Molecular identification of Palearctic members of Anopheles maculipennis in northern Iran

Background Members of Anopheles maculipennis complex are effective malaria vectors in Europe and the Caspian Sea region in northern Iran, where malaria has been re-introduced since 1994. The current study has been designed in order to provide further evidence on the status of species composition and to identify more accurately the members of the maculipennis complex in northern Iran. Methods The second internal transcribed spacer of ribosomal DNA (rDNA-ITS2) was sequenced in 28 out of 235 specimens that were collected in the five provinces of East Azerbayjan, Ardebil, Guilan, Mazandaran and Khorassan in Iran. Results The length of the ITS2 ranged from 283 to 302 bp with a GC content of 49.33 – 54.76%. No intra-specific variations were observed. Construction of phylogenetic tree based on the ITS2 sequence revealed that the six Iranian members of the maculipennis complex could be easily clustered into three groups: the An. atroparvus – Anopheles labranchiae group; the paraphyletic group of An. maculipennis, An. messeae, An. persiensis; and An. sacharovi as the third group. Conclusion Detection of three species of the An. maculipennis complex including An. atroparvus, An. messae and An. labranchiae, as shown as new records in northern Iran, is somehow alarming. A better understanding of the epidemiology of malaria on both sides of the Caspian Sea may be provided by applying the molecular techniques to the correct identification of species complexes, to the detection of Plasmodium composition in Anopheles vectors and to the status of insecticide resistance by looking to related genes.

main vector in Turkey and is, together with Anopheles superpictus and Anopheles pulcherrimus, the most important vector, of malaria in the former Soviet Union, although An. messeae has been implicated in the resurgence of malaria in Russia and the Ukraine [8]. Three species of the Maculipennis complex, An. atroparvus, An. labranchiae and An. sacharovi are known to be efficient current or historical vectors of malaria in the Palearctic region [9,10]. An. maculipennis s. s. has been identified as the major vector of malaria on the Caspian Sea coast area of Iran and An. sacharovi is considered the main vector in the central plateau of the country [11,12]. Djadid [13] reported that members of this species complex in northern Iran are active from May to September, with a peak in July and that they breed readily in rice fields, spring and clean standing water, and adult mosquitoes could be found in animal shelters (95%). These species were susceptible to dieldrin, malathion, deltamethrin and resistant to DDT. Their biting pattern on human and animals baits is more or less the same, starting at 19.00 hrs with a peak between 20.00 -21.00 hrs. Enzyme-linked immunosorbent assay (ELISA) carried out on 304 blood-fed mosquitoes collected from indoor resting sites revealed that they have fed predominantly on cattle, with fewer blood meals on sheep and poultry [13]. None of the mosquitoes had fed on human blood although a previous study by Manouchehri et al. [14] has shown an anthropophilic index for this species in northern Iran of 1.7-4.9%. Furthermore, Djadid [13] reported that hibernation in this species starts in October and that complete fat body could be seen in February. The following culicidae mosquitoes larvae have been found in An. maculipennis breeding places; Anopheles hyrcanus, Anopheles claviger, Culex pipiens, Culex mimeticus, Culex tritaeniorhynchus, Aedes vexans, Culiseta subochra, Uranotaenia unguiculata. Adult of Anopheles algeriensis and Anopheles hyrcanus also has been found in resting places of An. maculipennis [13].
From an operational point of view in malaria control, in the region to the north of the Zargors range of mountains, An. maculipennis, An. sacharivi and An. superpictus are recognized as malaria vectors. This region was malaria-free for more than 30 years. However, since 1994, malaria has been re-introduced to this area through Republic of Azerbaijan and Armenia [15,16].
Few studies have been carried out in northern areas of the country where malaria has reappeared and been introduced into several different provinces. Although, it has been postulated that members of An. maculipennis complex are responsible for malaria transmission, how many species within maculipennis complex are present in Caspian Sea region has not been defined yet. Do they exist as sympathric species and what if any, is, their role in malaria transmission? In view of the zoogeographical, ecological and social changes on both sides of Caspian Sea, this study was carried out in order to provide further evidence on the status of species composition and to identify more accurately the members of An. maculipennis complex in northern Iran. Field collection, morphological identification followed by amplification and sequencing of ITS2 region led to identification of six members of An. maculipennis complex and their comparison with those related sequences deposited in GenBank. The occurrence of three species of An. maculipennis complex including An. atroparvus, An. messae and An. labranchiae is reported for the first time from northern Iran. This proves the extended distribution of these species towards southern territory of the An. maculipennis complex. This is a pre-requisite for understanding the epidemiology of malaria on both sides of Caspian Sea, and attempting to prevent the re-introduction of malaria to malaria-free areas.

