DISCUSSION Year 2006 The general climate characteristics from year 2006 have influenced the mosquito fauna and they may be described as following: - very cold spring-time with unusual low-temperatures until end of May; - frequently abundant rainfalls and floods on spring-time and autumn; - very high relative humidity.

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In Slcioara from April to September, the anopheline fauna was dominated by Anopheles messeae. A small percentage of Anopheles atroparvus (20 %) has been identified at the beginning of spring time. The proportion of this species decreased until June when it was not longer traceable. Starting with July the percentage of this species increased steadily until it became the dominant species in October (Fig. 1.)

Salcioara 2006 100 90 80 70 60 50 40 30 20 10 0 Apr May Jun Aug Sept Oct %

Anopheles messeae

Anopheles atroparvus

Fig. 1. - Seasonal dynamics of the maculipennis group species in Slcioara in 2006 In Grindul Lupilor we observed a similar situation compared to Slcioara one peculiarity was the identification of some Anopheles maculipennis s.s specimens in May (Fig 8.). Year 2007 The general climate characteristics of 2007 revealed a drought-afflicted very hot year, following an almost rainless winter which determined the end of the hibernation period for the anopheline females earlier than in the previous year, 2006 (first egg-batches have been obtained in March). The Anopheles atroparvus species has been prospered by the climate conditions in Slcioara and Grindul Lupilor and as a result, this species dominated the anopheline fauna in March and April. Starting with May relationship between both species became similar to the corresponding relationship of the previous year in both locations (a big percentage of Anopheles messeae and a small percentage of Anopheles atroparvus until the end of summer) (Fig 2.) and Fig 3.). In Mila 26, 640 fed-females have been captured during the 2 year of the study length; out of these, 233 have laid egg batches. All specimen belong to the species Anopheles messeae which seems live here in pure populations. Our investigations on the anophelines in the Danube Delta and lagoon area could not provide evidence for the presence of Anopheles sacharovi. Estimation of the physiological age of females The anopheline females complete the gonotrophic cycle in 5 6 days and sometimes in about 10 days. The investigations provided evidence for only one and rarely two dilatations on the pedicel of ovarioles (this means only 1 or 2 egg batches deposition) in these anopheline females indicating their very short lifetime which does not permit the completion of sporogony (18 days) and apparearance of sporozoites in the salivary glands.

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Salcioara 2007

100 90 80 70 60 % 50 40 30 20 10 0 Mar Apr May Jun Jul Aug Sep

Anopheles messeae

Anopheles atroparvus

Fig. 2. - Seasonal dynamics of the maculipennis group species in Slcioara in 2007

Grindul Lupilor 2007

100 90 80 70 60
%

50 40 30 20 10 0 Mar Apr Jun Jul Aug Sep Anopheles messeae Anopheles atroparvus

Fig 3. - Seasonal dynamics of the maculipennis group species in Grindul Lupilor in 2007 CONCLUSIONS The presence of Anopheles messeae (which has been in the past the weakest malaria vector in our country) in pure or almost pure populations at Mila 26, the lack of Anopheles sacharovi (as the main vector for malaria in the past) and the weak representation of Anopheles atroparvus during the summer in the area of the Razim - Sinoe lagoonal complex, as well as the very short life span of anopheline females, indicate a very low risk of malaria re-emergence in these parts of RBDD.

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Acknowledgements. We are grateful to Dr. Vasile Otel and Dr. J. B. Kiss from Danube Delta National Institute for their support. This work was supported by EU grant GOCE-2003-010284 EDEN and was partially funded by Cantacuzino NIRDMI. The contents of this publication are the responsibility of the authors and don't necessarily reflect the views of the European Commission.
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Fig 4. - Indoor capturing using an entomological aspirators

Fig 5. - Outdoor capturing using a CDC light trap

Fig 6. - Anopheles messeaes egg batches

Fig 7. - Anopheles atroparvus egg batches

Grindul lupilor 2006

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50 40 30 20 10 0 May Jun Jul Aug Oct

Anopheles messeae Anopheles maculipennis

Anopheles atroparvus

Fig 8. - Seasonal dynamics of the maculipennis group species in Grindul Lupilor in 2006

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