Anopheles stephensi is a competent malaria vector in Iran, Afghanistan, China, the Middle-East, the Far-East and Indian subcontinent [2]. Besides, it has also been implicated recently in resurgence of urban malaria in Africa [9, 23] but with no details about species complex. Based on malaria transmission competence of three biological forms of An. stephensi, the Type form is reported as main malaria vector in urban areas of Indian subcontinent [24]. On the other hand, many reports have mentioned all these three forms as efficient vectors for malaria transmission in Iran [6,16]. Despite of efficient controlling strategies for malaria, this mosquito is increasing its geographic range. This may be attributed to the lack of precise recognition of vector Anopheles species complexes which is crucial in malaria surveillance, control, and eradication strategies [16]. Earlier studies used different methods to identify these complex forms of An. stephensi for example, counting ridges number on the eggs [18], cuticle hydrocarbons [25], chromosome karyotypes [3,26,27], ITS2, D3 loci, CO1 and COII markers [28]. But then again, the inconsistent range of ridge numbers, identical nucleotide sequences of ITS2 and D3 loci in type and mysorensis, and the identity of COII sequences between Indian type form and Iranian mysorensis suggest these markers are not suitable for the identification of biological forms of An. stepehnsi [24,28,29,30,31]. Our analysis also supports these results since we were unable to recognize (the biological form) our lab strain using COI, COII and ITS2. Taking together, our observations suggest these markers may be efficient in determining the inter-species (anopheles) differences but intra-species variations (sibling species of An. stephensi) can only be recognized with more specified markers i.e. OBP1. These markers will be crucial to help develop appropriate and reliable molecular keys (for sibling species identification) complementary to morphological keys [24]. Whereas morphology based identification may be an error prone (in case of overlapping ridges number in the intermediate biological form) process in a close resembled sibling species like An. stephensi. It was recently shown that Ansteobp1 intron I sequence could be used to solve this dilemma [16]. In a previous study, intron I sequences of OBP1 successfully distinguished the three biological forms despite 100% similarity in amino acid level [15]. Here, the morphological analysis of mosquito eggs with ridges number 12-13/egg corresponds to mysorensis form of An. stephensi which is in agreement with [3,18] who documented ridges number 10–15 for mysorensis. Likewise, phylogenetic analysis (Fig. 8) showed 100% similarity with mysorensis biological form (sibling C) reported from Iran and Afghanistan, the neighboring countries of China.
The COI and COII, ITS2 and D3 based analysis from Iran and India have revealed extensive gene flow among these variants [30,31] whereas other genetic studies (using microsatellite markers) have demonstrated a significant genetic differentiation and non-significant gene flow among them with type and intermediate being more closely related genetically [32]. These biotypes with taxonomic identities and differential vector competencies may contribute to the current epidemiological situations [24]. For example, invasion of An. stephensi has attributed significantly in the recent malaria outbreaks in non-endemic areas of India i.e. Kerala state and Lakshadweep islands [33]. Similarly, this species invaded Sri Lanka (from India via water carrying eggs in boats) through the narrow Palk Strait (southward) [33,34]. Here, the sequence analysis for COI, COII and ITS2 indicated our species 100%, 99.46% and 99.29% similar to other Chinese, Indian and Iranian strains of An. stephensi (Figs. 5, 6 and 7). In other words, it can be inferred that our lab strain (China) COI sequences as An. stephensi sibling C could be used to comparison of other An. stephensi COI sequences (Figs. 5 and 6).
Studies from adjoining countries of China i.e. Afghanistan, Pakistan, Saudi Arabia, Myanmar, Ethiopia and Iraq reported this variant (sibling C) of An. stephensi from rural areas, although it is an efficient vector (malaria) all over India and Iran [6,16,35]. As a precedent, the incursion of An. stephensi from India to Sri Lanka (sharing border), and within India to new areas, we are afraid of its invasion to China (already having An. sinensis, An. peditaeniatus and An. minimus (s.l.) as malaria vectors) in a similar trend. Recently, many studies have reported thousands of imported malaria cases in China (Hubei and Zhejiang Provinces, Tengchong, Yunnan, China-Myanmar and Tibet, China-India) [36]. Myanmar, where An. stephensi is the major malaria vector, may play a significant role in sharing this species to the contiguous parts of China. These scenarios are likely to impede the goal of malaria eradication from China. It is exciting to mention that earlier only two malaria parasites (Plasmodium falciparum and P. vivax) were prevailing but after control and elimination operations all the four parasites (P. falciparum, P. vivax, P. malariae, and P. ovale) are reported from China [37,38]. Similarly, the geographic distribution range/proportion of these vectors has also expanded/increased [36,37,38,39]. Therefore, we recommend further sustained entomological surveillance with precise species complexes before and after malaria elimination certification.