Materials and methods
Mosquitoes and ecological characteristics of collection sites 235 mosquito specimens of the An. maculipennis complex were collected by total catch in human and animal shelters in northern Iran including provinces of East Azerbayjan, Ardebil, Guilan, Mazandaran and Khorassan ( Figure  1) during three collections in May 1997 to September 1999 and May to September 2001.
Two mountainous provinces of East Azerbaijan and Ardebil sharing border line with republics of Azerbaijan and Armenia, while Guilan and Mazandran with Mediteranean climate are located in southern coast of Caspian Sea. Eastern corner of Mazandran and northern Khorassan are close to republic of Turkmenistan. However, the whole eastern part of Khorassan province is in border of Afghanistan. With respect to malaria transmission, all these five provinces have one peak during June-August. Details regarding the origin and number of specimens used for PCR amplification and sequencing are given in Table 1 and Figures 1, 2, 3, 4.

Mosquitoes' morphological identification
On arrival in the laboratory of malaria research group (MRG) in the Institut Pasteur of Iran, mosquitoes were identified by a morphological key of Iranian Anophelines [34] to distinguish An. maculipennis and An. sacharovi adults from other Anopheline species.

Mosquito genomic DNA extraction and PCR amplification
Mosquito genomic DNA was extracted using slight modification of the method described by Collins et al. [35]. The ITS2 region of rDNA gene was amplified using the universal primers of 5.8 s (5' ATC ACT CGG CTC GTG GAT CG 3') and 28 s (5' ATG CTT AAA TTT AGG GGG TAG TC 3'). All PCR reactions were performed in a total volume of 25 μl. The reaction mixture contained 50 ng of each of the specific primers of 5.8 s and 28 s, which are flanking the whole ITS2 and partial sequence of 5.8 s and 28 s regions at both ends [36], 0.5 unit of Taq polymerase, 0.1 mM each of dNTPs, 0.001% gelatine, 2.5 μl of 10X reaction buffer, 2 mM MgCl 2 . The amplification profile was as follows: denaturation at 94°C for 5 min, followed by 30 cycles of annealing at 53°C for 1 min and extension at 72°C for 1 min with 7 min extra extension time in the last cycle. The target amplified DNA was run on 1.5% agarose gel. Gels were stained with ethidium bromide and bands were visualized by UV transillumination.

Sequencing of PCR products
Sequencing was performed for selected specimens based on the size of their PCR product, collection site and prevalence of each species. Amplified fragments were purified by QIAquick ® (Germany) kit, and subjected to sequencing from both ends in an ABI-373 automatic sequencer by Primm Company (Italy), using the same amplification primers of 5.8 s or 28 s.

Data analysis
The sequencing signals in An. maculipennis specimens were double-checked and annotated followed by comparison with GenBank data and previously published [32,37] and unpublished sequences from the Malaria Research Group (MRG) in the Biotechnology Department at the Institut Pasteur of Iran. Because of the conserved nature of the partial sequence of 5.8 and 28 s, the alignment of sequences and construction of the phylogenetic tree were performed using only the whole ITS2 sequence in Gene Runner (version 3.05, 1994, Hastings Software Inc), Clus- Collection sites of Anopheles maculipennis complex specimens in Iran Figure 1 Collection sites of Anopheles maculipennis complex specimens in Iran. Numbers quoted in the map (1-5) corresponds to the study areas; East Azerbaijan (1), Ardebil (2), Guilan (3), Mazandran (4) and Khorassan (5) provinces, accordingly.

Results
The rDNA-ITS2 region was amplified by PCR from genomic DNA of 235 specimens from the five provinces of East Azerbaijan, Ardebil, Guilan, Mazandran and Khorassan in Iran ( Figure 1). Sequences for the ITS2 region were obtained from 28 specimens, which have been deposited in GenBank (  Figure  2. Presumptive boundaries of 5.8 s and 28 s genes were deduced from the comparison of alignments in the sequences of other mosquitoes [26,37,41]. The size of ITS2 sequences in six species ranged from 283 bp in An. maculipennis to 302 bp in An. sacharovi ( Figure 2). The ITS2 region in all six species identified molecularly in this study started with TTGACC except An. Atroparvus, which started with TTGATC. These sequences were well conserved and almost identical to those previously reported for Nearctic and Palearctic taxa of An. maculipennis complex [7,26,42]. No intra-specific variation was detected in the ITS2 sequences of either species.

