Efficiency of mitochondrial genes and nuclear Alu elements in detecting human DNA in blood meals of Anopheles stephensi mosquitoes: a time-course study
Parasites & Vectors volume 16, Article number: 284 (2023)
The time required for PCR detection of DNA in human blood meals in vector mosquitoes may vary, depending on the molecular markers used, based on the size and copy number of the amplicons. Detailed knowledge of the blood-feeding behavior of mosquito populations in nature is an essential component for evaluating their vectorial capacity and for assessing the roles of individual vertebrates as potential hosts involved in the transmission of vector-borne diseases.
Laboratory experiments were conducted to compare the time course of PCR detection of DNA in human blood meals from individual blood-fed Anopheles stephensi mosquitoes, using loci with different characteristics, including two mitochondrial DNA (mtDNA) genes, cytB (228 bp) and 16S ribosomal RNA (rRNA) (157 bp) and nuclear Alu-repeat elements (226 bp) at different time points after the blood meal.
Human DNA was detectable up to 84–120 h post-blood-feeding, depending on the length and copy number of the loci. Our results suggest that 16S rRNA and Alu-repeat markers can be successfully recovered from human DNA up to 5 days post-blood-meal. The 16S rDNA and Alu-repeat loci have a significantly (P = 0.008) slower decline rate than the cytB locus. Median detection periods (T50) for the amplicons were 117, 113 and 86.4 h for Alu-repeat, 16S rDNA and cytB, respectively, suggesting an inverse linear relationship between amplicon size/copy number and digestion time.
This comparative study shows that the Alu-repeat locus is the most efficient marker for time-course identification of human DNA from blood meals in female mosquitoes. It is also a promising tool for determining the anthropophilic index (AI) or human blood index (HBI), i.e. the proportion of blood meals from humans, which is often reported as a relative measure of anthropophagy of different mosquito vectors, and hence a measure of the vector competence of mosquito species collected in the field.
Insect-borne diseases are major global health problems, causing hundreds of thousands of deaths per year , and various mosquito species are among the most important vectors of disease, being responsible for the highest transmission of human and animal parasites and pathogens. Globally widespread, mosquito-transmitted diseases include malaria, lymphatic filariasis, dengue, chikungunya, Rift Valley fever, Japanese encephalitis, West Nile virus and Usutu virus . Hematophagy is present in most females of all mosquito species: they require a blood meal to produce mature eggs . The extent of vector contact with an infected host or reservoir is an important factor in the transmission of mosquito-borne diseases.
Different mosquito species have wide and variable host preferences, the most important of which include humans, cattle and other mammals, but also include birds and reptiles, amphibians, worms, leeches and fish . The host preference of each mosquito species shows an innate and specific pattern and may also be influenced by factors such as environmental conditions and host characteristics . By detecting the source of blood in blood-feeding mosquitoes, important information can be obtained, such as the degree of contact between vector and host populations and the insect’s host preferences under natural conditions. The anthropophagic index (percentage feeding on humans) is used as component of measuring vectorial capacity for human diseases, while knowledge of other hosts can provide a measure of the relative importance of animal reservoirs of vector-borne zoonotic or enzootic infections . Thus, by knowing the feeding patterns of mosquitoes on a particular host, it is possible to understand their life history, as well as the effect of host selection on their survival and reproduction and the ecology of diseases transmitted by mosquitoes. These data are also vital for operations associated with entomological surveillance and vector-monitoring, especially in the field of diseases whose etiology has an environmental component (environmental disorders) .
Various methods are available for determining the source of a mosquito blood meal. Previously, arthropod blood-feeding was detected by serological assays, such as the enzyme-linked immunosorbent (ELISA) method and the precipitin test [5,6,7]. While these assays are still used, they are insufficiently specific when attempting to identify the sources of a mosquito blood meal and often limited to determination of the order and family of the mosquitoes' host under study. New applications of PCR-based methods and the increasing amount of vertebrate DNA sequence data available in the public domain have allowed an increase in the specificity of blood-meal recognition down to the species or individual host level.
