Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

A novel assay to isolate and quantify third-stage Dirofilaria immitis and Brugia malayi larvae emerging from individual Aedes aegypti

A novel assay to isolate and quantify third-stage Dirofilaria immitis and Brugia malayi larvae... Background: Mosquitoes transmit filarial nematodes to both human and animal hosts, with worldwide health and economic consequences. Transmission to a vertebrate host requires that ingested microfilariae develop into infective third-stage larvae capable of emerging from the mosquito proboscis onto the skin of the host during blood-feeding. Determining the number of microfilariae that successfully develop to infective third-stage larvae in the mosquito host is key to understanding parasite transmission potential and to developing new strategies to block these worms in their vector. Methods: We developed a novel method to efficiently assess the number of infective third-stage filarial larvae that emerge from experimentally infected mosquitoes. Following infection, individual mosquitoes were placed in wells of a multi-well culture plate and warmed to 37 °C to stimulate parasite emergence. Aedes aegypti infected with Dirofilaria immitis were used to determine infection conditions and assay timing. The assay was also tested with Brugia malayi- infected Ae. aegypti. Results: Approximately 30% of Ae. aegypti infected with D. immitis and 50% of those infected with B. malayi pro- duced emerging third-stage larvae. Once D. immitis third-stage larvae emerged at 13 days post infection, the propor- tion of mosquitoes producing them and the number produced per mosquito remained stable until at least day 21. The prevalence and intensity of emerging third-stage B. malayi were similar on days 12–14 post infection. Increased uptake of D. immitis microfilariae increased the fitness cost to the mosquito but did not increase the number of emerging third-stage larvae. Conclusions: We provide a new assay with an associated set of infection conditions that will facilitate assessment of the filarial transmission potential of mosquito vectors and promote preparation of uniformly infectious third-stage larvae for functional assays. The ability to quantify infection outcome will facilitate analyses of molecular interac- tions between vectors and filariae, ultimately allowing for the establishment of novel methods to block disease transmission. Keywords: Aedes aegypti, Aedes albopictus, Dirofilaria immitis, Brugia malayi, Mosquito, Filaria, Transmission *Correspondence: [email protected] Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. McCrea et al. Parasites Vectors (2021) 14:30 Page 2 of 12 Background Arthropods serve as intermediate hosts and vectors of numerous human and animal infective filariae that con - tribute to a large disease burden worldwide. Indeed, mosquito-transmitted lymphatic filariasis, caused by Wuchereria bancrofti, Brugia malayi and B. timori, affects approximately 120 million people in 83 countries [1]. Mosquitoes are also responsible for transmission of animal infective filariae, the most studied being Dirofi - laria immitis, the causative agent of canine heartworm disease. Although detailed global numbers are not avail- able, there are an estimated 250,000–500,000 infected dogs in the USA, with some areas, such as the Mississippi River basin, reporting infection prevalence as high as 40% [2, 3]. Humans can also be incidentally infected by D. immitis, but they do not support the entire life cycle and typically present with only mild clinical signs [3]. Within the mosquito host, ingested microfilariae (mf ) migrate from the midgut to specific tissues specific to the mosquito species. The filarial agents of lymphatic filaria - sis migrate to the indirect flight muscles in the thorax, whereas D. immitis migrates into the Malpighian tubules (MT) [4]. In these tissues, the parasites develop intracellu- Fig. 1 Emergence of infective third-stage larvae (L3) from the tip larly, undergoing successive molts to form third-stage lar- of the mosquito proboscis. During blood-feeding, a subpopulation vae (L3). Some L3 migrate to the proboscis where they are of L3 in the labial sheath of the proboscis emerge from the poised for transmission (Additional file  1: Movie 1). Infec- proboscis, alighting on the skin of the host in a drop of the mosquito hemolymph (eL3). It is estimated that only approximately 10% of the tive L3 emerge most typically from the mosquito labellum eL3 survive on the skin and penetrate into it [26]; the fate of the L3 but they can also emerge from the labial sheath during that do not emerge during blood-feeding is not known. Emergence blood-feeding (Fig. 1) [5]. Larvae emerging from the mos- from the proboscis typically occurs at the labellum or distal portion quito are deposited onto the skin in a drop of hemolymph of the labial sheath and can be triggered by sensation of a thermal where they can enter the skin through the bite site [5]. The cue (red gradient). Other cues may also play a role, such as chemical compounds released by the host (green gradient) or a sensation of fate of the L3 that do not emerge is not known. mechanical forces caused by the deformation of the labial sheath Due to their importance to the disease cycle, much (orange gradient), which slides backward as the fascicle is inserted attention is focused on the transmission of infective L3. into the host skin. Adapted from Bancroft [36] Different methods have been employed to assay L3 in mosquitoes. A common approach, especially for field isolates, is the detection by PCR of larvae in the head In addition to assessing the intensity of transmission and other body parts of the mosquito following dissec- in field populations, an ability to enumerate and collect tion [6–8]. Although this method is not established for all L3 capable of emergence would greatly enhance func- filariae, stage-specific PCR has been used for detection tional assessment. Currently, studies of host immune of infectious L3 in W. bancrofti infected mosquitoes [9]. responses to L3, as well as in vitro and in vivo drug effi - PCR is sensitive and species specific and can be used on cacy studies, are typically carried out on L3 collected pools of mosquitoes in cases where infection prevalence from infected mosquitoes gently disrupted with a mor- is low. Another commonly used approach is to physi- tar and pestle and then separated by filtration though a cally examine larvae in the labial sheath of the proboscis mesh [10–12]. However, it is unknown whether all L3 or in dissected tissues of the mosquito. This approach is harvested by this method are mature and competent particularly useful in a laboratory setting to determine to infect the host. Furthermore, obtaining microfilaria the number of larvae at each developmental stage and to for mosquito infections requires access to an infected identify their tissue of residence. However, while each of animal or access to samples from another source, and these assays can reveal the number of L3 that have devel- although D. immitis microfilaria have been shown to be oped, specific assays to enumerate L3 capable of emerg - capable of cryopreservation [13], this method of stor- ing (eL3) when the mosquito blood-feeds have not been age is not commonly used. More typically, fresh sam- previously described. ples are shipped, despite being infective, little is known M cCrea et al. Parasites Vectors (2021) 14:30 Page 3 of 12 about how short-term storage during shipping affects with the guidelines of the Institutional Animal Care development to L3. and Use Committee of the University of Pennsylvania Blocking parasite development in the vector is a (IACUC, protocol 805059). The microfilarial load in novel approach being considered for controlling disease this dog was approximately 50,000 mf/ml. Blood con- transmission and requires a thorough understanding of taining B. malayi microfilariae (B. malayi microfilariae the molecular interactions between parasites and their in cat blood, live, FR3, NR-48887) was obtained from an vector. The means to quantify the prevalence of mos - experimentally infected cat containing with a microfi - quitoes with eL3 and determine the number of emerg- larial load of approximately 12,000 mf/ml (provided by ing L3 per mosquito would greatly facilitate this work. the NIH/NIAID FR3 for distribution by BEI Resources, Here we present a new assay to quantify infectious eL3 NIAID/NIH). In both cases, the blood was diluted with that works with different vector and filarial species. the appropriate volume of heparinized sheep blood to a We then use this assay to study the development and concentration of 4000 mf/ml. The sample was warmed characteristics of D. immitis eL3 in mosquitoes. The to 37 °C, and a 3.5-ml aliquot was placed in the inden- detailed infection parameters and assay conditions pre- tation on the outside of the bottom of a 300-ml plastic sented here to quantify and produce maximum yields baby bottle (Advent; Phillips, Amsterdam, the Neth- of eL3 could potentially facilitate both studies between erlands) and covered with Parafilm. The inside of the filariae and their mosquito and vertebrate hosts. bottle was filled with water at 37  °C. This assembly was placed with the membrane side down onto the top mesh of mosquito cages and the insects are allowed to Methods feed for 15  min (Fig.  2a). Immediately following the Mosquito strains and culture Aedes aegypti and Ae. albopictus strains were provided by the National Institutes of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID) Filariasis Research Reagent Resource Center (FR3) and the Malaria Research and Reference Reagent Resource Center (MR4) through distribution by the NIH/NIAID BEI Resources Repository. Aedes aegypti (Ae. aegypti, Strain Black Eye Liverpool, Eggs, FR3, NR-48921) is a Dirolfi aria immi - tis- and Brugia malayi-susceptible strain, and Ae. aegyp- ti (Ae. aegypti, Strain LVP-IB12, Eggs, MR4, MRA-735, contributed by David W. Severson) is a D. immitis- and B. malayi-refractory strain. Both strains were reared at 27  °C and 80% humidity under a 12/12-h photoperiod. NJ Aedes albopictus (Ae. albopictus Strain ATM-NJ95, Eggs, MR4, NR-48979) [14] was reared at 24 °C and 70% humidity under a 16/8-h (light/dark) photoperiod. Mos- quitoes were housed in 30-cm cages (BugDorm, Tai- chung, Taiwan) at a density of approximately 1000 per cage. Larvae were maintained at a density of 1 larva/3 ml. Larvae were fed a suspension of liver powder in water Fig. 2 Assay for eL3. a Infection and assay protocol followed in this (MP Biomedicals, Solon, OH, USA), and adults were study. Mosquitoes are fed on blood containing Dirofilaria immitis microfilariae. Uptake is measured in a small group, and the remaining maintained on 10% sucrose in water, which was changed mosquitoes are maintained until an emergence assay is performed. daily. Heparinized sheep blood (Hemostat, Dixon, CA, Immediately following the emergence assay with whole mosquitoes, USA) was provided using an artificial membrane feeder the Malpighian tubules are dissected and analyzed. The dissected at 37 °C for egg production. head and carcass are individually placed into an emergence assay to assay L3 that failed to emerge from intact mosquitoes. Additional file 2: Figure S1 shows images of dissected mosquitoes and Mosquito infection with Dirofilaria immitis and Brugia Malpighian tubules. b, c Mosquitoes are rinsed with 70% ethanol:30% malayi water for wetting, rinsed with water (b) and then placed individually The full protocol is described in greater detail by Pov - into wells of a 96-well plate (c). d Emerging eL3 from intact elones and McCrea [15]. Blood containing D. immitis mosquitoes or L3 from dissected heads and carcasses at the bottom each well are scored by microscopy. Additional file 3: Movie 2 shows microfilariae was obtained from an experimentally typical movement of eL3. e Larvae (white arrows) in live Malpighian infected dog according to Institutional Animal Care and tubules are scored by microscopy. Scale bar: 50 µm Use Committee-approved protocols and in accordance McCrea et al. Parasites Vectors (2021) 14:30 Page 4 of 12 feed, fully engorged blood-fed mosquitoes were sepa- tissue for at least 60 min and scored as described above. rated using CO anesthesia and housed under standard Only a portion of the mosquitoes, between 30 and 36 insectary conditions as described above. Any partial or from each replicate, used for the eL3 emergence assay unfed mosquitoes were discarded. were processed in this manner due to processing time constraints. Microfilarial uptake Within 1 hour of blood-feeding, fed mosquitoes were Emergence assay time course and microfilaria anesthetized with CO at room temperature. The entire concentration series undamaged midgut was dissected in deionized water Groups of 432–565 mosquitoes were fed on blood con- in a depression slide and immediately transferred to a taining D. immitis microfilariae at different concentra - standard microscope slide containing a 50-µl drop of tions and housed as described above. Mosquito mortality deionized water. Damaged midguts leaking blood dur- was monitored daily, as assessed by the fraction of the ing dissection were not used. The epithelium of the gut population that died each day. This was necessary since was separated from the blood bolus and moved through groups of 50 mosquitoes were removed for eL3 assays the water to remove any residual blood before it was as described above. Since the emergence prevalence was discarded. The blood was roughly dispersed using for - very low on day 12, emergence assays were performed ceps and then pipetted up-and-down with a 20-µl with only 25 mosquitoes per replicate saving more of the pipette. Microfilaria in the entire drop were counted population for later days where there would be a higher immediately without a coverslip. eL3 prevalence. For