This is the first time the host-seeking and reproductive response of An. stephensi to human blood group types has been undertaken. The findings of these laboratory studies suggest a strong preference in host-seeking of individuals with type B blood group with concomitant increased fecundity of mosquitoes feeding on that blood. Our results reflect those of other researchers who have found that the source of blood-fed on by mosquitoes of pest and public health importance influences fecundity and other aspects of their life cycle.
The amount and the quality of blood meal is a vital thing to female mosquitoes for their gonotrophic cycles and rate of egg production 27. However, the variation in the egg production rate and the number of females moving towards their first gonotrophic cycles is strictly linked with the amino acids and the protein contents in each blood meal 28. Djamila et al. (2016) evaluated the effects of two blood meals; the chicken and cattle blood, on the egg development rate of An. maculipennis and found fecundity were significantly lower with the chicken blood as compared with the cattle blood, while fertility is not associated with blood meals 29. In contrast, Ae. aegypti has significantly higher fecundity and fertility when fed on human blood as compared to the pig and sheep blood 30. The hatching rate of the eggs was also associated with the type and the size of blood meals 30. The fertility, fecundity, hatching rate, developmental time of the larvae of Cx. theileri is strongly associated with the source of blood meals. The fecundity of females fed on chicken blood was significantly higher than the females fed on human and cow blood 31.
We have analyzed the rate of digestion of different blood types by An. stephensi female mosquitoes by several methods; observational studies, precipitin test, and benzidine test 32–34. The visual and the tests indicated that the blood group O is the blood that could easily be digested by females in all three replications. This may be because of the chemical structure of the O blood group; it is simpler than the other blood groups in case of antigen absence.
All the anautogenous female mosquitoes required blood meals to start and complete their oogenesis or egg development mechanism 35. Most of the previous studies concluded that the proteins contained within the blood meal have a vital role in the start of oogenesis 35. However, not all the blood meals are equal in energy for all the anautogenous species of mosquitoes, and switching sources of blood meals from preferred hosts may adversely impact reproductive capacity in some mosquito species. The female Ae. aegypti fed on bovine blood starts early oogenesis than fed on human blood 8, while the fecundity of the Cx. pipiens fed on chicken blood is significantly higher than the females fed on animal blood 13. The female Anopheles mosquitoes fed on a rich liquid diet (amino acids, vitamin, carbohydrates, proteins, cholesterol, and phagostimulant) shows the oogenesis at a comparable rate to the females fed on blood 36. Several methods are used to study the egg development mechanism or oogenesis in female mosquitoes. But most important and widely used of them are a light microscope, scanning electron microscope and fluorescent confocal microscopy 37–39. In this study, we demonstrated that SEM was an effective method to study the ultrastructure of ovaries of female mosquitoes fed on different blood groups.
Experiments on females fed on ABO blood groups and control reveal the different levels of changes in the structure of reproductive organs that lead to the series of changes in cell structure. Blood type B elucidated the significantly higher changes in the cell structure and ovaries development. One of the protuberant changes in the cell structure of females fed on blood group B a large number of un-oriented microvilli in the area of oocytes (Fig. 3). The same type of microvilli was also observed in the case of females fed on blood groups O and AB but few in numbers and small in size. Results indicated that these microvilli are not uniform in length, and these are not regularly present in all females fed on different blood groups. This type of results is in line with the Brandt 40 microvilli are not uniform in length 39. There are two clusters of ovarioles identified under an SEM in the ovaries of females mosquitoes fed on blood group B, and slightly smaller clusters are detected in the case of blood groups O and AB. Females ingested the blood type A showed the contrary results; there are no or very few microvilli are identified all the females of all the replications. Sponge-like fat bodies surround these clusters of ovarioles. The results are also in line with the scanning electron microscopy results obtained by Clements 35. The total numbers of ovarioles were ranged from 120 to 650 in the case of blood group B and the 60 to 400 in the case of O and AB blood groups. There are only 20–30 ovarioles were identified in the case of females fed on blood group A. The results are also in agreement with Roth and Porter 41, who described the oogenesis in Ae. aegypti.