The efficacy and the success of PCR amplification are affected by several factors, including time since blood meal, PCR amplicon size, locus sequence/gene copy number, DNA extraction procedure, preservation method, storage time and blood-meal size [8,9,10,11,12,13]. Among these factors, longer time, increased amplicon size and low gene/locus copy number have inverse effects on the success of the PCR, as has been demonstrated extensively in studies designed to identify blood meals in hematophagous arthropods [14, 15].
Different DNA-based molecular markers have been used to identify ingested blood in arthropods. Mitochondrial genes (mtDNA) have been used extensively as reliable common markers to identify the source of the blood meal ingested by arthropods [16,17,18,19,20,21]. Consequently, mtDNA has been used widely in entomological, forensic and genetic studies because of their valuable features, such as lack of recombination, presence of very high copy numbers in each cell, maternal inheritance, absence of introns, existence of single-copy orthologous genes and high mutation rate [22,23,24,25]. Among the mtDNAs, cytochrome b (cytB) and 16S ribosomal RNA (rRNA) are the most applicable and are therefore appropriate for use in arthropod blood-meal identification in terms of determining the host species in phylogenetic and biodiversity studies and in identifying human individuals in forensic investigations [26,27,28,29,30,31,32,33,34].
Single-copy nuclear genes can also be used to identify the source of an arthropod blood meal . However, when working with such a small amount of starting material, the amplification of target DNA can be more challenging with single-copy genes, thus a preferred approach is the use of Alu elements, which are transposable elements (TEs) that exist only in primates . Alu repeats consist of short interspersed nuclear elements (SINEs) that replicate through LINE (long interspersed nuclear element)-mediated reverse transcription of an RNA polymerase III transcript . They are the most abundant individual feature in the human genome, forming 10% of the human genome mass, with over one million copies per genome; each Alu element is approximately 280 bp long and always comes after a poly(A) tail of varying length. Although Alu repeats are not known to have a specific biological function, they have been extensively studied due to their many branches and copies in the human genome [37, 38]. There have been many reports on the use of Alu-based PCR amplification as a very sensitive and powerful tool for human genomic DNA identification and quantitation in forensic laboratories .
The objectives of this study were to compare the effects of elapsed time, copy number and amplicon size on the effective use of three different loci (mitochondrial cytB and 16S rRNA genes, and nuclear Alu-repeat elements) in PCR assays for tracking human DNA in blood meals of the Anopheles stephensi mosquito. Anopheles stephensi was selected for study is one of the most important malaria vectors in Asian countries and, currently, in the Horn of Africa.
Anopheles stephensi mosquitoes were maintained at 28 °C ± 2 °C, 60 ± 10% humidity, under a 12:12-h light:dark photoperiod, and were fed only on a 10% sucrose solution before the experiments. The colony of An. stephensi used in this study was maintained in the insectarium at the School of Public Health, Tehran University of Medical Sciences. Eggs were hatched in about 1 l of tap water that was continuously supplemented with a few flakes of fish food until the larvae pupated. Pupae were collected and separated according to age. Adult mosquitoes aged 5–7 days were blood-fed, either artificially on membrane blood feeders or directly on male or female human volunteer hands and forearms in a cage under laboratory conditions .
The exclusively human blood-fed mosquitoes were transferred to a cage supplied with 10% sucrose solution to reduce mortality. Individual mosquitoes were randomly chosen, anesthetized and killed by freezing at 0, 6, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120 and 132 h post-feeding. Knockdown and killing times of < 10 s was achieved for each specimen. Starved male mosquitoes and ddH2O were used as negative controls. Specimens were stored dry in micro-tubes at − 20 °C. Prior to the experiment on PCR success rate, the head and legs were removed from each dead mosquito to reduce potential PCR inhibitors [41, 42] and to avoid high concentrations of nontarget DNA, respectively.