the concentration-series experi- ments, a blood dilution containing 32,000 mf/ml was created and then subjected to twofold serial dilution to Emergence assay for eL3 and other larval stages create feeding doses of 16,000, 8000 and 4000 mf/mL. At The full protocol is described in greater detail by Pov - each concentration, five mosquitoes of each strain were elones and McCrea [16], which is modified from a used to measure uptake, as described above. For these previously published method [17]. Briefly, eL3 were experiments, mortality was monitored daily until day 17 assayed by placing live mosquitoes in 70% ethanol when an assay for eL3 was performed on the remaining in water for 1  min, following which they were rinsed mosquitoes. Kaplan–Meier survival analysis was per- twice in deionized water and then placed individually formed between adjacent doses within each mosquito into wells of a 96-well plate containing 200 µl Dulbec- strain and between the same doses across the mosquito co’s Modified Eagle’s Medium (DMEM) supplemented strains. with l -glutamine, a high level of glucose and sodium pyruvate (Corning Mediatech, Manassas, VA, USA). At this point, the mosquitoes are still alive and can be Results observed moving in the buffer, but they cannot stand Novel assay for filarial nematode L3 emerging on the surface due to the wetting procedure. The plate from individual mosquitoes was then placed in a 37  °C incubator with 5% C O for In our assay, outlined in Fig.  2a, we infect mosquitoes 60 min, and the number of eL3 were determined with- using an artificial membrane feeder and then determine out removing the mosquitoes using an inverted micro- the number of microfilariae ingested immediately after - scope with a ×4 objective. In some experiments, after wards [15]. The microfilarial uptake represents the the - the eL3 emergence assay, mosquitoes were removed oretical maximum number of parasites in the assay. To from the plate individually for assaying other larvae. assay the L3 capable of emerging from the mosquitoes, First, the Malpighian tubules were dissected by remov- we simulate the thermal cue that would be experienced ing the posterior two abdominal segments. The set of by a mosquito landing on a mammalian vertebrate host five Malpighian tubules was removed from the midgut by placing mosquitoes in buffer and warming the buffer and transferred to a microscope slide containing 15 µl to 37  °C [16]. Mosquitoes are placed individually into phosphate buffered  saline and covered with a 22-mm wells of a 96-well plate after the wetting procedure coverslip. The larvae were counted, and although dif - (Fig. 2b, c), and care is taken to minimize damage to the ferent stages were present we did not categorize them mosquito during transfer. Upon warming, competent (Additional file  4: Figure S2). The head was then sepa - L3 emerge from the mosquito and sink to the bottom rated from the body, and the dissected head and car- of the well, where they can be counted (Fig.  2d; Addi- cass fragment were placed separately into fresh wells tional file  3: Movie 2). eL3 collected by this method are of a 96-well plate prepared and incubated as described capable of molting to the fourth larval stage in  vitro above. L3 were allowed to migrate out of the dissected [18] and are infectious to dogs [17], and we believe M cCrea et al. Parasites Vectors (2021) 14:30 Page 5 of 12 they represent the number of L3 that emerge onto the could not unambiguously characterize them as L3. We skin of the host. After the emergence assay, the mos- found that the number of eL3 was significantly lower quitoes are still viable and can move, but they cannot than the total L3 (Fig. 3c). Therefore, we concluded that escape the buffer due to the wetting procedure. Nota - only a fraction of the larvae that develop to L3 emerge bly, following the emergence assay, further analyses can during our emergence assay, consistent with previous be carried out to determine the number and location observations [5]. of larvae remaining within the mosquito. In our study, both dissected Malpighian tubules (Fig.  2e), and the Once eL3 have developed, their intensity and prevalence head and remaining carcass were assayed separately, are constant as described in the following section (Additional file  2: To determine the optimal day to perform the emergence Figure S1). assay, we wanted to identify a time point at which eL3 recovery was maximized but mosquito mortality was still minimal. To do this, we infected populations ranging Microfilariae robustly migrate into the Malpighian tubules from 432 to 565 individuals, and then removed groups but only a fraction develop to eL3 of approximately 50 mosquitoes on consecutive days for To determine the efficiency of eL3 development we an emergence assay. Data from four independent experi- infected D. immitis susceptible (Ae. aegypti ) or refrac- ments were pooled since it was not possible to obtain tory (Ae. aegypti ) strains and performed an emergence all time points from a single infection. We assessed the assay 14  dpi. We found that Ae. aegypti released no variation in eL3 number over the 10-day period from 12 eL3, despite having a median uptake of 12 mf/mosquito to 21 dpi. The main emergence phase started at 13 dpi, (Fig.  3a). This is consistent with previous observations as we observed only a single eL3 in 48 mosquitoes at 12 that ingested microfilariae arrest their development dpi. Once L3 begin to emerge from mosquitoes, both the soon after invading cells of the Malpighian tubules number that emerge per mosquito as well as the preva- [19–23]. In the susceptible strain (Ae. aegypti ), 81 lence of mosquitoes with at least one emerging L3 are of the 252 mosquitoes assayed (32%) had at least one relatively constant from 13 to 21 dpi (Fig.  4). These data eL3 when given a median uptake of 15 mf/mosquito suggest that larvae develop synchronously and that once (Fig.  3b). A portion of the susceptible mosquitoes used they reach the L3 stage, their numbers remain relatively for the emergence assay were dissected, and a substan- stable over time. Beyond 13 dpi, mosquito mortality con- tial number had L3 remaining in the dissected heads tinues and eL3 prevalence or intensity is not increased, (26/102; 25%) and carcasses (48/102; 47%) (Fig.  3b). Therefore, to maintain population sizes and maximize The majority of larvae assayed at 14 dpi were present in eL3 yield we typically assay mosquitoes 14–17 dpi. the Malpighian tubules, and nearly all of the dissected mosquitoes (93/96; 97%) had at least one larva present Increasing the number of Dirofilaria immitis microfilariae in the Malpighian tubules (Fig. 3b). In a few cases, Mal- ingested does not increase eL3 prevalence pighian tubules were damaged during the dissection To optimize the number of eL3 recovered from the mos- and excluded from this and subsequent analyses. Pool- quito, we next determined if increasing the number of ing all larvae present across all tissues revealed that the microfilariae in the blood meal improved the recovery median total number of larvae per mosquito was eight. of eL3. For these experiments we used the standard con- Therefore, at 14 dpi, 53% of the 15 ingested microfilar - centration of 4000 mf/ml and included three additional iae could be accounted for as larvae in the mosquito concentrations of 8000, 16,000 and 32,000 mf/ml. We (Fig. 3b). In the dissected Malpighian tubules, we found found that increased concentrations of microfilaria in the larvae in various stages of development, but we did membrane feeder had a consequent impact on the num- not determine their stage (Additional file  4: Figure S2). ber present in the mosquito midgut immediately after We note, however, that larvae found in the Malpighian blood-feeding, with no significant difference between S R tubules, even the most stunted sausage forms, are via- Ae. aegypti and Ae. aegypti (Fig.  5a). At each successive ble, as we observed them to be capable of movement. concentration, microfilariae present in the blood meal Previous studies suggest that not all L3 emerge when increased approximately twofold. We monitored mosquito infected mosquitoes blood-feed on an animal [5]. Since survival for 17 days, at which time an emergence assay was this previous observation was not quantitative, we ana- performed. We chose day 17 to allow more time to moni- lyzed the data from the emergence assay (Fig.  3b) to tor mosquito survival. The prevalence of eL3 in Ae. aegypti compare the number of eL3 to the total L3 (eL3 + L3 was not significantly different, regardless of the concentra - in dissected heads, and dissected carcasses). We did not tion of microfilariae fed (Fig.  5b). On a population level, the include larvae found in the Malpighian tubules since we number of eL3 produced per mosquito was not increased McCrea et al. Parasites Vectors (2021) 14:30 Page 6 of 12 Fig. 3 A fraction of ingested microfilariae develops into eL3 in Aedes aegypti (a D. immitis- and Brugia malayi-susceptible strain). a Dots indicate the number of microfilariae present in the midgut of individual Ae. aegypti (a D. immitis- and B. malayi-resistant strain) immediately after feeding on infected blood (Uptake) and the number of eL3 assayed 14 days post infection (dpi). The black line indicates the median. b Dots indicate the number of microfilariae present in the midgut of individual Ae. aegypti immediately after feeding on infected blood (Uptake), the number of eL3 assayed 14 dpi (eL3), the number of L3 emerging from the dissected heads (Head) or carcass (Carcass) and the number present in the Malpighian tubules (MT ). The Total is sum of all parasites of any stage found in any tissue or assay on 14 dpi. The red line indicates the median. c Dots are the number of eL3 from individual Ae. aegypti and all third-stage larvae assayed ( Total; sum of eL3, Head, and Carcass) (data taken from panel b). The red line is the median number. The asterisks indicate a Mann–Whitney P value < 0.001. These data are the sum of three independent biological replicates. The number of mosquitoes assayed is shown below each column by feeding concentrations of microfilariae greater than uninfected blood to 4000 mf/ml and 16,000 to 32,000 mf/ 4000 mf/ml (Fig.  5c). Increasing the concentration of D. ml comparisons were significantly different (Fig.  5e). When S S R immitis microfilaria used to infect Ae. aegypti increased we compared the survival of Ae. aegypti and Ae. aegypti mosquito mortality in a dose-dependent manner (Fig.  5d). at the different concentrations of microfilariae, there was Each increase in microfilaria concentration significantly no difference in survival at the lowest concentration (Addi - increased mortality from the previous one, including the tional file  5: Figure S3). There was a modest difference at lowest concentration, which elevated mortality compared 8000 mf/ml, with Ae. aegypti showing greater mortality, to those fed on uninfected blood. A similar dose–response and this difference further increased at the two highest was observed in the refractory strain; however, only the concentrations. S S Fig. 4 The number of D. immitis eL3 from Ae. aegypti are constant between 13 and 21 dpi. a Graph of the average prevalence in Ae. aegypti of D. immitis eL3 assayed 12–21 dpi. Error bars indicate the standard deviation (SD). b Dots are the number of D. immitis eL3 from individual mosquitoes assayed from 12 to 21 dpi. This experiment was performed four times. Two replicates were assayed on 12 to 18 dpi; the other two replicates were assayed on 14 to 21 dpi. Each replicate was performed with approximately 50 mosquitoes. The replicates on 12 dpi were performed with fewer mosquitoes since our preliminary data suggested that emergence was negligible on this day M cCrea et al. Parasites Vectors (2021) 14:30 Page 7 of 12 Stored blood containing microfilariae retains its ability fresh microfilaremic blood and ones fed on an aliquot of to infect mosquitoes and produce eL3 the same blood sample stored for 1 day at 4 °C (Fig.  6a). All D. immitis experiments described above used freshly However, there were significant decreases in eL3 preva - isolated microfilaremic blood. However, there are times lence in mosquitoes fed on microfilaremic blood stored when it is not possible to use this material immediately, for 2 days at 4° C compared to mosquitoes fed on blood such as when blood is shipped from another location. We stored at 4° C for 1 day and to mosquitoes fed on fresh wanted to compare the number of eL3 produced when blood. Similar trends were observed in infection inten- mosquitoes were fed infected blood stored at 4  °C for 1 sity (Fig. 6b). Our data show that although microfilaremic or 2 days. For these experiments, we fed mosquitoes with blood stored at 4 °C for 2 days can still infect mosquitoes, freshly isolated infected blood and on the next 2 consec- there is a trend towards lower numbers of eL3 follow- utive days fed new cohorts with the same sample that had ing blood storage, suggesting that refrigerated samples been stored at 4  °C. We performed an emergence assay should be used as soon as possible for maximum infec- at day 17 of each infection. There was no significant dif - tion potency. ference in the prevalence of eL3 between mosquitoes fed Fig. 5 Increasing the dose of microfilariae does not increase numbers of eL3 but increases mosquito mortality. a Dots indicate the number of D. S R immitis microfilariae present in midguts of Ae. aegypti (blue) and Ae. aegypti (red) immediately following blood-feeding on the indicated doses of microfilariae. Data in each column are normally distributed, and black lines and numbers indicate the mean. No significant differences were S R found when Ae. aegypti and Ae. aegypti were compared at the different doses using an unpaired t-test. Data are from two independent biological replicates. b Graph of the average prevalence