To incriminate a mosquito vector in an endemic area of filariasis, it is necessary to confirm the susceptibility rate in a laboratory-bred, clean mosquito colony, which has been fed on carrier blood containing microfilariae. By using this criterion, the susceptibility test in an experimental laboratory is an efficient classical tool when suspecting the potential vector of a certain mosquito species. Nonetheless, susceptibility alone does not imply an important role in the transmission of disease in nature, whereas a refractory one can rule out the significance of a vector entirely .
Investigation on the susceptibility of eight species members of the Thai An. hyrcanus group to nocturnally subperiodic B. malayi indicated that An. peditaeniatus, An. crawfordi, An. nigerrimus, An. argyropus and An. pursati were high potential vectors. An. paraliae and An. sinensis were low potential vectors, while An. nitidus was a refractory vector. However, a crucial question regarding the susceptibility level determined in this study might be raised, due to the artificial feeding of mosquitoes on blood containing B. malayi microfilariae, which was not as natural as direct feeding on cat- and/or jird-infected B. malayi. Nevertheless, previous reports  confirmed that these two feeding techniques could be used robustly for routine screening of potential mosquito vectors of filarial parasites, since they did not differ significantly. This was despite the artificial feeding technique yielding slightly higher infective rates and parasite loads than the direct feeding method, presumably due to the effect of anticoagulant (10 units of heparin/1 ml of blood).
Among the five high potential vectors, An. peditaeniatus, An. crawfordi and An. nigerrimus were found to be abundant and widely distributed in Thailand and other countries [India (Assam, Bihar and Punjab), Sri Lanka, Bangladesh, China (Hainan Island), Myanmar, Cambodia, Vietnam, Malaysia (Malaysian Peninsular, Sabah and Sarawak), Indonesia (Java and Sumatra) and Brunei], and were proven as outdoor-biters of humans in certain localities of Thailand [24, 25]. Regarding vector competence, An. peditaeniatus and An. nigerrimus have been incriminated so far as suspected vectors of P. vivax in Thailand [26–28], as well as An. nigerrimus as a potential natural vector of W. bancrofti in Phang Nga province, southern Thailand , and An. peditaeniatus as a secondary vector of Japanese encephalitis virus in China and India [29, 30]. Beneficial results reported herein emphasize the potential role of An. peditaeniatus, An. crawfordi, An. nigerrimus, An. argyropus and An. pursati in transmitting nocturnally subperiodic B. malayi in southern Thailand as well as other countries, in which these anopheline species and filarial parasite were found sympatrically and/or co-endemic with malaria and Japanese encephalitis. The list of these potential vector-species could be used as a promising guideline for the field approach to incriminate natural vectors in endemic areas of Brugian filariasis. Remarkably, An. sinensis has been incriminated as an important vector of nocturnally periodic B. malayi in China, Korea and Japan , but in this study, it was proven as a low potential vector of nocturnally subperiodic B. malayi. It is interesting to note that the An. sinensis strain from Korea and China was compatible genetically and/or nearly identical to that from Thailand, based on the crossing experiments and comparative sequence analyses of the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2), and mitochondrial cytochrome c oxidase subunit I (COI) and subunit II (COII) . This evidence appeared to support the high specificity between B. malayi physiological races and the An. sinensis vector.
It has been known for refractoriness of certain mosquito species towards filarial parasites to occur in the forgut (cibarial and pharyngeal amartures), midgut (fast blood coagulation) or thoracic muscles (direct toxicity and melanotic encapsulation) [32–34]. Regarding refractoriness in the thoracic muscle, large numbers of B. malayi and B. pahangi microfilariae exsheathed in refractory Ae. albopictus after gaining entry into the mosquitoes, and subsequently migrated to the thoracic muscles without further development [35, 36]. The results revealed that the factor(s) in the thoracic muscles of Ae. albopictus conferred with the refractoriness. Evidence of refractoriness to B. pahangi microfilariae infection is of additional interest, as it could be induced in normally susceptible Ae. tabu by rearing female mosquitoes on sugar solution containing thoracic homogenate of refractory Ae. malayansis mosquitoes . This result agreed with a subsequent study in that the high inhibition of B. pahangi larval development could be induced in the thoracic muscle of susceptible Ae. togoi. This was performed by intrathoracic injection of crude thoracic homogenate (CTH) from refractory Ae. albopictus into susceptible Ae. togoi prior to feeding on blood containing B. pahangi microfilariae . Thus, these two pieces of evidence seem to reflect the inhibitory effect that might be due to direct toxicity of the homogenate on developing larvae. Furthermore, the melanization of immune responses in various insects against a wide-range of invading pathogens and parasites has been documented [34, 38–40]. The immune response of mosquitoes is put into effect through the plasma components of both the hemolymph, i.e., the humoral response, and hemocytes, the cellular response . The authors also suggested that the intracellular melanotic encapsulation of filarial developing stages, as observed in specific mosquito organs, may be caused by exposure to low-molecular-weight immune molecules, which are carried in the hemolymph (plasma) and can penetrate the basement membrane covering the cells of specific organs. This concept suggested that the same mechanisms controlling melanotic encapsulation reactions (immune response) extracellularly in the hemocoel also control them intracellularly in specific organs of the host in which the parasite develops. Subsequent evidence from using RNAi methodology to knock-down PAH (phenylalanine hydroxylase) expression in the mosquitoes, Ae. aegypti and Armigeres subalbatus, demonstrated that limitation in the amount of tyrosine, available for tyrosinase-mediated hydroxylation, significantly reduces the effectiveness of melanization reactions against inoculated filarial parasites . Additionally, at least four specific enzymes [DCE (dopachrome conversion enzyme), DDC (dopa decarboxylase), PO (phenoloxidase) and TH (tyrosine hydroxylase)] were concerned in the biosynthesis of melanin . Current studies on the possible factors affecting the difference in susceptibility levels of eight An. hyrcanus species to nocturnally subperiodic B. malayi revealed that at least two refractory mechanisms (direct toxicity and/or melanotic encapsulation) were involved in the refractoriness of thoracic muscles for parasite development. Variations in the percentages of melanotic encapsulation and degenerated L1 larvae recovered in the thoracic muscles of Ae. togoi (0% and 6.85%), An. peditaeniatus (0% and 3.66%), An. crawfordi (0% and 2.67%), An. paraliae (47.19% and 29.21%), An. sinensis (32.05% and 52.57%) and An. nitidus (94.44% and 5.56%), were good supportive evidence.