DNA extraction and quantification
Genomic DNA was extracted using the protocol of the Vivantis GF-1 nucleic acid extraction kit (Vivantis Technologies, Singapore). The final elution was carried out twice in the same 20 μl of elution buffer to maximize the amount of recovered DNA. Positive controls were 0.1–3 μl human blood samples, roughly equal to the minimum and maximum amount, respectively, of a blood meal in a mosquito′s midgut . These samples were taken with a lancet from a female and male donor separately and poured directly into a 1.5-ml micro-tube. Using the Vivantis GF-1 extraction kit, DNA was extracted from the male mosquito (negative controls), the human blood (positive controls) and 10 individual female mosquitoes from each post-feeding time point. DNA was quantified on a Nano-Drop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
Primers and PCR reactions
Three human-specific primer pairs (Table 1) for the cytB, 16S rRNA and Alu-repeat loci were used to confirm the absence of human DNA from ddH2O and male mosquitoes (the negative controls) and the presence of human DNA in the fed female mosquitoes and in the blood stains (positive controls). Various volumes (0.1, 1.0 or 2.0 μl) of DNA from human blood stains, as positive controls, were used to test the sensitivity and reproducibility of the three loci amplification. To test possible inhibitory substances (exocuticle and thorax) that might affect the PCR amplification, a serial dilution of human blood was performed in volumes of 0.1, 1.0, and 2.0 μl, plus a male mosquito in each sample.
PCR amplifications for all genes or loci were performed according to the protocol for Taq DNA Polymerase Master Mix (2×) (Ampliqon A/S, Odense M, Denmark). The PCR reaction was carried out in a 25-μl reaction volume consisting of 12.5 µl of Master Mix, 1 µl of each primer and 10 μl of template or ddH2O. The Master Mix is a premixed solution containing HotStarTaq DNA polymerase, PCR buffer and deoxynucleotide triphosphates (dNTPs), with a final concentration of 2 mM MgCl2 and 200 mM of each dNTP. Details of the thermal cycling of each protocol are listed in Table 1. All amplicons were visualized under ultraviolet light using a UV-transilluminator after electrophoresis at 85 V in a 3% agarose gel and staining with Safe stain. Negative and positive controls were included in each PCR.
The success rates of the PCR assay for each locus were subjected to regression analysis, and the median detection period (T50)  was designed for each amplicon using the regression equation y = mx + c, where x = time and m and c are constants. T50 is defined as the point at which 50% of amplification is expected to be successful, as predicted by the regression line. In this experiment we calculated regression lines only for critical time points, which included the last two time points with 100% success amplification, followed by the time points with < 100% success up to the lack of amplicons (0%). Regression lines were subsequently subjected to comparison by analysis of covariance (ANCOVA; general linear model [GLM]) using SPSS version 26 software package (SPSS IBM Corp., Armonk, NY, USA) to compare the rate of waning of the slopes of the three amplicons. To control the experiment, a subset of PCR products from each locus was randomly sequenced by the same forward and reverse primers used for the PCR and their identity was then determined by BLASTn searches against the GenBank nucleic acid sequence database (http://www.ncbi.nlm.nih.gov/BLAST/).
The sensitivity and reproducibility tests of the PCR for the three loci showed that all of the DNA samples (0.1, 1.0 and 2.0 µl) were positive for the expected bands of 226, 228 and 157 bp for the Alu-repeat, cytB and 16S rRNA, respectively, validating the DNA extraction process and showing the sensitivity and reproducibility of the amplification of the three loci using the minimum and maximum blood volumes (0.1 and 2.0 µl, respectively; Additional file 1: Fig. S1). No PCR product was obtained with the negative controls. A test was performed for the presence of potential PCR inhibitors in the mosquito DNA and for the frequency of false-negative PCR reactions in DNA from blood-fed mosquitoes. All the samples (a mix of DNA from human blood and a decapitated and amputated male mosquito) produced bands of the expected sizes corresponding to the three loci, confirming that any possible PCR inhibitors had been removed (Additional file 2: Table S1). In addition, BLASTn searches against the GenBank nucleic acid sequence database showed that the blood meals identified from mosquitoes were identical (> 99%) to human sequences.
The results of this experiment showed that the success rate of the amplification of human DNA decreased with increasing elapsed time post-feeding. Specifically, human DNA could be traced in the blood meals of An. stephensi using the cytB gene for up to 84 h post-feeding; the 16S rRNA locus could be used up to 120 h post-feeding; and the Alu-repeats in human DNA could be detected up to 120 h post-feeding (Figs. 1, 2, 3). The maximum period of detection was similar (120 h) for the shortest amplicon (16S rRNA, 157 bp) and the amplicon with the highest copy number (Alu-repeat, 226 bp).