of D. immitis eL3 in mosquitoes feeding on blood with increasing concentrations of microfilariae, assayed 17 dpi. Error bars indicate the SD. There was no significant difference comparing the columns using analysis of variance. c Dots are the number of D. immitis eL3 from individual mosquitoes assayed 17 dpi. No significant differences were found when we compared all groups to each other or compared 4000 mf/mL to the other groups using a Kruskal–Wallis test with Dunn’s correction for multiple comparisons. Data for panels b S R and c are pooled from four separate biological replicates. d, e Kaplan–Meier survival plot for Ae. aegypti (d) or Ae. aegypti (e) fed with uninfected blood (BF) or blood containing different concentrations of microfilariae (4000, 8000, 16,000, 32,000 mf/ml). Pairs of adjacent treatment groups were analyzed by Kaplan–Meier, and relationships with significant differences are indicated with asterisks in Additional file 5: Figure S3 and in Additional file 6: Table S1 McCrea et al. Parasites Vectors (2021) 14:30 Page 8 of 12 Emergence assay works in a natural vector of Dirofilaria immitis Since Ae. aegypti is not considered to be an important natural vector of D. immitis, we wanted to determine whether eL3 could be obtained from a mosquito species associated with D. immitis transmission. For these exper- iments we used a strain of Ae. albopictus isolated from NJ Keyport, NJ, USA (Ae. albopictus ). We found that eL3 were present at 17 dpi, but their prevalence and number were lower than those present in Ae. aegypti (Fig.  7a, b) when the mosquitoes were reared and housed in our standard insectary conditions (27  °C with 80% rela- tive humidity [RH] and a 12:12-h photoperiod). When NJ we tested Ae. albopictus reared and infected under its Fig. 6 Infected blood stored at 4 °C produces eL3. Infected blood ideal temperature and humidity conditions (24  °C, 70% was used fresh (0) or after storage for 1 or 2 days (1, 2) at 4 °C. a RH, and a 16:8-h [light/dark] photoperiod), the preva- Graph of the average prevalence of D. immitis eL3 at 17 dpi in Ae. lence and number of eL3 were increased, although they aegypti . The error bar indicates the SD. The number of mosquitoes (n) were still lower than was typical for Ae. aegypti (Fig. 7c, analyzed is indicated for each sample. Asterisks indicate a significant NJ d), likely because the strain of Ae. albopictus used did difference at P < 0.05 using Fisher’s exact test. b Blue dots are the number of D. immitis eL3 emerging from individual mosquitoes not feed to repletion from an artificial membrane feeder assayed at 17 dpi. Asterisks indicate significant differences in under any conditions that we tested. Even though we intensity using Kruskal–Wallis test with Dunn’s correction for multiple were not able to make quantitative comparisons to Ae. comparisons (*P < 0.05; **P < 0.01). Data are pooled from two aegypti , our data show that the emergence assay works independent experiments with different mosquito species. Individual mosquito emergence assay for transmission residing in the mosquito emerge when stimulated either stage Brugia malayi by warming the mosquito or by direct contact with a To determine whether the emergence assay will be mammalian host, even if already residing in the head. informative for enumerating eL3 for other filarial nem - Our method provides a more accurate indication of a atodes, we infected  mosquitoes with B. malayi. These particular vector species’ potential for pathogen trans- parasites have a shorter developmental period in the mission. For example, our results show that not all strains mosquito [24], so we performed our emergence assay at of Ae. aegypti or Ae. albopictus support eL3 develop- S R 12–14 dpi in both Ae. aegypti and Ae. aegypti mosqui- ment, even though microfilariae invade the Malpighian toes. Despite taking up a similar number of microfilar - tubules. In other studies, we used this assay to show that iae in the blood meal (Fig.  8a), no eL3 were produced activation of the Toll immune signaling pathway restricts by Ae. aegypti , showing that this strain is refractory to development of D. immitis and B. malayi eL3 [19, 25]. both B. malayi and D. immitis (Fig.  8b). In contrast, a In addition, this assay could also permit assessment of significant proportion of Ae. aegypti mosquitoes devel- manipulations of the mosquito host or larvae that result oped eL3, showing that this strain is susceptible to both in an inability of L3 to emerge in response to thermal B. malayi and D. immitis (Fig.  8b). There was no sig - stimuli or due to motor defects that could be missed by nificant difference in either the prevalence of mosqui - current methods. Finally, the results of our assays sug- toes producing eL3 or in the number of eL3 produced gest that quantitating all L3 by dissection will likely result per mosquito between the days we assayed (Fig.  8b-c). in overestimating the number of larvae that are capable These data indicate that despite their different life-cycle of emerging from the mosquito. This is particularly true in the mosquito, eL3 of human filariae can be measured in the case where the number of L3 is derived by mos- using our emergence assay. quito homogenization, as L3 from the body cavity and those present in the Malpighian tubules are likely to be Discussion included in this method. The development of an assay for eL3 may be valuable for Overall, we found that 32% of the D. immitis-infected studying mosquito–filaria interactions, thereby provid - and approximately 50% of B. malayi-infected mosqui- ing a clearer indication of vector competence and vec- toes develop at least one eL3, which is similar to previ- tor transmission intensity. As our data and a previously ous observations measuring emergence of L3 [26–28]. reported observation [5] indicate, only a portion of L3 Since mosquitoes typically blood-feed more than once, it M cCrea et al. Parasites Vectors (2021) 14:30 Page 9 of 12 Fig. 7 Aedes albopictus supports eL3 development. a, c Graphs of the average prevalence of D. immitis eL3 emerging at 17 dpi in Ae. aegypti (blue S NJ NJ NJ bar, Ae. aeg ) or Ae. albopictus (purple bar, Ae. albo ) reared and maintained under standard insectary conditions (a) or in Ae. albopictus (purple NJ bar, Ae. albo ) reared and maintained at 24 °C, 70% relative humidity and 16:8-h (light/dark) photoperiod (c). Error bars indicate the SD. The number of mosquitoes (n) analyzed is indicated for each sample. b, d Blue and purple dots are the number of D. immitis eL3 emerging at 17 dpi from S S NJ NJ individual Ae. aegypti (Ae. aeg ) or Ae. albopictus (Ae. albo ) mosquitoes, respectively, reared and maintained under standard insectary conditions (b) or at 24 °C, 70% relative humidity and 16:8-h (light/dark) photoperiod (d) Fig. 8 Emergence assay can be used to quantify B. malayi eL3. a Dots indicate the number of Brugia malayi microfilariae present in midguts of S R Ae. aegypti (blue) and Ae. aegypti (red) immediately following blood-feeding. Data in each column are not normally distributed; the black line is the median. The number of mosquitoes (n) analyzed is indicated for each sample. No significant difference was found using a Mann–Whitney test S R (P = 0.09). b Graph of the average prevalence of B. malayi eL3 assayed 12–14 dpi in Ae. aegypti (blue bars) and at 12 and 14 dpi in Ae. aegypti (red bars). Error bars indicate the SD. The number of mosquitoes (n) analyzed is indicated for each sample. No emerging parasites were observed for Ae. R S S aegypti . There were no significant differences between the days assayed for Ae. aegypti , and all Ae. aegypti days assayed were significantly different from both Ae. aegypti on the two days assayed using an analysis of variance with Tukey’s multiple comparisons test, as indicated by asterisks (P < 0.001). c Blue dots are the number of B. malayi eL3 from individual mosquitoes assayed 12–14 dpi. No significant differences were found when all groups were compared to each other or when day 12 post infection was compared to the other groups using a Kruskal–Wallis test with Dunn’s correction for multiple comparisons. Data were pooled from three independent experiments is interesting to speculate that the L3 that do not emerge resources in the vector or a parasite strategy to control may enter the proboscis and emerge in a subsequent the density of L3 to limit damage to the vector. blood meal, thereby increasing the chance of transmis- Results of our experiments to optimize recovery of eL3 sion. On the other hand, it is also possible that the eL3 by increasing the dose of D. immitis microfilariae given we observe are the only ones competent to emerge and to mosquitoes reveal that microfilarial uptake and eL3 infect another host. In this case, the failure of the remain- emergence are uncoupled, as we did not observe signifi - ing L3 to emerge could be due to competition for limited cant increases in the prevalence of mosquitoes with eL3 McCrea et al. Parasites Vectors (2021) 14:30 Page 10 of 12 or the number of eL3 per mosquito at the doses tested. into hemolymph within the mosquito body cavity, where While it is likely that microfilarial uptake and eL3 emer - they could be eliminated or sequestered. As the pos- gence are coupled at lower concentrations of micro- sibilities outlined above are not mutually exclusive, we filariae, we did not test this since our main goal was to believe they could collectively account for the differ - establish conditions where eL3 recovery is maximized ential between microfilarial uptake and total parasites to support future study of transmission-blocking strate- ultimately detected. Interestingly, it was recently shown gies. Varying the timing of the assay revealed that at 13 using field populations of Ae. albopictus, a natural vec - dpi there is no significant difference in the prevalence or tor of D. immitis, that in areas of greater infection preva- number of eL3. Taken together, our data suggest that the lence, larval damage to the mosquito is better tolerated, Malpighian tubules have a limited capacity to support D. benefitting both the vector and the parasite [33]. immitis development and that no additional eL3 are pro- We used our emergence assay to analyze the effects of duced once the limit is reached. However, we observed storing microfilaremic blood at 4  °C and showed that it a range of developmental phenotypes in the Malpighian is possible to produce eL3 after a storage time of at least tubules of mosquitoes 14 days after D. immitis infection, 2 days, but likely even longer. However, while delays in raising the possibility that larval damage to the tubules initiating mosquito infection do not preclude experi- prevents further development, as all larvae are viable at mentation, the strongest prevalence and intensity of eL3 this time point, even those displaying delayed develop- production was observed in mosquitoes infected through ment phenotypes. While a substantial number of midgut the ingestion of fresh microfilaremic blood. Given the epithelial cells are invaded during infection by ookinetes loss of eL3 production with blood storage time, it is not of Plasmodium, the causative agent of malaria, damaged recommended to make comparisons within an experi- cells are eliminated and replaced through tissue regen- ment using a blood sample stored for different lengths of eration [29, 30]. As larval development is blocked and time. However, internal comparisons are reasonable, as is mosquito fitness is negatively impacted over time, this the goal of using the stored blood to produce eL3. process suggests that damaged cells of the Malpighian Because L3 obtained in our assay have responded to tubules persist and are not replaced. In addition, the con- physiological conditions, L3 produced via an emergence tinuing metabolism of the increased number of larvae or assay may provide a more easily accessible source of an accumulation of larval waste products may also con- infective larvae than L3 obtained from gently crushed tribute to the increased mosquito mortality we observed and sieved mosquitoes [34]. While we developed this in Ae. aegypti at later timepoints. emergence assay for use on single mosquitoes, it is By quantifying microfilarial uptake in individual mos - also suitable for use on populations en masse, and eL3 quitoes and comparing it to all parasites that emerge obtained this way are known to be infective [17]. As such, from either the whole mosquito, the head or the car- eL3 may be quite valuable for future identification and cass or to those present in the Malpighian tubules at 14 testing of novel heartworm preventatives, which requires dpi, we found that approximately 60% of the ingested infectious D. immitis L3, as well as for studies of the host microfilariae are accounted for at 14 dpi. Although we immune response to infection, which often require infec- are accounting for a majority of the parasites ingested by tious L3. Similarly, in vitro L3 to L4 molting assays, such the mosquitoes, it is nevertheless interesting to specu- as the newly developed genetic transformation protocol late about the fate of those that are not assayed. Some L3 for B. malayi [35], may benefit from L3 produced using remain in the proboscis of dissected heads, even after the our method. Finally, given the widespread nature of vec- primary emergence assay and the subsequent emergence tor-borne filarial diseases and their potential to spread assay after dissection. However, we did not systematically due to changing environmental conditions, new meth- attempt to quantify these, and it remains possible that L3 ods like this could help assess transmission potential in may also be retained in the dissected carcasses. In addi- the field and to develop methods in the laboratory to tion, because some microfilariae may be damaged by the block transmission. This easy, efficient and cost-effective mosquito