Phylogenetic analysis
The presumptive nucleotide sequences of the rDNA-ITS2 region in nine Palearctic members of the An. maculipennis species complex generated in this study and by others [7,26,29,30,45,46] and also three Nearctic taxa [42] were examined for phylogenetic analysis.  [42] and in seven Palearctic members of An. maculipennis complex about 280-300 bp [26]. However, the sequence of each species from Iran and other parts of the world within this complex is highly conserved, with about 99-100% similarity.
In a previous study by Marinucci et al. [26], the phylogenetic relationships among the members of the An. maculipennis complex inferred by maximum parsimony analysis of the PAUP programme and neighbour joining and maximum likelihood analysis of the PHYLIP program. All the trees obtained were almost identical in topology although the relationships among the three species i.e. An. maculipennis, An. messeae and An. melanoon, remained unresolved. Perhaps due to the differentiation of these species from neighbouring taxa within a brief evolutionary timeframe that dispensed insufficient differences to support these individuals' lineages [26]. Recently rDNA-ITS2 sequences of three other Palearctic members of the An. maculipennis complex have been reported from Romania and Russia, namely An. daciae, An. artemievi and An. bek-lemishevi [29,30,45,46]. Kampen analysed An. beklemishevi specimens from Russia by their ITS2 ribosomal DNA sequences to amend and to specify the phylogenetic tree of the An. maculipennis species complex [30]. The results generally correspond with the data presented in the current study except that the constructed trees do not include An. persinsis, and that An. messeae and An. atroparvus are not as close as demonstrated by Kampen [30]. He showed that An. beklemishevi is in a closer relationship to the Nearctic rather than to the Palearctic sibling species, which is in concordance with the demonstration of final phylogenetic tree drawn from this study by including An. beklemishevi sequence (GenBank: AY593958) to other sequences ( Figure 4). However, An. sacharovi as a Palearctic member of An. maculipennis complex perhaps due to its common evolutionary speciation, seems to be the closest taxa to both the Nearctic species and An. beklemishevi.
The phylogenetic tree generated in the current study has separated Nearctic members of An. maculipennis complex into two distinct lineages ( Figure 3). In agreement with Marrinucci et al. [26], An. occidentalis was placed in a sister group in relation to An. hermsi-An. freeborni. An. sacharovi placed in a sister group to the remainder of the Palearctic group. The An. labranchiae-An. atroparvus clade was the sister group to An. maculipennis, An. persiensis and An. messeae. However, interestingly, the newly described member of complex, An. perciensis is closer to An. sacharovi, perhaps revealing its common evolutionary background with this species as compared to other members of An. maculipennis (Figures 3 and 4).
With regards to the presence of six members of An. maculipennis complex, further complementary ecological studies are needed in order to determine the role of each species in malaria transmission in different areas of northern Iran. To achieve this goal, some field experiments were carried out. The preliminary results of dissection of An. sacharovi (species identification confirmed by ITS2 sequence analysis) collected during the suspected hibernation period (October-March) showed that this species will go into hibernation with gonotrophic dissociation. In this case, all dissected mosquitoes have shown no dilatation proving that the female adult mosquitoes over winter as nuliparus and the last blood they took will be used for producing the fat body allowing the female to start the next generation in the beginning of next seasonal activity.

Conclusion
Beklemishev [54] listed eight major factors that determine epidemiological efficacy of malaria vectors in Russia: susceptibility of mosquitoes to Plasmodium parasites, sporozoite survival in salivary glands, female feeding behavior, absolute and relative number of mosquitoes, seasonal dynamics of mosquito densities, survival rate and infective period of mosquito females, ambient temperature and winter diapauses of adult females in a state of gonotrophic dissociation. Besides previous studies indicated that the sibling species are not equally important as vectors for malaria parasites because of their feeding preferences and their differential susceptibility to infection, as described in complex species of Anopheles culicifacies, Anopheles gambiae, Anopheles fluviatilis [10,[55][56][57][58][59]. This may be grounds for re-considering the importance of other previously identified species in this region and their role in malaria transmission.
Nowadays, the most important vectors are considered to be An. sacharovi, which is responsible for the majority of Plasmodium vivax transmissions in the Asian part of Turkey [60], and An. labranchiae, formerly was the main vector of malaria in Italy [9]. An. atroparvus is the most efficient vector in Britain [43]. An. maculipennis, An. messeae and An. sacharovi are capable of transmitting malaria; however, they exhibit different vector capacities [8,60,61]. In this regard, detection of three species of An. maculipennis complex including An. atroparvus, An. messae and An. labranchiae, as new records in northern Iran, is a case for concern because of their potential in malaria transmission and more important, the extent of their geographical distribution towards southern territory of the An. maculipennis complex.
A better understanding on the epidemiology of malaria on both sides of the Caspian Sea may be provided by applying the molecular techniques to enable the correct identification of species complexes, the detection of plasmodium composition in anopheles vectors and the status of insecticide resistance by looking at related genes. In addition, it is worth remembering that as the An. maculipennis complex is distributed over an area that covers central Iran to the Caspian Sea region and up to Sweden, an international effort is required to prevent the re-introduction of malaria to those areas where "anophelism without malaria" prevails.