The success of PCR amplification was 100% for up to 84-, 84- and 72-h post-feeding for 16S rRNA, the Alu-repeat and the cytB gene, respectively. The time points at which 50% of the PCR amplification is expected to be successful (T50), as calculated by the regression lines for the critical time points, were 117 h for the Alu-repeat (226 bp), 113 h for the 16S rRNA (157 bp) and 86.4 h for the cytB (228 bp) amplicons (Fig. 4). A comparison between the regressions for the two mitochondrial amplicons (cytB and 16S rRNA), which are assumed to have the same copy number in the cells, showed the PCR success rate of the smaller amplicon (16S rRNA) to be significantly higher (GLM, P = 0.008, F = 11.497, R2 = 82.5%). A comparison between the regressions for the cytB and Alu-repeat amplicons showed that the PCR success rate of the higher copy-number amplicon (Alu-repeat) was also significantly higher than that for the cytB amplicon (GLM, P = 0.008, F = 11.687, R2 = 79.5%). However, the comparison between the regressions for the 16S rRNA and Alu-repeat amplicons showed that the PCR success rate of the higher copy-number amplicon (Alu-repeat) was only slightly higher than that of the 16S rRNA amplicon, with the difference lacking statistical significance (GLM, P = 0.638, F = 0.236, R2 = 79.7%). The logistical regression found that the probability of obtaining successful amplification for cytB, 16S rRNA and Alu-repeat loci was, respectively, 30%, 18.3% and 17.4% less for every 12-h increase in post-feeding interval at their critical time points.
Here we propose a method for identifying human DNA in the blood meal of An. stephensi mosquitoes that involves amplification of the human-specific regions of cytB, 16S rRNA and Alu repeats, without the need to sequence the PCR products. We show that the success of the amplification over time is mainly dependent on amplicon size and locus copy number, with smaller amplicons (16S rRNA) and higher copy number loci (Alu-repeats) being detectable for longer periods of time after blood-feeding.
The PCR amplification of either mitochondrial 16S rRNA and or nuclear Alu-repeats in this study proved to be very efficient for identifying the origin of mosquito blood meals. We were able to trace human DNA in An. stephensi mosquito blood meals up to 120 h post-feeding. The use of this mitochondrial marker thus has the potential to be extremely useful for use in species determination and in individual human identification, for epidemiological entomology studies and also for forensic purposes [25, 46, 47]. In addition, the Alu repeats are a naturally amplified source of human genetic information and robust tools for sensitive human DNA detection and quantitation, as has previously been demonstrated in a forensic study .
In the present study, we demonstrated that human DNA can potentially be amplified by PCR, even when it has been degraded by digestion in the mosquito gut, because human DNA was detectable in An. stephensi for up to 84–120 h post-feeding, depending upon the locus used. The stage of blood digestion progressed from the fully fed period (fresh) to the semi-gravid period and then to fully gravid period within the first 2–3 days (48–72 h) post-feeding under the laboratory conditions (28 °C ± 2 °C), and visual validation of blood in the mosquito abdomen was also possible during the fresh and semi-gravid periods. However, our results show that the source of the blood meal could be identified from DNA, even when the blood-meal residue in the abdomen was no longer distinguishable by eye. This result is consistent with the findings reported by Replogle et al. , who showed that it was possible to successfully genotype individual human blood donors using mtDNA obtained from excrement of the pubic louse (Pthirus pubis L.) (Phthiraptera, Pthiridae), even when no DNA from the blood meals remained in the gut, thus demonstrating the potential of the method for the amplification of DNA degraded by digestion.