immune system or fail to follow sensory cues, method has the potential to be a valuable tool for the they may fail to invade the Malpighian tubules and may quantitative analysis of mosquito transmission of filariae, be voided into the hindgut and then the feces [4, 31, 32]. especially to researchers in countries most affected by Likewise, the activity of microfilariae and larvae in the these parasites where resources are often constrained. Malpighian tubule cells may damage these cells, some- times to the point where microfilariae are released into Conclusions the tubule lumen, where they also could be eliminated as We have established a novel assay to determine the waste. Alternatively, if the basal side of the Malpighian number of infectious L3 filarial larvae capable of tubule cell is compromised, parasites could be released emerging from individual mosquitoes, which we refer M cCrea et al. Parasites Vectors (2021) 14:30 Page 11 of 12 Authors’ contributions to as eL3. We have shown this assay works with both MP was responsible for the study conception. ARM, EBE, GTO, FMO and MP different mosquito and filarial nematode species. As were responsible for acquisition of data. ARM, EBE, GTO, FMO, TJN, JBL and MP such, our method is likely to be suitable for assess- were responsible for the analysis and interpretation of data. MP and ARM were responsible for drafting and critical revision of the manuscript. EBE, GTO, FMO, ment eL3 of other mosquito transmitted filariae, such TJN and JBL were responsible for critical revision of the manuscript. All authors as Dirofilaria repens, a canine and human skin-dwell - read and approved the final manuscript. ing relative of D. immitis, and Wuchereria bancrofti, Funding which is responsible for approximately 90% of human This work was supported by a University Research Foundation grant (URF- lymphatic filariasis and can be transmitted by several 2017), intramural funds, and NIH grant AI139060 to MP. JBL was supported by genera of mosquitoes (Culex, Aedes and Anopheles). NIH grants AI050668 and AI44572. It remains to be determined if our assay will work for Availability of data and materials filariae transmitted by other arthropod vectors, such All data generated or analyzed during this study are included in this published as biting flies, mites, fleas and ticks. In summary, the article and its supplementary information files. method we describe here will greatly facilitate current Ethics approval and consent to participate efforts to block transmission of arthropod transmitted Blood containing D. immitis microfilariae was obtained from an experimen- filariae by enabling quantitative analysis in the vector as tally infected dog according to Institutional Animal Care and Use Committee approved protocols and in accordance with the guidelines of the Institutional well as providing a more reliable way to harvest infec- Animal Care and Use Committee of the University of Pennsylvania (IACUC, tious larvae to evaluate new preventatives, treatments protocol 805059). or vaccines. Consent for publication Not applicable. Supplementary information The online version contains supplementary material available at https ://doi. Competing interests org/10.1186/s1307 1-020-04529 -w. The authors declare that they have no competing interests. Received: 15 August 2020 Accepted: 7 December 2020 Additional file 1: Movie 1. Third-stage larvae visible moving in the proboscis of Ae. aegypti 14 days after infection with D. immitis. Additional file 2: Figure S1. Mosquito dissection for analysis after emergence assay. After the emergence assay from the whole body (a), the head (b) and carcass (c) were placed individually into separate wells. The References number of L3 larvae emerging from the dissected head and carcass were 1. Taylor MJ, Hoerauf A, Bockarie M. Lymphatic filariasis and onchocer - assayed after incubation at 37 °C. The Malpighian tubules were removed ciasis. Lancet. 2010;376(9747):1175–85. https ://doi.org/10.1016/S0140 from the carcass prior to placing it in the well. Mosquito after removing -6736(10)60586 -7. head with fine forceps (d) and carcass after the Malpighian tubules (arrow) 2. Companion Animal Parasite Council. 2012. https ://capcv et.org/ are dissected out (e). The scale bars in a–c and d, e are 500 and 1000 µm, maps/#2019/all/heart worm-canin e/dog/unite d-state s/. Accessed Dec respectively. 2019. 3. McCall JW, Genchi C, Kramer LH, Guerrero J, Venco L. Heartworm disease Additional file 3: Movie 2. Two eL3 in a well of a 96-well plate following in animals and humans. Adv Parasitol. 2008;66:193–285. https ://doi. an emergence assay performed on Ae. aegypti 14 days after infection with org/10.1016/S0065 -308X(08)00204 -2. D. immitis. Another eL3 is visible at the top of the frame. 4. Beerntsen BT, James AA, Christensen BM. Genetics of mosquito vector Additional file 4: Figure S2. Example of D. immitis larval lengths scored competence. Microbiol Mol Biol Rev. 2000;64(1):115–37. in Malpighian tubules. Malpighian tubules dissected following an 5. McGreevy PB, Theis JH, Lavoipierre MM, Clark J. Studies on filariasis. III. emergence assay contain viable larvae of different lengths indicated with Dirofilaria immitis: emergence of infective larvae from the mouthparts of asterisks. Different stage larvae are typically present. Aedes aegypti. J Helminthol. 1974;48 4:221–8. S 6. Bradley TJ, Nayar JK. An ultrastructural study of Dirofilaria immitis infec- Additional file 5: Figure S3. Ae. aegypti have greater mortality than Ae. R tion in the Malpighian tubules of Anopheles quadrimaculatus. J Parasitol. aegypti following D. immitis infection. Pairs of adjacent treatment groups 1987;73(5):1035–43. from Fig. 5d, e were analyzed by Kaplan–Meier and relationships, with 7. McKay T, Bianco T, Rhodes L, Barnett S. Prevalence of Dirofilaria immitis significant differences indicated with asterisks here and in Additional file 6: S R (Nematoda: Filarioidea) in mosquitoes from northeast Arkansas, the Table S1. Aedes aegypti and Ae. aegypti are indicated in blue and red, United States. J Med Entomol. 2013;50(4):871–8. https ://doi.org/10.1603/ respectively. me121 97. Additional file 6: Table S1. Pairwise comparisons of mosquito survival 8. Ledesma N, Harrington L. Mosquito vectors of dog heartworm in following blood feeding on different doses of D. immitis microfilariae. the United States: vector status and factors influencing transmission efficiency. Top Companion Anim Med. 2011;26(4):178–85. https ://doi. org/10.1053/j.tcam.2011.09.005. Abbreviations 9. Laney SJ, Ramzy RM, Helmy HH, Farid HA, Ashour AA, Weil GJ, et al. BE, susceptible, Ae. aegypti : Aedes aegypti Blackeye Liverpool strain; LVP, Detection of Wuchereria bancrofti L3 larvae in mosquitoes: a reverse refractory, Ae. aegypti : Aedes aegypti Liverpool strain; mf: Microfilariae; MT: transcriptase PCR assay evaluating infection and infectivity. PLoS Malpighian tubules; L3: Third-stage larvae; eL3: Emerging third-stage larvae. neglected tropical diseases. 2010;4(2):e602; https ://doi.org/10.1371/journ al.pntd.00006 02. Acknowledgements 10. Blagburn BL, Arther RG, Dillon AR, Butler JM, Bowles JV, von Simson We thank Leslie King who helped with the critical reading of the manuscript C, et al. Efficacy of four commercially available heartworm preven- and the Filariasis Research Reagent Resource center for providing Brugia tive products against the JYD-34 laboratory strain of Dirofilaria malayi and the Aedes aegypti Black Eye Liverpool strain. immitis. Parasites Vectors. 2016;9:191. https ://doi.org/10.1186/s1307 1-016-1476-7. McCrea et al. Parasites Vectors (2021) 14:30 Page 12 of 12 11. Bowman DD, McTier TL, Adams EL, Mahabir SP, Login JA, Bidgood T, 25. McCrea AR, Jimenez Castro PD, Kaplan RM, Povelones M. Activation of the et al. Evaluation of the efficacy of ProHeart 6 (moxidectin) against a Toll pathway in Aedes aegypti blocks the development of emerging third- resistant isolate of Dirofilaria immitis (JYD-34) in dogs. Parasites Vectors. stage larvae of drug-resistant Dirofilaria immitis. Vet Parasitol. 2020. https 2017;10(Suppl 2):502. https ://doi.org/10.1186/s1307 1-017-2431-y.://doi.org/10.1016/j.vetpa r.2020.10910 0. 12. Kryda K, Six RH, Walsh KF, Holzmer SJ, Chapin S, Mahabir SP, et al. 26. Grieve RB, Lok JB, Glickman LT. Epidemiology of canine heartworm infec- Laboratory and field studies to investigate the efficacy of a novel, orally tion. Epidemiol Rev. 1983;5:220–46. https ://doi.org/10.1093/oxfor djour administered combination product containing moxidectin, sarolaner nals.epire v.a0362 60. and pyrantel for the prevention of heartworm disease (Dirofilaria immitis) 27. Gasarasi DB. The transmission dynamics of bancroftian filariasis: the in dogs. Parasites Vectors. 2019;12(1):445; https ://doi.org/10.1186/s1307 distribution of the infective larvae of Wuchereria bancrofti in Culex 1-019-3702-6. quinquefasciatus and Anopheles gambiae and its effect on parasite escape 13. Lok JB, Mika-Grieve M, Grieve RB. Cryopreservation of Dirofilaria immitis from the vector. Trans R Soc Trop Med Hyg. 2000;94(3):341–7. https ://doi. microfilariae and third-stage larvae. J Helminthol. 1983;57(4):319–24. org/10.1016/s0035 -9203(00)90349 -3. https ://doi.org/10.1017/s0022 149x0 00110 19. 28. Paily KP, Hoti SL, Manonmani AM, Balaraman K. Longevity and migration 14. Marcombe S, Farajollahi A, Healy SP, Clark GG, Fonseca DM. Insecticide of Wuchereria bancrofti infective larvae and their distribution pattern in resistance status of United States populations of Aedes albopictus relation to the resting and feeding behaviour of the vector mosquito and mechanisms involved. PLoS One. 2014;9 7:e101992; https ://doi. Culex quinquefasciatus. Ann Trop Med Parasitol. 1995;89(1):39–47. https :// org/10.1371/journ al.pone.01019 92.doi.org/10.1080/00034 983.1995.11812 927. 15. Povelones M, McCrea AR. Infecting and mosquitoes with Dirofilaria immi- 29. Han YS, Barillas-Mury C. Implications of time bomb model of ookinete tis and Brugia malayi. 2020; doi: 10.17504/protocols.io.bb7birin invasion of midgut cells. Insect Biochem Mol Biol. 2002;32(10):1311–6. 16. Povelones M, McCrea AR. Dirofilaria immitis and Brugia malayi emergence 30. Vlachou D, Zimmermann T, Cantera R, Janse CJ, Waters AP, Kafatos FC. assay for individual or pools of mosquitoes. 2020; doi: 10.17504/protocols. Real-time, in vivo analysis of malaria ookinete locomotion and mosquito io.bb7airie midgut invasion. Cell Microbiol. 2004;6(7):671–85. 17. Lok JB, Knight DH, Wang GT, Doscher ME, Nolan TJ, Hendrick MJ, et al. 31. Christensen BM. Observations on the immune response of Aedes Activity of an injectable, sustained-release formulation of moxidectin trivittatus against Dirofilaria immitis. Trans R Soc Trop Med Hyg. administered prophylactically to mixed-breed dogs to prevent infection 1981;75(3):439–43. with Dirofilaria immitis. Am J Vet Res. 2001;62(11):1721–6. 32. Bartholomay LC. Infection barriers and responses in mosquito–filarial 18. Lok JB, Mika-Grieve M, Grieve RB, Chin TK. In vitro development of worm interactions. Curr Opinion Insect Sci. 2014;3:37–42. https ://doi. third- and fourth-stage larvae of Dirofilaria immitis: comparison of basal org/10.1016/j.cois.2014.08.006. culture media, serum levels and possible serum substitutes. Acta Trop. 33. Dharmarajan G, Walker KD, Lehmann T. Variation in tolerance to 1984;41(2):145–54. parasites affects vectorial capacity of natural Asian tiger mosquito 19. Edgerton EB, McCrea AR, Berry CT, Kwok JY, Thompson LK, Watson B, et al. populations. Curr Biol. 2019;29(22):3946-52.e5. https ://doi.org/10.1016/j. Activation of mosquito immunity blocks the development of transmis- cub.2019.09.047. sion-stage filarial nematodes. Proc Natl Acad Sci USA. 2020. https ://doi. 34. Evans CC, Moorhead AR, Storey BE, Blagburn BL, Wolstenholme AJ, org/10.1073/pnas.19093 69117 . Kaplan RM. Evaluation of the larval migration inhibition assay for detect- 20. Kartman L. Factors influencing infection of the mosquito with Dirofi- ing macrocyclic lactone resistance in Dirofilaria immitis. Vet Parasitol. laria immitis (Leidy, 1856). Exp Parasitol. 1953;2(1):27–78. https ://doi. 2017;246:76–81. https ://doi.org/10.1016/j.vetpa r.2017.09.003. org/10.1016/0014-4894(53)90005 -8. 35. Liu C, Mhashilkar AS, Chabanon J, Xu S, Lustigman S, Adams JH, et al. 21. McGreevy PB, McClelland GA, Lavoipierre MM. Inheritance of susceptibil- Development of a toolkit for piggyBac-mediated integrative transfec- ity to Dirofilaria immitis infection in Aedes aegypti. Ann Trop Med Parasitol. tion of the human filarial parasite Brugia malayi. PLoS Negl Trop Dis. 1974;68(1):97–109. 2018;12(5):e0006509. https ://doi.org/10.1371/journ al.pntd.00065 09. 22. Nayar JK, Knight JW, Bradley TJ. Further characterization of refractoriness 36. Bancroft TL. Report LXXXV: Some further observations on the life-history in Aedes aegypti (L.) to infection by Dirofilaria immitis (Leidy). Experimen- of Filaria Immitis Leidy. Br Med J. 1904;1(2258):822–3. tal parasitology. 1988;66(1):124–31. http://dx.doi.org/10.1016/0014- 4894(88)90057 -4. Publisher’s Note 23. Sauerman DM Jr, Nayar JK. Characterization of refractoriness in Aedes Springer Nature remains neutral with regard to jurisdictional claims in pub- Aegypti (Diptera: Culicidae) to infection by Dirofilaria immitis. J Med Ento - lished maps and institutional affiliations. mol. 1985;22(1):94–101. https ://doi.org/10.1093/jmede nt/22.1.94. 24. Erickson SM, Xi Z, Mayhew GF, Ramirez JL, Aliota MT, Christensen BM, et al. Mosquito infection responses to developing filarial worms. PLoS Negl Trop Dis. 2009;3 10:e529; https ://doi.org/10.1371/journ al.pntd.00005 29. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Parasites & Vectors Springer Journals

A novel assay to isolate and quantify third-stage Dirofilaria immitis and Brugia malayi larvae emerging from individual Aedes aegypti

Download PDF
12 pages

Loading next page...