Most of the autogenous mosquitoes detect the volatile semiochemicals by the use of olfactory neurons present on antennae. Earlier studies indicated the EAG response of An. gambiae towards the volatile compounds of human sweat and the carboxylic acid from the cheese 42,43. About eight volatile compounds were identified from the chicken feces that provoked the electroantennogram response from the Cx. quinquefasciatus, 44. These volatiles also derived the behavior of female Cx. quinquefasciatus under control conditions. The female Ae. albopictus showed the inverse dose-dependent EAG response against certain fatty acids and alcohols derived from the human skin emanations 45.
In the case of the present study, substantial electroantennogram responses were displayed by the antennae of halted mosquitoes against known stimulants L-lactic acid, 1-octen-3-01, and isovaleric acid. After confirming the sensitivity and response of antennae and the EAG towards the human-specific known stimulants, treatment stimuli ABO blood groups were applied and demonstrated a positive response of An. stephensi to the blood group B. The results from the electroantennogram response clearly stated that the blood groups have significant effects on the behavior of female An. stephensi; the blood group A is the least attractive. The unique negative behavior was also observed when a blend of odors of human blood was released; it indicates that the females might be able to differentiate the odor of different blood groups and also alter the response accordingly. However, it will be crucial to understand the relatedness of blood type and mosquito attractiveness given there may be additional considerations, such as skin biota or other skin-associated chemicals, beyond blood group type that may influence the host-seeking behavior of mosquitoes.
Artificial blood-feeders with collagen membranes are an effective method to evaluate attraction responses of blood and the chemical compounds released by the human skin and the sweat. The chemical volatiles permeated from the collagen membrane attracted the female mosquitoes and indicated the landing behavior in choice assays. In most of the previous studies, the collagen membrane was used in artificial membrane feeders to study the hematophagous behavior of female mosquitoes 46. The blood emitted chemical volatiles that attracted the hungry female mosquitoes. Female Cx. quinquefasciatus and Ae. aegypti showed different levels of attraction towards the avian and bovine 46. Blood composition and concentration are the key drivers of this type of behavior in female mosquitoes, mainly in a wide choice. Along with the blood second, the most important driver is sweat volatiles; these volatiles affects the behavior and landing response of An. gambiae and Ae. albopictus 43,45. Both the electroantennogram and wind tunnel bioassays were conducted based on the strength of the anemotactic response of An. stephensi. In the case of the present study, both tests conducted in the olfactometer indicated that blood groups have a substantial impact on the behavior and fertility and fecundity of the female An. stephensi mosquitoes. In both, cases blood group B acted as a mosquito magnet so that the human-specific blood groups may act as an important part for the identification of autogenous female mosquitoes such as An. stephensi.
In conclusion, the present study supports the hypothesis that blood groups have effects on the fertility, fecundity, and behavior of female An. stephensi, just like An. albimanus 46. An. stephensi is a well-known vector involved in the transmission of malarial parasites (P. falciparum and P. vivax) and given blood from different vertebrates affects the behavior, fertility, and fecundity of mosquitoes, our results suggest this may be due to differences in the chemical composition and concentration of blood of vertebrate 29,46. Significant differences were observed in both the host-seeking behavior in response to differing ABO blood group types together with concomitant differences in fecundity following blood-feeding by An. stephensi in a laboratory setting. The results of this study represent an important breakthrough in the field of parasitology and malaria control. It provides clear insights about the behavior of female mosquitoes that not only may have implications for determining in those in the community most at risk of exposure to malaria parasites, and consequently possibly prioritized for anti-malarial medication. It opens potential opportunities for the development and adaptation of novel mosquito control and surveillance strategies that exploit the host-seeking behaviours demonstrated here.