We showed here that short amplicons with high copy number can be recovered from a mosquito blood meal up to 120 h (5 days) post-feeding. This is consistent with a short amplicon (103–118 bp) assay for the identification of sand fly blood meal, which could detect various small amounts of the host DNA up to 120 h after blood-feeding . However, the use of longer amplicons generally permits detection of host DNA for 1–4 days post-feeding [8, 35, 50,51,52,53,54]. In forensic studies, short amplicons of short tandem repeat (STR) alleles (normally < 200 bp) are used to identify an individual from blood or tissue isolated from insects [37, 38]. However, the use of short STR amplicons has only been shown to allow DNA recovery for 15–88 h post-blood-meal [50, 55]. Our data suggests that exploring the use of shorter STR alleles with higher copy numbers could increase the recovery time of human DNA from mosquitoes for criminal investigation.
The results of this study demonstrate that longer amplicons are more subject to degradation than short amplicons (Fig. 4). In the An. stephensi blood meal, the DNA degradation rate of the short 16S rRNA amplicon is comparable with that of the longer, high copy number Alu-repeat, suggesting that the high copy number of the Alu-repeat compensates for its longer amplicon size.
In addition to the time post-blood meal, the locus copy number and the amplicon size, other factors may affect the success of blood-meal source identification using PCR. These include the PCR-inhibitory substances present in insect tissue, especially those in the cuticle, head and thorax [56, 57], as well as those in blood, such as heme . In this study we used a combination of male mosquitoes and human blood to test the inhibitory effects of these materials on our PCR reactions; it is evident from the absence of false negatives that the PCR reactions were not hindered by those materials present in the insects or the blood. It appears that across the time course of the experiment, the combined activities of digestive enzymes and nucleases play the main roles in degrading the ingested human blood, so that human DNA could not be obtained after 5 days post-blood meal.
We describe here an assay with greatly enhanced sensitivity and specificity for identifying the source of the blood meals of hematophagous mosquitoes, which allows detection of human DNA in a single round of PCR. We demonstrate for the first time that the use of 16S rRNA and/or Alu-repeat elements permits the identification of human blood sources from An. stephensi mosquitoes up to 5 days post-blood meal. It is evident that selecting short amplicons and high copy number loci of the human host will increase the longer-term success rate of PCR. However, since DNA degradation over time is a concern, we recommend exploring combinations of high copy number loci and shorter amplicons to maximize the recovery of human DNA from hematophagous insects for longer times post-blood meal.
Availability of data and materials
The datasets supporting the findings of this article are included within the article.
WHO. World malaria report 2022. 2022. https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2022. Accessed 27 March 2023
Takken W, Verhulst NO. Host preferences of blood-feeding mosquitoes. Annu Rev Entomol. 2013;58:433–53.
Santos CS, Pie MR, da Rocha TC, Navarro-Silva MA. Molecular identification of blood meals in mosquitoes (Diptera, Culicidae) in urban and forested habitats in southern Brazil. PLoS ONE. 2019;14:e0212517.
Boakye DA, Tang J, Truc P, Merriweather A, Unnasch TR. Identification of blood-meals in haematophagous Diptera by cytochrome B heteroduplex analysis. Med Vet Entomol. 1999;13:282–7.
Kent RJ. Molecular methods for arthropod blood-meal identification and applications to ecological and vector-borne disease studies. Mol Ecol Res. 2009;9:4–18.
Baum M, Ribeiro MC, Lorosa ES, Damasio GA, Castro EA. Eclectic feeding behavior of Lutzomyia (Nyssomyia) intermedia (Diptera, Psychodidae, Phlebotominae) in the transmission area of American cutaneous leishmaniasis, state of Paraná. Rev Soc Bras Med Trop. 2013;46:560–5.
Afonso MMS, Miranda Chaves SA, Rangel EF. Evaluation of feeding habits of haematophagous insects, with emphasis on Phlebotominae (Diptera: Psychodidae), vectors of Leishmaniasis-review. Trends Entomol. 2012;8:125–36.
Oshaghi MA, Chavshin AR, Vatandoost H, Yaaghoobi F, Mohtarami F, Noorjah N. Effects of post-ingestion and physical conditions on PCR amplification of host blood meal DNA in mosquitoes. Exp Parasitol. 2006;112:232–6.
Lim L, Ab Majid AH. Human profiling from STR and SNP analysis of tropical bed bug, Cimex hemipterus, for forensic science. Sci Rep. 2023;13:1506.