 
/lp/springer-journals/a-novel-assay-to-isolate-and-quantify-third-stage-dirofilaria-immitis-yGQ5rj2l4a

References (50)

Publisher
Springer Journals
Copyright
Copyright © The Author(s) 2021
eISSN
1756-3305
DOI
10.1186/s13071-020-04529-w
Publisher site
See Article on Publisher Site

Abstract

Background: Mosquitoes transmit filarial nematodes to both human and animal hosts, with worldwide health and economic consequences. Transmission to a vertebrate host requires that ingested microfilariae develop into infective third-stage larvae capable of emerging from the mosquito proboscis onto the skin of the host during blood-feeding. Determining the number of microfilariae that successfully develop to infective third-stage larvae in the mosquito host is key to understanding parasite transmission potential and to developing new strategies to block these worms in their vector. Methods: We developed a novel method to efficiently assess the number of infective third-stage filarial larvae that emerge from experimentally infected mosquitoes. Following infection, individual mosquitoes were placed in wells of a multi-well culture plate and warmed to 37 °C to stimulate parasite emergence. Aedes aegypti infected with Dirofilaria immitis were used to determine infection conditions and assay timing. The assay was also tested with Brugia malayi- infected Ae. aegypti. Results: Approximately 30% of Ae. aegypti infected with D. immitis and 50% of those infected with B. malayi pro- duced emerging third-stage larvae. Once D. immitis third-stage larvae emerged at 13 days post infection, the propor- tion of mosquitoes producing them and the number produced per mosquito remained stable until at least day 21. The prevalence and intensity of emerging third-stage B. malayi were similar on days 12–14 post infection. Increased uptake of D. immitis microfilariae increased the fitness cost to the mosquito but did not increase the number of emerging third-stage larvae. Conclusions: We provide a new assay with an associated set of infection conditions that will facilitate assessment of the filarial transmission potential of mosquito vectors and promote preparation of uniformly infectious third-stage larvae for functional assays. The ability to quantify infection outcome will facilitate analyses of molecular interac- tions between vectors and filariae, ultimately allowing for the establishment of novel methods to block disease transmission. Keywords: Aedes aegypti, Aedes albopictus, Dirofilaria immitis, Brugia malayi, Mosquito, Filaria, Transmission *Correspondence: [email protected] Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. McCrea et al. Parasites Vectors (2021) 14:30 Page 2 of 12 Background Arthropods serve as intermediate hosts and vectors of numerous human and animal infective filariae that con - tribute to a large disease burden worldwide. Indeed, mosquito-transmitted lymphatic filariasis, caused by Wuchereria bancrofti, Brugia malayi and B. timori, affects approximately 120 million people in 83 countries [1]. Mosquitoes are also responsible for transmission of animal infective filariae, the most studied being Dirofi - laria immitis, the causative agent of canine heartworm disease. Although detailed global numbers are not avail- able, there are an estimated 250,000–500,000 infected dogs in the USA, with some areas, such as the Mississippi River basin, reporting infection prevalence as high as 40% [2, 3]. Humans can also be incidentally infected by D. immitis, but they do not support the entire life cycle and typically present with only mild clinical signs [3]. Within the mosquito host, ingested microfilariae (mf ) migrate from the midgut to specific tissues specific to the mosquito species. The filarial agents of lymphatic filaria - sis migrate to the indirect flight muscles in the thorax, whereas D. immitis migrates into the Malpighian tubules (MT) [4]. In these tissues, the parasites develop intracellu- Fig. 1 Emergence of infective third-stage larvae (L3) from the tip larly, undergoing successive molts to form third-stage lar- of the mosquito proboscis. During blood-feeding, a subpopulation vae (L3). Some L3 migrate to the proboscis where they are of L3 in the labial sheath of the proboscis emerge from the poised for transmission (Additional file  1: Movie 1). Infec- proboscis, alighting on the skin of the host in a drop of the mosquito hemolymph (eL3). It is estimated that only approximately 10% of the tive L3 emerge most typically from the mosquito labellum eL3 survive on the skin and penetrate into it [26]; the fate of the L3 but they can also emerge from the labial sheath during that do not emerge during blood-feeding is not known. Emergence blood-feeding (Fig. 1) [5]. Larvae emerging from the mos- from the proboscis typically occurs at the labellum or distal portion quito are deposited onto the skin in a drop of hemolymph of the labial sheath and can be triggered by sensation of a thermal where they can enter the skin through the bite site [5]. The cue (red gradient). Other cues may also play a role, such as chemical compounds released by the host (green gradient) or a sensation of fate of the L3 that do not emerge is not known. mechanical forces caused by the deformation of the labial sheath Due to their importance to the disease cycle, much (orange gradient), which slides backward as the fascicle is inserted attention is focused on the transmission of infective L3. into the host skin. Adapted from Bancroft [36] Different methods have been employed to assay L3 in mosquitoes. A common approach, especially for field isolates, is the detection by PCR of larvae in the head In addition to assessing the intensity of transmission and other body parts of the mosquito following dissec- in field populations, an ability to enumerate and collect tion [6–8]. Although this method is not established for all L3 capable of emergence would greatly enhance func- filariae, stage-specific PCR has been used for detection tional assessment. Currently, studies of host immune of infectious L3 in W. bancrofti infected mosquitoes [9]. responses to L3, as well as in vitro and in vivo drug effi - PCR is sensitive and species specific and can be used on cacy studies, are typically carried out on L3 collected pools of mosquitoes in cases where infection prevalence from infected mosquitoes gently disrupted with a mor- is low. Another commonly used approach is to physi- tar and pestle and then separated by filtration though a cally examine larvae in the labial sheath of the proboscis mesh [10–12]. However, it is unknown whether all L3 or in dissected tissues of the mosquito. This approach is harvested by this method are mature and competent particularly useful in a laboratory setting to determine to infect the host. Furthermore, obtaining microfilaria the number of larvae at each developmental stage and to for mosquito infections requires access to an infected identify their tissue of residence. However, while each of animal or access to samples from another source, and these assays can reveal the number of L3 that have devel- although D. immitis microfilaria have been shown to be oped, specific assays to enumerate L3 capable of emerg - capable of cryopreservation [13], this method of stor- ing (eL3) when the mosquito blood-feeds have not been age is not commonly used. More typically, fresh sam- previously described. ples are shipped, despite being infective, little is known M cCrea et al. Parasites Vectors (2021) 14:30 Page 3 of 12 about how short-term storage during shipping affects with the guidelines of the Institutional Animal Care development to L3. and Use Committee of the University of Pennsylvania Blocking parasite development in the vector is a (IACUC, protocol 805059). The microfilarial load in novel approach being considered for controlling disease this dog was approximately 50,000 mf/ml. Blood con- transmission and requires a thorough understanding of taining B. malayi microfilariae (B. malayi microfilariae the molecular interactions between parasites and their in cat blood, live, FR3, NR-48887) was obtained from an vector. The means to quantify the prevalence of mos - experimentally infected cat containing with a microfi - quitoes with eL3 and determine the number of emerg- larial load of approximately 12,000 mf/ml (provided by ing L3 per mosquito would greatly facilitate this work. the NIH/NIAID FR3 for distribution by BEI Resources, Here we present a new assay to quantify infectious eL3 NIAID/NIH). In both cases, the blood was diluted with that works with different vector and filarial species. the appropriate volume of heparinized sheep blood to a We then use this assay to study the development and concentration of 4000 mf/ml. The sample was warmed characteristics of D. immitis eL3 in mosquitoes. The to 37 °C, and a 3.5-ml aliquot was placed in the inden- detailed infection parameters and assay conditions pre- tation on the outside of the bottom of a 300-ml plastic sented here to quantify and produce maximum yields baby bottle (Advent; Phillips, Amsterdam, the Neth- of eL3 could potentially facilitate both studies between erlands) and covered with Parafilm. The inside of the filariae and their mosquito and vertebrate hosts. bottle was filled with water at 37  °C. This assembly was placed with the membrane side down onto the top mesh of mosquito cages and the insects are allowed to Methods feed for 15  min (Fig.  2a). Immediately following the Mosquito strains and culture Aedes aegypti and Ae. albopictus strains were provided by the National Institutes of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID) Filariasis Research Reagent Resource Center (FR3) and the Malaria Research and Reference Reagent Resource Center (MR4) through distribution by the NIH/NIAID BEI Resources Repository. Aedes aegypti (Ae. aegypti, Strain Black Eye Liverpool, Eggs, FR3, NR-48921) is a Dirolfi aria immi - tis- and Brugia malayi-susceptible strain, and Ae. aegyp- ti (Ae. aegypti, Strain LVP-IB12, Eggs, MR4, MRA-735, contributed by David W. Severson) is a D. immitis- and B. malayi-refractory strain. Both strains were reared at 27  °C and 80% humidity under a 12/12-h photoperiod. NJ Aedes albopictus (Ae. albopictus Strain ATM-NJ95, Eggs, MR4, NR-48979) [14] was reared at 24 °C and 70% humidity under a 16/8-h (light/dark) photoperiod. Mos- quitoes were housed in 30-cm cages (BugDorm, Tai- chung, Taiwan) at a density of approximately 1000 per cage. Larvae were maintained at a density of 1 larva/3 ml. Larvae were fed a suspension of liver powder in water Fig. 2 Assay for eL3. a Infection and assay protocol followed in this (MP Biomedicals, Solon, OH, USA), and adults were study. Mosquitoes are fed on blood containing Dirofilaria immitis microfilariae. Uptake is measured in a small group, and the remaining maintained on 10% sucrose in water, which was changed mosquitoes are maintained until an emergence assay is performed. daily. Heparinized sheep blood (Hemostat, Dixon, CA, Immediately following the emergence assay with whole mosquitoes, USA) was provided using an artificial membrane feeder the Malpighian tubules are dissected and analyzed. The dissected at 37 °C for egg production. head and carcass are individually placed into an emergence assay to assay L3 that failed to emerge from intact mosquitoes. Additional file 2: Figure S1 shows images of dissected mosquitoes and Mosquito infection with Dirofilaria immitis and Brugia Malpighian tubules. b, c Mosquitoes are rinsed with 70% ethanol:30% malayi water for wetting, rinsed with water (b) and then placed individually The full protocol is described in greater detail by Pov - into wells of a 96-well plate (c). d Emerging eL3 from intact elones and McCrea [15]. Blood containing D. immitis mosquitoes or L3 from dissected heads and carcasses at the bottom each well are scored by microscopy. Additional file 3: Movie 2 shows microfilariae was obtained from an experimentally typical movement of eL3. e Larvae (white arrows) in live Malpighian infected dog according to Institutional Animal Care and tubules are scored by microscopy. Scale bar: 50 µm Use Committee-approved protocols and in accordance McCrea et al. Parasites Vectors (2021) 14:30 Page 4 of 12 feed, fully engorged blood-fed mosquitoes were sepa- tissue for at least 60 min and scored as described above. rated using CO anesthesia and housed under standard Only a portion of the mosquitoes, between 30 and 36 insectary conditions as described above. Any partial or from each replicate, used for the eL3 emergence assay unfed mosquitoes were discarded. were processed in this manner due to processing time constraints. Microfilarial uptake Within 1 hour of blood-feeding, fed mosquitoes were Emergence assay time course and microfilaria anesthetized with CO at room temperature. The entire concentration series undamaged midgut was dissected in deionized water Groups of 432–565 mosquitoes were fed on blood con- in a depression slide and immediately transferred to a taining D. immitis microfilariae at different concentra - standard microscope slide containing a 50-µl drop of tions and housed as described above. Mosquito mortality deionized water. Damaged midguts leaking blood dur- was monitored daily, as assessed by the fraction of the ing dissection were not used. The epithelium of the gut population that died each day. This was necessary since was separated from the blood bolus and moved through groups of 50 mosquitoes were removed for eL3 assays the water to remove any residual blood before it was as described above. Since the emergence prevalence was discarded. The blood was roughly dispersed using for - very low on day 12, emergence assays were performed ceps and then pipetted up-and-down with a 20-µl with only 25 mosquitoes per replicate saving more of the pipette. Microfilaria in the entire drop were counted population for later days where there would be a higher immediately without