Mukabana WR, Taken W, Seda P, Kileen GF, Hawley WA, Knols BJG. Extent of digestion affects the success of amplifying human DNA from blood-meals of Anopheles gambiae (Diptera: Culicidae). Bull Entomol Res. 2002;92:233–9.
Davey JS, Casey CS, Burgess IF, Cable J. DNA detection rates of host mtDNA in blood-meals of human body lice (Pediculus humanus L., 1758). Med Vet Entomol. 2007;21:293–6.
Bellekom B, Bailey A, England M, Langlands Z, Lewis OT, Hackett TD. Effects of storage conditions and digestion time on DNA amplification of biting midge (Culicoides) blood meals. Parasit Vectors. 2023;16:13.
Martínez-de la Puente J, Ruiz S, Soriguer R, Figuerola J. Effect of blood meal digestion and DNA extraction protocol on the success of blood meal source determination in the malaria vector Anopheles atroparvus. Malar J. 2013;12:109.
Adler PH, McCreadie JW. Black flies (Simuliidae). In: Mullen GR, Durden LA, editors. Medical and veterinary entomology. 3rd Edition. San Diego: Elsevier; 2019. p. 237–59.
Reeves LE, Holderman CJ, Gillett-Kaufman JL, Kawahara AY, Kaufman PE. Maintenance of host DNA integrity in field-preserved mosquito (Diptera: Culicidae) blood meals for identification by DNA barcoding. Parasit Vectors. 2016;9:503.
Gebresilassie A, Abbasi I, Aklilu E, Yared S, Kirstein OD, Moncaz A, et al. Host-feeding preference of Phlebotomus orientalis (Diptera: Psychodidae) in an endemic focus of visceral leishmaniasis in northern Ethiopia. Parasit Vectors. 2015;8:270.
Maleki-Ravasan N, Oshaghi M, Javadian E, Rassi Y, Sadraei J, Mohtarami F. Blood Meal Identification in field-captured sand flies: comparison of PCR-RFLP and ELISA assays. Iran J Arthropod Borne Dis. 2009;3:8–18.
Steuber S, Abdel-Rady A, Clausen PH. PCR-RFLP analysis: a promising technique for host species identification of blood meals from tsetse flies (Diptera: Glossinidae). Parasitol Res. 2005;97:247–54.
Oshaghi MA, Chavshin AR, Vatandoost H. Analysis of mosquito blood-meals using RFLP markers. Exp Parasitol. 2006;114:259–64.
Khanzadeh F, Khaghaninia S, Maleki-Ravasan N, Koosha M, Oshaghi MA. Molecular detection of Dirofilaria spp. and host blood-meal identification in the Simulium turgaicum complex (Diptera: Simuliidae) in the Aras River Basin, northwestern Iran. Parasit Vectors. 2020;13:548.
Apperson CS, Hassan HK, Harrison BA, Savage HM, Aspen SE, Farajollahi AR, et al. Host feeding patterns of established and potential mosquito vectors of West Nile virus in the eastern United States. Vector Borne Zoonotic Dis. 2004;4:71–82.
Castro JA, Picornell A, Ramon M. Mitochondrial DNA: a tool for populational genetics studies. Int Microbiol. 1998;1:327–32.
Oshaghi MA. mtDNA inheritance in the mosquitoes of Anopheles stephensi. Mitochondrion. 2005;5:266–71.
Yan L, She Y, Elzo MA, Zhang C, Fang X, Chen H. Exploring genetic diversity and phylogenic relationships of Chinese cattle using gene mtDNA 16S rRNA. Arch Anim Breed. 2019;62:325.
Al-Nayili HJ, Al-Dahmoshi HO. Mitochondrial 16S rRNA gene-dependent blood typing as a forensic tool. Indian J Forens Med Toxicol. 2020;14:1594–602.
Kocher TD, Thomas WK, Meyer A, Edwards SV, Pääbo S, Villablanca FX, et al. Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc Natl Acad Sci USA. 1989;86:6196–200.
Irwin DM, Kocher TD, Wilson AC. Evolution of the cytochrome b gene of mammals. J Mol Evol. 1991;32:128–44.
Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. Molecular biology of the cell (third edition). New York & London: Garland Publishing.