a coverslip. eL3 prevalence. For the concentration-series experi- ments, a blood dilution containing 32,000 mf/ml was created and then subjected to twofold serial dilution to Emergence assay for eL3 and other larval stages create feeding doses of 16,000, 8000 and 4000 mf/mL. At The full protocol is described in greater detail by Pov - each concentration, five mosquitoes of each strain were elones and McCrea [16], which is modified from a used to measure uptake, as described above. For these previously published method [17]. Briefly, eL3 were experiments, mortality was monitored daily until day 17 assayed by placing live mosquitoes in 70% ethanol when an assay for eL3 was performed on the remaining in water for 1  min, following which they were rinsed mosquitoes. Kaplan–Meier survival analysis was per- twice in deionized water and then placed individually formed between adjacent doses within each mosquito into wells of a 96-well plate containing 200 µl Dulbec- strain and between the same doses across the mosquito co’s Modified Eagle’s Medium (DMEM) supplemented strains. with l -glutamine, a high level of glucose and sodium pyruvate (Corning Mediatech, Manassas, VA, USA). At this point, the mosquitoes are still alive and can be Results observed moving in the buffer, but they cannot stand Novel assay for filarial nematode L3 emerging on the surface due to the wetting procedure. The plate from individual mosquitoes was then placed in a 37  °C incubator with 5% C O for In our assay, outlined in Fig.  2a, we infect mosquitoes 60 min, and the number of eL3 were determined with- using an artificial membrane feeder and then determine out removing the mosquitoes using an inverted micro- the number of microfilariae ingested immediately after - scope with a ×4 objective. In some experiments, after wards [15]. The microfilarial uptake represents the the - the eL3 emergence assay, mosquitoes were removed oretical maximum number of parasites in the assay. To from the plate individually for assaying other larvae. assay the L3 capable of emerging from the mosquitoes, First, the Malpighian tubules were dissected by remov- we simulate the thermal cue that would be experienced ing the posterior two abdominal segments. The set of by a mosquito landing on a mammalian vertebrate host five Malpighian tubules was removed from the midgut by placing mosquitoes in buffer and warming the buffer and transferred to a microscope slide containing 15 µl to 37  °C [16]. Mosquitoes are placed individually into phosphate buffered  saline and covered with a 22-mm wells of a 96-well plate after the wetting procedure coverslip. The larvae were counted, and although dif - (Fig. 2b, c), and care is taken to minimize damage to the ferent stages were present we did not categorize them mosquito during transfer. Upon warming, competent (Additional file  4: Figure S2). The head was then sepa - L3 emerge from the mosquito and sink to the bottom rated from the body, and the dissected head and car- of the well, where they can be counted (Fig.  2d; Addi- cass fragment were placed separately into fresh wells tional file  3: Movie 2). eL3 collected by this method are of a 96-well plate prepared and incubated as described capable of molting to the fourth larval stage in  vitro above. L3 were allowed to migrate out of the dissected [18] and are infectious to dogs [17], and we believe M cCrea et al. Parasites Vectors (2021) 14:30 Page 5 of 12 they represent the number of L3 that emerge onto the could not unambiguously characterize them as L3. We skin of the host. After the emergence assay, the mos- found that the number of eL3 was significantly lower quitoes are still viable and can move, but they cannot than the total L3 (Fig. 3c). Therefore, we concluded that escape the buffer due to the wetting procedure. Nota - only a fraction of the larvae that develop to L3 emerge bly, following the emergence assay, further analyses can during our emergence assay, consistent with previous be carried out to determine the number and location observations [5]. of larvae remaining within the mosquito. In our study, both dissected Malpighian tubules (Fig.  2e), and the Once eL3 have developed, their intensity and prevalence head and remaining carcass were assayed separately, are constant as described in the following section (Additional file  2: To determine the optimal day to perform the emergence Figure S1). assay, we wanted to identify a time point at which eL3 recovery was maximized but mosquito mortality was still minimal. To do this, we infected populations ranging Microfilariae robustly migrate into the Malpighian tubules from 432 to 565 individuals, and then removed groups but only a fraction develop to eL3 of approximately 50 mosquitoes on consecutive days for To determine the efficiency of eL3 development we an emergence assay. Data from four independent experi- infected D. immitis susceptible (Ae. aegypti ) or refrac- ments were pooled since it was not possible to obtain tory (Ae. aegypti ) strains and performed an emergence all time points from a single infection. We assessed the assay 14  dpi. We found that Ae. aegypti released no variation in eL3 number over the 10-day period from 12 eL3, despite having a median uptake of 12 mf/mosquito to 21 dpi. The main emergence phase started at 13 dpi, (Fig.  3a). This is consistent with previous observations as we observed only a single eL3 in 48 mosquitoes at 12 that ingested microfilariae arrest their development dpi. Once L3 begin to emerge from mosquitoes, both the soon after invading cells of the Malpighian tubules number that emerge per mosquito as well as the preva- [19–23]. In the susceptible strain (Ae. aegypti ), 81 lence of mosquitoes with at least one emerging L3 are of the 252 mosquitoes assayed (32%) had at least one relatively constant from 13 to 21 dpi (Fig.  4). These data eL3 when given a median uptake of 15 mf/mosquito suggest that larvae develop synchronously and that once (Fig.  3b). A portion of the susceptible mosquitoes used they reach the L3 stage, their numbers remain relatively for the emergence assay were dissected, and a substan- stable over time. Beyond 13 dpi, mosquito mortality con- tial number had L3 remaining in the dissected heads tinues and eL3 prevalence or intensity is not increased, (26/102; 25%) and carcasses (48/102; 47%) (Fig.  3b). Therefore, to maintain population sizes and maximize The majority of larvae assayed at 14 dpi were present in eL3 yield we typically assay mosquitoes 14–17 dpi. the Malpighian tubules, and nearly all of the dissected mosquitoes (93/96; 97%) had at least one larva present Increasing the number of Dirofilaria immitis microfilariae in the Malpighian tubules (Fig. 3b). In a few cases, Mal- ingested does not increase eL3 prevalence pighian tubules were damaged during the dissection To optimize the number of eL3 recovered from the mos- and excluded from this and subsequent analyses. Pool- quito, we next determined if increasing the number of ing all larvae present across all tissues revealed that the microfilariae in the blood meal improved the recovery median total number of larvae per mosquito was eight. of eL3. For these experiments we used the standard con- Therefore, at 14 dpi, 53% of the 15 ingested microfilar - centration of 4000 mf/ml and included three additional iae could be accounted for as larvae in the mosquito concentrations of 8000, 16,000 and 32,000 mf/ml. We (Fig. 3b). In the dissected Malpighian tubules, we found found that increased concentrations of microfilaria in the larvae in various stages of development, but we did membrane feeder had a consequent impact on the num- not determine their stage (Additional file  4: Figure S2). ber present in the mosquito midgut immediately after We note, however, that larvae found in the Malpighian blood-feeding, with no significant difference between S R tubules, even the most stunted sausage forms, are via- Ae. aegypti and Ae. aegypti (Fig.  5a). At each successive ble, as we observed them to be capable of movement. concentration, microfilariae present in the blood meal Previous studies suggest that not all L3 emerge when increased approximately twofold. We monitored mosquito infected mosquitoes blood-feed on an animal [5]. Since survival for 17 days, at which time an emergence assay was this previous observation was not quantitative, we ana- performed. We chose day 17 to allow more time to moni- lyzed the data from the emergence assay (Fig.  3b) to tor mosquito survival. The prevalence of eL3 in Ae. aegypti compare the number of eL3 to the total L3 (eL3 + L3 was not significantly different, regardless of the concentra - in dissected heads, and dissected carcasses). We did not tion of microfilariae fed (Fig.  5b). On a population level, the include larvae found in the Malpighian tubules since we number of eL3 produced per mosquito was not increased McCrea et al. Parasites Vectors (2021) 14:30 Page 6 of 12 Fig. 3 A fraction of ingested microfilariae develops into eL3 in Aedes aegypti (a D. immitis- and Brugia malayi-susceptible strain). a Dots indicate the number of microfilariae present in the midgut of individual Ae. aegypti (a D. immitis- and B. malayi-resistant strain) immediately after feeding on infected blood (Uptake) and the number of eL3 assayed 14 days post infection (dpi). The black line indicates the median. b Dots indicate the number of microfilariae present in the midgut of individual Ae. aegypti immediately after feeding on infected blood (Uptake), the number of eL3 assayed 14 dpi (eL3), the number of L3 emerging from the dissected heads (Head) or carcass (Carcass) and the number present in the Malpighian tubules (MT ). The Total is sum of all parasites of any stage found in any tissue or assay on 14 dpi. The red line indicates the median. c Dots are the number of eL3 from individual Ae. aegypti and all third-stage larvae assayed ( Total; sum of eL3, Head, and Carcass) (data taken from panel b). The red line is the median number. The asterisks indicate a Mann–Whitney P value < 0.001. These data are the sum of three independent biological replicates. The number of mosquitoes assayed is shown below each column by feeding concentrations of microfilariae greater than uninfected blood to 4000 mf/ml and 16,000 to 32,000 mf/ 4000 mf/ml (Fig.  5c). Increasing the concentration of D. ml comparisons were significantly different (Fig.  5e). When S S R immitis microfilaria used to infect Ae. aegypti increased we compared the survival of Ae. aegypti and Ae. aegypti mosquito mortality in a dose-dependent manner (Fig.  5d). at the different concentrations of microfilariae, there was Each increase in microfilaria concentration significantly no difference in survival at the lowest concentration (Addi - increased mortality from the previous one, including the tional file  5: Figure S3). There was a modest difference at lowest concentration, which elevated mortality compared 8000 mf/ml, with Ae. aegypti showing greater mortality, to those fed on uninfected blood. A similar dose–response and this difference further increased at the two highest was observed in the refractory strain; however, only the concentrations. S S Fig. 4 The number of D. immitis eL3 from Ae. aegypti are constant between 13 and 21 dpi. a Graph of the average prevalence in Ae. aegypti of D. immitis eL3 assayed 12–21 dpi. Error bars indicate the standard deviation (SD). b Dots are the number of D. immitis eL3 from individual mosquitoes assayed from 12 to 21 dpi. This experiment was performed four times. Two replicates were assayed on 12 to 18 dpi; the other two replicates were assayed on 14 to 21 dpi. Each replicate was performed with approximately 50 mosquitoes. The replicates on 12 dpi were performed with fewer mosquitoes since our preliminary data suggested that emergence was negligible on this day M cCrea et al. Parasites Vectors (2021) 14:30 Page 7 of 12 Stored blood containing microfilariae retains its ability fresh microfilaremic blood and ones fed on an aliquot of to infect mosquitoes and produce eL3 the same blood sample stored for 1 day at 4 °C (Fig.  6a). All D. immitis experiments described above used freshly However, there were significant decreases in eL3 preva - isolated microfilaremic blood. However, there are times lence in mosquitoes fed on microfilaremic blood stored when it is not possible to use this material immediately, for 2 days at 4° C compared to mosquitoes fed on blood such as when blood is shipped from another location. We stored at 4° C for 1 day and to mosquitoes fed on fresh wanted to compare the number of eL3 produced when blood. Similar trends were observed in infection inten- mosquitoes were fed infected blood stored at 4  °C for 1 sity (Fig. 6b). Our data show that although microfilaremic or 2 days. For these experiments, we fed mosquitoes with blood stored at 4 °C for 2 days can still infect mosquitoes, freshly isolated infected blood and on the next 2 consec- there is a trend towards lower numbers of eL3 follow- utive days fed new cohorts with the same sample that had ing blood storage, suggesting that refrigerated samples been stored at 4  °C. We performed an emergence assay should be used as soon as possible for maximum infec- at day 17 of each infection. There was no significant dif - tion potency. ference in the prevalence of eL3 between mosquitoes fed Fig. 5 Increasing the dose of microfilariae does not increase numbers of eL3 but increases mosquito mortality. a Dots indicate the number of D. S R immitis microfilariae present in midguts of Ae. aegypti (blue) and Ae. aegypti (red) immediately following blood-feeding on the indicated doses of microfilariae. Data in each column are normally distributed, and black lines and numbers indicate the mean. No significant differences were S R found when Ae. aegypti and Ae. aegypti were compared at the different doses using an unpaired t-test. Data are from two independent