Parson W, Pegoraro K, Niederstätter H, Föger M, Steinlechner M. Species identification by means of the cytochrome b gene. Int J Legal Med. 2000;114:23–8.
Hsieh HM, Chiang HL, Tsai LC, Lai SY, Huang NE, Linacre A, et al. Cytochrome b gene for species identification of the conservation animals. Forensic Sci Int. 2001;122:7–18.
Ouso DO, Otiende MY, Jeneby MM, Oundo JW, Bargul JL, Miller SE, et al. Three-gene PCR and high-resolution melting analysis for differentiating vertebrate species mitochondrial DNA for biodiversity research and complementing forensic surveillance. Sci Rep. 2020;10:4741.
Logue K, Keven JB, Cannon MV, Reimer L, Siba P, Walker ED, et al. Unbiased characterization of anopheles mosquito blood meals by targeted high-throughput sequencing. PLoS Negl Trop Dis. 2016;10:e0004512.
Roellig DM, Gomez-Puerta LA, Mead DG, Pinto J, Ancca-Juarez J, Calderon M, et al. Chagas disease workgroup in Arequipa. Hemi-nested PCR and RFLP methodologies for identifying blood meals of the Chagas disease vector Triatoma infestans. PLoS One. 2013;11:e74713.
Petersen FT, Meier R, Kutty SN, Wiegmann BM. The phylogeny and evolution of host choice in the Hippoboscoidea (Diptera) as reconstructed using four molecular markers. Mol Phylogenet Evol. 2007;45:111–22.
Talebzadeh F, Raoofian R, Ghadipasha M, Moosa-Kazemi SH, Akbarzadeh K, Oshaghi MA. Sex-typing of ingested human blood meal in Anopheles stephensi mosquito based on the amelogenin gene. Exp Parasitol. 2023;248:108517.
Batzer MA, Deininger PL. Alu repeats and human genomic diversity. Nat Rev Genet. 2002;3:370–9.
Price AL, Eskin E, Pevzner PA. Whole-genome analysis of Alu repeat elements reveals complex evolutionary history. Genome Res. 2004;14:2245–52.
Schmid CW. Alu: a parasite’s parasite? Nat Genet. 2003;35:15–6.
Walker JA, Kilroy GE, Xing J, Shewale J, Sinha SK, Batzer MA. Human DNA quantitation using Alu element-based polymerase chain reaction. Anal Biochem. 2003;315:122–8.
Anjomruz M, Oshaghi MA, Pourfatollah AA, Sedaghat MM, Raeisi A, Vatandoost H, et al. Preferential feeding success of laboratory reared Anopheles stephensi mosquitoes according to ABO blood group status. Acta Trop. 2014;140:118–23.
Beckmann JF, Fallon AM. Decapitation improves detection of Wolbachia pipientis (Rickettsiales: Anaplasmataceae) in Culex pipiens (Diptera: Culicidae) mosquitoes by the polymerase chain reaction. J Med Entomol. 2012;49:1103–8.
Boncristiani H, Li J, Evans J, Pettis J, Chen Y. Scientific note on PCR inhibitors in the compound eyes of honey bees Apis mellifera. Apidologie. 2011;42:457–60.
Graumans W, Heutink R, van Gemert GJ, van de Vegte-Bolmer M, Bousema T, Collins KA. A mosquito feeding assay to examine Plasmodium transmission to mosquitoes using small blood volumes in 3D printed nano-feeders. Parasit Vectors. 2020;13:401.
Hoogendoorn M, Heimpel GE. PCR-based gut content analysis of insect predators: using ribosomal ITS-1 fragments from prey to estimate predation frequency. Mol Ecol. 2001;10:2059-67.
Chang MC, Teng HJ, Chen CF, Chen YC, Jeng CR. The resting sites and blood-meal sources of Anopheles minimus in Taiwan. Malar J. 2008;7:1–8.
Caenazzo L, Ceola F, Ponzano E, Novelli E. Human identification analysis to forensic purposes with two mitochondrial markers in polyacrylamide mini gel. Forens Sci Int Genet Suppl Ser. 2008;1:266–8.