biological replicates. b Graph of the average prevalence of D. immitis eL3 in mosquitoes feeding on blood with increasing concentrations of microfilariae, assayed 17 dpi. Error bars indicate the SD. There was no significant difference comparing the columns using analysis of variance. c Dots are the number of D. immitis eL3 from individual mosquitoes assayed 17 dpi. No significant differences were found when we compared all groups to each other or compared 4000 mf/mL to the other groups using a Kruskal–Wallis test with Dunn’s correction for multiple comparisons. Data for panels b S R and c are pooled from four separate biological replicates. d, e Kaplan–Meier survival plot for Ae. aegypti (d) or Ae. aegypti (e) fed with uninfected blood (BF) or blood containing different concentrations of microfilariae (4000, 8000, 16,000, 32,000 mf/ml). Pairs of adjacent treatment groups were analyzed by Kaplan–Meier, and relationships with significant differences are indicated with asterisks in Additional file 5: Figure S3 and in Additional file 6: Table S1 McCrea et al. Parasites Vectors (2021) 14:30 Page 8 of 12 Emergence assay works in a natural vector of Dirofilaria immitis Since Ae. aegypti is not considered to be an important natural vector of D. immitis, we wanted to determine whether eL3 could be obtained from a mosquito species associated with D. immitis transmission. For these exper- iments we used a strain of Ae. albopictus isolated from NJ Keyport, NJ, USA (Ae. albopictus ). We found that eL3 were present at 17 dpi, but their prevalence and number were lower than those present in Ae. aegypti (Fig.  7a, b) when the mosquitoes were reared and housed in our standard insectary conditions (27  °C with 80% rela- tive humidity [RH] and a 12:12-h photoperiod). When NJ we tested Ae. albopictus reared and infected under its Fig. 6 Infected blood stored at 4 °C produces eL3. Infected blood ideal temperature and humidity conditions (24  °C, 70% was used fresh (0) or after storage for 1 or 2 days (1, 2) at 4 °C. a RH, and a 16:8-h [light/dark] photoperiod), the preva- Graph of the average prevalence of D. immitis eL3 at 17 dpi in Ae. lence and number of eL3 were increased, although they aegypti . The error bar indicates the SD. The number of mosquitoes (n) were still lower than was typical for Ae. aegypti (Fig. 7c, analyzed is indicated for each sample. Asterisks indicate a significant NJ d), likely because the strain of Ae. albopictus used did difference at P < 0.05 using Fisher’s exact test. b Blue dots are the number of D. immitis eL3 emerging from individual mosquitoes not feed to repletion from an artificial membrane feeder assayed at 17 dpi. Asterisks indicate significant differences in under any conditions that we tested. Even though we intensity using Kruskal–Wallis test with Dunn’s correction for multiple were not able to make quantitative comparisons to Ae. comparisons (*P < 0.05; **P < 0.01). Data are pooled from two aegypti , our data show that the emergence assay works independent experiments with different mosquito species. Individual mosquito emergence assay for transmission residing in the mosquito emerge when stimulated either stage Brugia malayi by warming the mosquito or by direct contact with a To determine whether the emergence assay will be mammalian host, even if already residing in the head. informative for enumerating eL3 for other filarial nem - Our method provides a more accurate indication of a atodes, we infected  mosquitoes with B. malayi. These particular vector species’ potential for pathogen trans- parasites have a shorter developmental period in the mission. For example, our results show that not all strains mosquito [24], so we performed our emergence assay at of Ae. aegypti or Ae. albopictus support eL3 develop- S R 12–14 dpi in both Ae. aegypti and Ae. aegypti mosqui- ment, even though microfilariae invade the Malpighian toes. Despite taking up a similar number of microfilar - tubules. In other studies, we used this assay to show that iae in the blood meal (Fig.  8a), no eL3 were produced activation of the Toll immune signaling pathway restricts by Ae. aegypti , showing that this strain is refractory to development of D. immitis and B. malayi eL3 [19, 25]. both B. malayi and D. immitis (Fig.  8b). In contrast, a In addition, this assay could also permit assessment of significant proportion of Ae. aegypti mosquitoes devel- manipulations of the mosquito host or larvae that result oped eL3, showing that this strain is susceptible to both in an inability of L3 to emerge in response to thermal B. malayi and D. immitis (Fig.  8b). There was no sig - stimuli or due to motor defects that could be missed by nificant difference in either the prevalence of mosqui - current methods. Finally, the results of our assays sug- toes producing eL3 or in the number of eL3 produced gest that quantitating all L3 by dissection will likely result per mosquito between the days we assayed (Fig.  8b-c). in overestimating the number of larvae that are capable These data indicate that despite their different life-cycle of emerging from the mosquito. This is particularly true in the mosquito, eL3 of human filariae can be measured in the case where the number of L3 is derived by mos- using our emergence assay. quito homogenization, as L3 from the body cavity and those present in the Malpighian tubules are likely to be Discussion included in this method. The development of an assay for eL3 may be valuable for Overall, we found that 32% of the D. immitis-infected studying mosquito–filaria interactions, thereby provid - and approximately 50% of B. malayi-infected mosqui- ing a clearer indication of vector competence and vec- toes develop at least one eL3, which is similar to previ- tor transmission intensity. As our data and a previously ous observations measuring emergence of L3 [26–28]. reported observation [5] indicate, only a portion of L3 Since mosquitoes typically blood-feed more than once, it M cCrea et al. Parasites Vectors (2021) 14:30 Page 9 of 12 Fig. 7 Aedes albopictus supports eL3 development. a, c Graphs of the average prevalence of D. immitis eL3 emerging at 17 dpi in Ae. aegypti (blue S NJ NJ NJ bar, Ae. aeg ) or Ae. albopictus (purple bar, Ae. albo ) reared and maintained under standard insectary conditions (a) or in Ae. albopictus (purple NJ bar, Ae. albo ) reared and maintained at 24 °C, 70% relative humidity and 16:8-h (light/dark) photoperiod (c). Error bars indicate the SD. The number of mosquitoes (n) analyzed is indicated for each sample. b, d Blue and purple dots are the number of D. immitis eL3 emerging at 17 dpi from S S NJ NJ individual Ae. aegypti (Ae. aeg ) or Ae. albopictus (Ae. albo ) mosquitoes, respectively, reared and maintained under standard insectary conditions (b) or at 24 °C, 70% relative humidity and 16:8-h (light/dark) photoperiod (d) Fig. 8 Emergence assay can be used to quantify B. malayi eL3. a Dots indicate the number of Brugia malayi microfilariae present in midguts of S R Ae. aegypti (blue) and Ae. aegypti (red) immediately following blood-feeding. Data in each column are not normally distributed; the black line is the median. The number of mosquitoes (n) analyzed is indicated for each sample. No significant difference was found using a Mann–Whitney test S R (P = 0.09). b Graph of the average prevalence of B. malayi eL3 assayed 12–14 dpi in Ae. aegypti (blue bars) and at 12 and 14 dpi in Ae. aegypti (red bars). Error bars indicate the SD. The number of mosquitoes (n) analyzed is indicated for each sample. No emerging parasites were observed for Ae. R S S aegypti . There were no significant differences between the days assayed for Ae. aegypti , and all Ae. aegypti days assayed were significantly different from both Ae. aegypti on the two days assayed using an analysis of variance with Tukey’s multiple comparisons test, as indicated by asterisks (P < 0.001). c Blue dots are the number of B. malayi eL3 from individual mosquitoes assayed 12–14 dpi. No significant differences were found when all groups were compared to each other or when day 12 post infection was compared to the other groups using a Kruskal–Wallis test with Dunn’s correction for multiple comparisons. Data were pooled from three independent experiments is interesting to speculate that the L3 that do not emerge resources in the vector or a parasite strategy to control may enter the proboscis and emerge in a subsequent the density of L3 to limit damage to the vector. blood meal, thereby increasing the chance of transmis- Results of our experiments to optimize recovery of eL3 sion. On the other hand, it is also possible that the eL3 by increasing the dose of D. immitis microfilariae given we observe are the only ones competent to emerge and to mosquitoes reveal that microfilarial uptake and eL3 infect another host. In this case, the failure of the remain- emergence are uncoupled, as we did not observe signifi - ing L3 to emerge could be due to competition for limited cant increases in the prevalence of mosquitoes with eL3 McCrea et al. Parasites Vectors (2021) 14:30 Page 10 of 12 or the number of eL3 per mosquito at the doses tested. into hemolymph within the mosquito body cavity, where While it is likely that microfilarial uptake and eL3 emer - they could be eliminated or sequestered. As the pos- gence are coupled at lower concentrations of micro- sibilities outlined above are not mutually exclusive, we filariae, we did not test this since our main goal was to believe they could collectively account for the differ - establish conditions where eL3 recovery is maximized ential between microfilarial uptake and total parasites to support future study of transmission-blocking strate- ultimately detected. Interestingly, it was recently shown gies. Varying the timing of the assay revealed that at 13 using field populations of Ae. albopictus, a natural vec - dpi there is no significant difference in the prevalence or tor of D. immitis, that in areas of greater infection preva- number of eL3. Taken together, our data suggest that the lence, larval damage to the mosquito is better tolerated, Malpighian tubules have a limited capacity to support D. benefitting both the vector and the parasite [33]. immitis development and that no additional eL3 are pro- We used our emergence assay to analyze the effects of duced once the limit is reached. However, we observed storing microfilaremic blood at 4  °C and showed that it a range of developmental phenotypes in the Malpighian is possible to produce eL3 after a storage time of at least tubules of mosquitoes 14 days after D. immitis infection, 2 days, but likely even longer. However, while delays in raising the possibility that larval damage to the tubules initiating mosquito infection do not preclude experi- prevents further development, as all larvae are viable at mentation, the strongest prevalence and intensity of eL3 this time point, even those displaying delayed develop- production was observed in mosquitoes infected through ment phenotypes. While a substantial number of midgut the ingestion of fresh microfilaremic blood. Given the epithelial cells are invaded during infection by ookinetes loss of eL3 production with blood storage time, it is not of Plasmodium, the causative agent of malaria, damaged recommended to make comparisons within an experi- cells are eliminated and replaced through tissue regen- ment using a blood sample stored for different lengths of eration [29, 30]. As larval development is blocked and time. However, internal comparisons are reasonable, as is mosquito fitness is negatively impacted over time, this the goal of using the stored blood to produce eL3. process suggests that damaged cells of the Malpighian Because L3 obtained in our assay have responded to tubules persist and are not replaced. In addition, the con- physiological conditions, L3 produced via an emergence tinuing metabolism of the increased number of larvae or assay may provide a more easily accessible source of an accumulation of larval waste products may also con- infective larvae than L3 obtained from gently crushed tribute to the increased mosquito mortality we observed and sieved mosquitoes [34]. While we developed this in Ae. aegypti at later timepoints. emergence assay for use on single mosquitoes, it is By quantifying microfilarial uptake in individual mos - also suitable for use on populations en masse, and eL3 quitoes and comparing it to all parasites that emerge obtained this way are known to be infective [17]. As such, from either the whole mosquito, the head or the car- eL3 may be quite valuable for future identification and cass or to those present in the Malpighian tubules at 14 testing of novel heartworm preventatives, which requires dpi, we found that approximately 60% of the ingested infectious D. immitis L3, as well as for studies of the host microfilariae are accounted for at 14 dpi. Although we immune response to infection, which often require infec- are accounting for a majority of the parasites ingested by tious L3. Similarly, in vitro L3 to L4 molting assays, such the mosquitoes, it is nevertheless interesting to specu- as the newly developed genetic transformation protocol late about the fate of those that are not assayed. Some L3 for B. malayi [35], may benefit from L3 produced using remain in the proboscis of dissected heads, even after the our method. Finally, given the widespread nature of vec- primary emergence assay and the subsequent emergence tor-borne filarial diseases and their potential to spread assay after dissection. However, we did not systematically due to changing environmental conditions, new meth- attempt to quantify these, and it remains possible that L3 ods like this could help assess transmission potential in may also be retained in the dissected carcasses. In addi- the field and to develop methods in the laboratory to tion, because some microfilariae may be damaged by the block transmission. This easy, efficient and cost-effective mosquito immune system or fail to follow sensory cues, method has the potential to be a valuable tool for the they may fail to invade the Malpighian tubules and may quantitative analysis of mosquito transmission of filariae, be voided into the hindgut and then the feces [4, 31, 32]. especially to researchers in countries most affected by Likewise, the activity of microfilariae and larvae in the these parasites where resources are often constrained. Malpighian tubule cells may damage these cells, some- times to the point where microfilariae are released into Conclusions the tubule lumen, where they also could be eliminated as We have established a novel assay to determine the waste. Alternatively, if the basal side of the Malpighian number of infectious L3 filarial larvae capable of tubule cell is compromised, parasites could be released emerging from individual mosquitoes, which we refer M cCrea et al. Parasites Vectors (2021) 14:30 Page 11 of 12 Authors’ contributions to as eL3. We have shown this assay works with both MP was responsible for the study conception. ARM, EBE, GTO, FMO and MP different mosquito and filarial nematode species. As were responsible for acquisition of data. ARM, EBE, GTO, FMO, TJN, JBL and MP such, our method is likely to be suitable for assess- were responsible for the analysis and interpretation of data. MP and ARM were responsible for drafting and critical revision of the manuscript. EBE, GTO, FMO, ment eL3 of other mosquito transmitted filariae, such TJN and JBL were responsible for critical revision of the manuscript. All authors as Dirofilaria repens, a canine and human skin-dwell - read and approved the final manuscript. ing relative of D. immitis, and Wuchereria bancrofti, Funding which is responsible for approximately 90% of human This work was supported by a University Research Foundation grant (URF- lymphatic filariasis and can be transmitted by several 2017), intramural funds, and NIH grant AI139060 to MP. JBL was supported by genera of mosquitoes (Culex, Aedes and Anopheles). NIH grants AI050668 and AI44572. It remains to be determined if our assay will work for Availability of data and materials filariae transmitted by other arthropod vectors, such All data generated or analyzed during this study are included in this published as biting flies, mites, fleas and ticks. In summary, the article and its supplementary information files. method we describe here will greatly facilitate current Ethics approval and consent to participate efforts to block transmission of arthropod transmitted Blood containing D. immitis microfilariae was obtained from an experimen- filariae by enabling quantitative analysis in the vector as tally infected dog according to Institutional Animal Care and Use Committee approved protocols and in accordance with the guidelines of the Institutional well as providing a more reliable way to harvest infec- Animal Care and Use Committee of the University of Pennsylvania (IACUC, tious larvae to evaluate new preventatives, treatments protocol 805059). or vaccines. Consent for publication Not applicable. Supplementary information The online version contains supplementary material available at https ://doi. Competing interests org/10.1186/s1307 1-020-04529 -w. The authors declare that they have no competing interests. Received: 15 August 2020 Accepted: 7 December 2020 Additional file 1: Movie 1. Third-stage larvae visible moving in the proboscis of Ae. aegypti 14 days after infection with D. immitis. Additional file 2: Figure S1. Mosquito dissection for analysis after emergence assay. After the emergence assay from the whole body (a), the head (b) and carcass (c) were placed individually into separate wells. The References number of L3 larvae emerging from the dissected head and carcass were 1. Taylor MJ, Hoerauf A, Bockarie M. Lymphatic filariasis and onchocer - assayed after incubation at 37 °C. The Malpighian tubules were removed ciasis. Lancet. 2010;376(9747):1175–85. https ://doi.org/10.1016/S0140 from the carcass prior to placing it in the well. Mosquito after removing -6736(10)60586 -7. head with fine forceps (d) and carcass after the Malpighian tubules (arrow) 2. Companion Animal Parasite Council. 2012. https ://capcv et.org/ are dissected out (e). The scale bars in a–c and d, e are 500 and 1000 µm, maps/#2019/all/heart worm-canin e/dog/unite d-state s/. Accessed Dec respectively. 2019. 3. McCall JW, Genchi C, Kramer LH, Guerrero J, Venco L. Heartworm disease Additional file 3: Movie 2. Two eL3 in a well of a 96-well plate following in animals and humans. Adv Parasitol. 2008;66:193–285. https ://doi. an emergence assay performed on Ae. aegypti 14 days after infection with org/10.1016/S0065 -308X(08)00204 -2. D. immitis. Another eL3 is visible at the top of the frame. 4. Beerntsen BT, James AA, Christensen BM. Genetics of mosquito vector Additional file 4: Figure S2. Example of D. immitis larval lengths scored competence. Microbiol Mol Biol Rev. 2000;64(1):115–37. in Malpighian tubules. Malpighian tubules dissected following an 5. McGreevy PB, Theis JH, Lavoipierre MM, Clark J. Studies on filariasis. III. emergence assay contain viable larvae of different lengths indicated with Dirofilaria immitis: emergence of infective larvae from the mouthparts of asterisks. Different stage larvae are typically present. Aedes aegypti. J Helminthol. 1974;48 4:221–8. S 6. Bradley TJ, Nayar JK. An ultrastructural study of Dirofilaria immitis infec- Additional file 5: Figure S3. Ae. aegypti have greater mortality than Ae. R tion in the Malpighian tubules of Anopheles quadrimaculatus. J Parasitol. aegypti following D. immitis infection. Pairs of adjacent treatment groups 1987;73(5):1035–43. from Fig. 5d, e were analyzed by Kaplan–Meier and relationships, with 7. McKay T, Bianco T, Rhodes L, Barnett S. Prevalence of Dirofilaria immitis significant differences indicated with asterisks here and in Additional file 6: S R (Nematoda: Filarioidea) in mosquitoes from northeast Arkansas, the Table S1. Aedes aegypti and Ae. aegypti are indicated in blue and red, United States. J Med Entomol. 2013;50(4):871–8. https ://doi.org/10.1603/ respectively. me121 97. Additional file 6: Table S1. Pairwise comparisons of mosquito survival 8. Ledesma N, Harrington L. Mosquito vectors of dog heartworm in following blood feeding on different doses of D. immitis microfilariae. the United States: vector status and factors influencing transmission efficiency. Top Companion Anim Med. 2011;26(4):178–85. https ://doi. org/10.1053/j.tcam.2011.09.005. Abbreviations 9. Laney SJ, Ramzy RM, Helmy HH, Farid HA, Ashour AA, Weil GJ, et al. BE, susceptible, Ae. aegypti : Aedes aegypti Blackeye Liverpool strain; LVP, Detection of Wuchereria bancrofti L3 larvae in mosquitoes: a reverse refractory, Ae. aegypti : Aedes aegypti Liverpool strain; mf: Microfilariae; MT: transcriptase PCR assay evaluating infection and infectivity. PLoS Malpighian tubules; L3: Third-stage larvae; eL3: Emerging third-stage larvae. neglected tropical diseases. 2010;4(2):e602; https ://doi.org/10.1371/journ al.pntd.00006 02. Acknowledgements 10. Blagburn BL, Arther RG, Dillon AR, Butler JM, Bowles JV, von Simson We thank Leslie King who helped with the critical reading of the manuscript C, et al. Efficacy of four commercially available heartworm preven- and the Filariasis Research Reagent Resource center for providing Brugia tive products against the JYD-34 laboratory strain of Dirofilaria malayi and the Aedes aegypti Black Eye Liverpool strain. immitis. Parasites Vectors. 2016;9:191. https ://doi.org/10.1186/s1307 1-016-1476-7. McCrea et al. Parasites Vectors (2021) 14:30 Page 12 of 12 11. Bowman DD, McTier TL, Adams EL, Mahabir SP, Login JA, Bidgood T, 25. McCrea AR, Jimenez Castro PD, Kaplan RM, Povelones M. Activation of the et al. Evaluation of the efficacy of ProHeart 6 (moxidectin) against a Toll pathway in Aedes aegypti blocks the development of emerging third- resistant isolate of Dirofilaria immitis (JYD-34) in dogs. Parasites Vectors. stage larvae of drug-resistant Dirofilaria immitis. Vet Parasitol. 2020. https 2017;10(Suppl 2):502. https ://doi.org/10.1186/s1307 1-017-2431-y.://doi.org/10.1016/j.vetpa r.2020.10910 0. 12. Kryda K, Six RH, Walsh KF, Holzmer SJ, Chapin S, Mahabir SP, et al. 26. Grieve RB, Lok JB, Glickman LT. Epidemiology of canine heartworm infec- Laboratory and field studies to investigate the efficacy of a novel, orally tion. Epidemiol Rev. 1983;5:220–46. https ://doi.org/10.1093/oxfor djour administered combination product containing moxidectin, sarolaner nals.epire v.a0362 60. and pyrantel for the prevention of heartworm disease (Dirofilaria immitis) 27. Gasarasi DB. The transmission dynamics of bancroftian filariasis: the in dogs. Parasites Vectors. 2019;12(1):445; https ://doi.org/10.1186/s1307 distribution of the infective larvae of Wuchereria bancrofti in Culex 1-019-3702-6. quinquefasciatus and Anopheles gambiae and its effect on parasite escape 13. Lok JB, Mika-Grieve M, Grieve RB. Cryopreservation of Dirofilaria immitis from the vector. Trans R Soc Trop Med Hyg. 2000;94(3):341–7. https ://doi. microfilariae and third-stage larvae. J Helminthol. 1983;57(4):319–24. org/10.1016/s0035 -9203(00)90349 -3. https ://doi.org/10.1017/s0022 149x0 00110 19. 28. Paily KP, Hoti SL, Manonmani AM, Balaraman K. Longevity and migration 14. Marcombe S, Farajollahi A, Healy SP, Clark GG, Fonseca DM. Insecticide of Wuchereria bancrofti infective larvae and their distribution pattern in resistance status of United States populations of Aedes albopictus relation to the resting and feeding behaviour of the vector mosquito and mechanisms involved. PLoS One. 2014;9 7:e101992; https ://doi. Culex quinquefasciatus. Ann Trop Med Parasitol. 1995;89(1):39–47. https :// org/10.1371/journ al.pone.01019 92.doi.org/10.1080/00034 983.1995.11812 927. 15. Povelones M, McCrea AR. Infecting and mosquitoes with Dirofilaria immi- 29. Han YS, Barillas-Mury C. Implications of time bomb model of ookinete tis and Brugia malayi. 2020; doi: 10.17504/protocols.io.bb7birin invasion of midgut cells. Insect Biochem Mol Biol. 2002;32(10):1311–6. 16. Povelones M, McCrea AR. Dirofilaria immitis and Brugia malayi emergence 30. Vlachou D, Zimmermann T, Cantera R, Janse CJ, Waters AP, Kafatos FC. assay for individual or pools of mosquitoes. 2020; doi: 10.17504/protocols. Real-time, in vivo analysis of malaria ookinete locomotion and mosquito io.bb7airie midgut invasion. Cell Microbiol. 2004;6(7):671–85. 17. Lok JB, Knight DH, Wang GT, Doscher ME, Nolan TJ, Hendrick MJ, et al. 31. Christensen BM. Observations on the immune response of Aedes Activity of an injectable, sustained-release formulation of moxidectin trivittatus against Dirofilaria immitis. Trans R Soc Trop Med Hyg. administered prophylactically to mixed-breed dogs to prevent infection 1981;75(3):439–43. with Dirofilaria immitis. Am J Vet Res. 2001;62(11):1721–6. 32. Bartholomay LC. Infection barriers and responses in mosquito–filarial 18. Lok JB, Mika-Grieve M, Grieve RB, Chin TK. In vitro development of worm interactions. Curr Opinion Insect Sci. 2014;3:37–42. https ://doi. third- and fourth-stage larvae of Dirofilaria immitis: comparison of basal org/10.1016/j.cois.2014.08.006. culture media, serum levels and possible serum substitutes. Acta Trop. 33. Dharmarajan G, Walker KD, Lehmann T. Variation in tolerance to 1984;41(2):145–54. parasites affects vectorial capacity of natural Asian tiger mosquito 19. Edgerton EB, McCrea AR, Berry CT, Kwok JY, Thompson LK, Watson B, et al. populations. Curr Biol. 2019;29(22):3946-52.e5. https ://doi.org/10.1016/j. Activation of mosquito immunity blocks the development of transmis- cub.2019.09.047. sion-stage filarial nematodes. Proc Natl Acad Sci USA. 2020. https ://doi. 34. Evans CC, Moorhead AR, Storey BE, Blagburn BL, Wolstenholme AJ, org/10.1073/pnas.19093 69117 . Kaplan RM. Evaluation of the larval migration inhibition assay for detect- 20. Kartman L. Factors influencing infection of the mosquito with Dirofi- ing macrocyclic lactone resistance in Dirofilaria immitis. Vet Parasitol. laria immitis (Leidy, 1856). Exp Parasitol. 1953;2(1):27–78. https ://doi. 2017;246:76–81. https ://doi.org/10.1016/j.vetpa r.2017.09.003. org/10.1016/0014-4894(53)90005 -8. 35. Liu C, Mhashilkar AS, Chabanon J, Xu S, Lustigman S, Adams JH, et al. 21. McGreevy PB, McClelland GA, Lavoipierre MM. Inheritance of susceptibil- Development of a toolkit for piggyBac-mediated integrative transfec- ity to Dirofilaria immitis infection in Aedes aegypti. Ann Trop Med Parasitol. tion of the human filarial parasite Brugia malayi. PLoS Negl Trop Dis. 1974;68(1):97–109. 2018;12(5):e0006509. https ://doi.org/10.1371/journ al.pntd.00065 09. 22. Nayar JK, Knight JW, Bradley TJ. Further characterization of refractoriness 36. Bancroft TL. Report LXXXV: Some further observations on the life-history in Aedes aegypti (L.) to infection by Dirofilaria immitis (Leidy). Experimen- of Filaria Immitis Leidy. Br Med J. 1904;1(2258):822–3. tal parasitology. 1988;66(1):124–31. http://dx.doi.org/10.1016/0014- 4894(88)90057 -4. Publisher’s Note 23. Sauerman DM Jr, Nayar JK. Characterization of refractoriness in Aedes Springer Nature remains neutral with regard to jurisdictional claims in pub- Aegypti (Diptera: Culicidae) to infection by Dirofilaria immitis. J Med Ento - lished maps and institutional affiliations. mol. 1985;22(1):94–101. https ://doi.org/10.1093/jmede nt/22.1.94. 24. Erickson SM, Xi Z, Mayhew GF, Ramirez JL, Aliota MT, Christensen BM, et al. Mosquito infection responses to developing filarial worms. PLoS Negl Trop Dis. 2009;3 10:e529; https ://doi.org/10.1371/journ al.pntd.00005 29. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

Journal

Parasites & Vectors – Springer Journals

Published: Jan 7, 2021

There are no references for this article.