Guha S, Kashyap VK. Development of novel heminested PCR assays based on mitochondrial 16s rRNA gene for identification of seven pecora species. BMC Genet. 2005;6:1–7.
Replogle J, Lord WD, Budowle B, Meinking TL, Taplin D. Identification of host DNA by amplified fragment length polymorphism analysis: preliminary analysis of human crab louse (Anoplura: Pediculidae) excreta. J Med Entomol. 1994;31:686–90.
Sales KG, Costa PL, de Morais RC, Otranto D, Brandão-Filho SP, Cavalcanti Mde P, et al. Identification of phlebotomine sand fly blood meals by real-time PCR. Parasit Vectors. 2015;8:230.
Curic G, Hercog R, Vrselja Z, Wagner J. Identification of person and quantification of human DNA recovered from mosquitoes (Culicidae). Forens Sci Int Genet. 2014;8:109–12.
Kent RJ, Norris DE. Identification of mammalian blood meals in mosquitoes by a multiplexed polymerase chain reaction targeting cytochrome b. Am J Trop Med Hyg. 2005;73:336–42.
Haouas N, Pesson B, Boudabous R, Dedet JP, Babba H, Ravel C. Development of a molecular tool for an identification of Leishmania reservoir host by blood meal analysis in the vectors. Am J Trop Med Hyg. 2007;77:1054–9.
Abbasi I, Cunio R, Warburg A. Identification of blood-meals imbibed by phlebotomine sand flies using cytochrome b PCR and reverse line blotting. Vector Borne Zoonotic Dis. 2009;9:79–86.
Garlapati RB, Abbasi I, Warburg A, Poché D, Poché R. Identification of blood-meals in wild caught blood fed Phlebotomus argentipes (Diptera: Psychodidae) using cytochrome b PCR and reverse line blotting in Bihar. India J Med Entomol. 2012;49:515–21.
Coulson RM, Curtis CF, Ready PD, Hill N, Smith DF. Amplification and analysis of human DNA present in mosquito bloodmeals. Med Vet Entomol. 1990;4:357–66.
Beckmann JF, Fallon AM. Decapitation improves detection of Wolbachia pipientis (Rickettsiales: Anaplasmataceae) in Culex pipiens (Diptera: Culicidae) mosquitoes by the polymerase chain reaction. J Med Entomol. 2012;49:1103–8.
Lévy J, Hancock J, Ravindran A, Gross D, Tamborindeguy C, Pierson E. Methods for rapid and effective PCR-based detection of 'Candidatus Liberibacter solanacearum’ from the insect vector Bactericera cockerelli: streamlining the DNA extraction/purification process. J Econ Entomol. 2013;106:1440–5.
The manuscript was edited for English language by the ICGEB Editing service (email: email@example.com).
Research was supported by the Tehran University of Medical Sciences, Iran (Grant nos. 57115 and 50450).
Ethics approval and consent to participate
All protocols were reviewed and approved by the Ethical Board of Tehran University of Medical Sciences, Iran (Ethics code: IR.TUMS.SPH.REC.1401.115).
Consent for publication
The authors declare that they have no competing interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
The sensitivity and reproducibility tests of the PCR for the three loci with DNA samples 2 μl (No. 1 above the wells) and 0.1 μl (No. 2 above the wells), No 3 above the wells is negative control (ddH20). Panel A: cytB (228 bp), panel B: Alu-repeat (226 bp), and panel C: 16S rRNA (157 bp).
Results of PCR amplification of Alu-repeat, 16S rRNA and CytB loci for validating probable PCR inhibitors in the human blood and mosquito. HB, Human blood; DC-AP, decapitated and amputated; Mos, mosquito.
About this article
Cite this article
Talebzadeh, F., Ghadipasha, M., Gharehdaghi, J. et al. Efficiency of mitochondrial genes and nuclear Alu elements in detecting human DNA in blood meals of Anopheles stephensi mosquitoes: a time-course study. Parasites Vectors 16, 284 (2023). https://doi.org/10.1186/s13071-023-05884-0
- 16S rRNA
- Time course
- Blood meal
- Anopheles stephensi
- Forensic entomology
- vector capacity