Susceptibility of wild and colonized Anopheles stephensi to Plasmodium vivax infection

Susceptibility of wild and colonized Anopheles stephensi to Plasmodium vivax infection Background: As much as 80% of global Plasmodium vivax infections occur in South Asia and there is a shortage of direct studies on infectivity of P. vivax in Anopheles stephensi, the most common urban mosquito carrying human malaria. In this quest, the possible effects of laboratory colonization of mosquitoes on infectivity and development of P. vivax is of interest given that colonized mosquitoes can be genetically less divergent than the field population from which they originated. Methods: Patient-derived P. vivax infected blood was fed to age-matched wild and colonized An. stephensi. Such a comparison requires coordinated availability of same-age wild and colonized mosquito populations. Here, P. vivax infection are studied in colonized An. stephensi in their 66th–86th generation and fresh field-caught An. stephensi. Wild mosquitoes were caught as larvae and pupae and allowed to develop into adult mosquitoes in the insectary. Parasite development to oocyst and sporozoite stages were assessed on days 7/8 and 12/13, respectively. Results: While there were batch to batch variations in infectivity of individual patient-derived P. vivax samples, both wild and colonized An. stephensi were roughly equally susceptible to oocyst stage Plasmodium infection. At the level of sporozoite development, significantly more mosquitoes with sporozoite load of 4 + were seen in wild than in colo- nized populations. Background experimental Plasmodium infections [2, 4–10]. However, Plasmodium-Anopheles interactions in malaria endemic it is also known that when colonized mosquitoes (and research sites are widely studied using colonized mos- other insects) are maintained in the laboratory for gen- quito populations [1–3]. Laboratory-adapted mosqui- erations, may not accurately reflect the genetic make-up toes offer significant advantages in logistics, ease of of a wild population due to founder effects, inbreeding, maintenance, flexibility of scaling up and reproducibil - genetic drift, and accumulation of traits that favour their ity of experimental infections. Many Anopheles species, survival in artificial breeding conditions [ 6, 11–14]. Colo- especially the Plasmodium vectors Anopheles gambiae, nized mosquito populations can also lose alleles that are Anopheles dirus, Anopheles albimanus, and Anoph- required in the wild. eles darlingi have been colonized and are used for Studies in Drosophila show that the laboratory bred populations have lower fitness and are less adaptable compared to the outbred population from which they *Correspondence: ashwani07@gmail.com originated [15, 16]. Transcriptome analysis of wild and Ajeet Kumar Mohanty and Praveen Balabaskaran Nina contributed laboratory An. gambiae indicate substantive divergence, equally to this work with elevated expression of genes involved in insecticide National Institute of Malaria Research, Field Unit, Campal, Goa 403001, India resistance, immunity and olfaction in wild mosquitoes, Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Mohanty et al. Malar J (2018) 17:225 Page 2 of 10 while metabolism and protein synthesis genes were emergence, the adult mosquitoes were fed 10% glucose expressed higher in colonized population [14]. In colo- soaked in a cotton pad. Adult mosquitoes were identi- nized An. gambiae, microsatellite DNA polymorphisms fied using standard keys by trained experts. On day 5 were lower compared to wild populations [17]. Simi- post-emergence, 10 female mosquitoes were starved larly, in An. albimanus colonized for 20 years, significant over-night (14–16 h) and then allowed to feed on human genetic differentiation was found in the mitochondrial blood in a membrane feeder. A single fed female mos- gene cytb gene between laboratory and field populations. quito was kept inside the cage for oviposition, and its Even after just 21 generations of colonization, low to eggs were used for the continuous cyclic population of moderate genetic diversity was observed in An. darlingi pure An. stephensi colony. Larvae hatched from these [18]. The interaction of Plasmodium with Anopheles in eggs were reared in reverse-osmosis purified water under the wild must constantly evolve, and a successful vector- the same laboratory conditions described above. The parasite association must depend on the ability of para- colonized mosquitoes used in these experiments were in sites to continuously adapt to the changing ecosystem. their 66th–86th generation. How mosquito genetic differentiation affects the suscep - tibility of colonized mosquitoes to Plasmodium infec- Wild Anopheles stephensi larvae collection tion is poorly understood. One way to study the effect of and maintenance genetic diversity on vector-parasite interactions in wild Larvae and pupae of wild An. stephensi were collected and colonized mosquitoes would be to feed the same by dipping technique from the curing waters of natural patient-derived Plasmodium blood to age-matched wild breeding habitats in construction sites around the city of and laboratory mosquitoes, and track infection kinetics. Ponda in Goa, India. Along with larvae and pupae, the Anopheles stephensi is the major urban malaria vec- surrounding water was also transferred to plastic bowls, tor in India and is a dominant vector in Goa, where the and brought to MESA insectary at the NIMR Goa field MESA-ICEMR (Malaria Evolution in South Asia-Inter- station. In the laboratory, the third and fourth stage lar- national Center of Excellence for Malaria Research) vae were separated from the first and second, and were study site is located [19]. Anopheles stephensi was colo- allowed to develop separately. The field collected pupae nized from a single wild-caught female, and currently were kept in a separate 500  ml plastic bowl containing the colony is in its 86th generation. Infections in a high approximately 200  ml of tap water, and the bowl was passaged An. stephensi line were compared with wild An. kept in a cage for emergence. Temperature, humidity stephensi, after challenge with patient isolates of P. vivax. and feeding protocols were the same as described above To get age matched wild mosquitoes, field-caught larvae for the pure colony. The emerged wild adult mosquitoes and pupae of An. stephensi were allowed to develop into were species-verified using standard morphological keys adult mosquitoes and immediately used for comparison by trained experts [20, 21]. with colonized An. stephensi of the same age (day 6 or 7). The study directly compares the susceptibility of wild and Approvals for the study colonized An. stephensi when fed many different local All necessary approvals for collecting blood from malaria patient isolates of P. vivax from South Asia. patients and conducting the study were obtained from the Institutional Ethics Committee of Goa Medical Col- Methods lege and Hospital (GMC), the University of Washing- Establishment and maintenance of pure colony ton Institutional Review Board, NIH/NIAID Division of Anopheles stephensi of Microbiology and Infectious Disease (DMID), Health Anopheles stephensi larvae were collected by dipping Ministry Screening Committee (HMSC) of the Govern- technique from the curing waters, which are intended ment of India and by the Government of Goa Public for curing the cement slabs in the construction sites Health Department. in North Goa, India in January, 2012. The larvae were reared in plastic trays containing curing water col- Collection of Plasmodium vivax infected blood lected from the breeding habitat and set in the labora- Plasmodium vivax-positive patients, identified by tory at temperature 27 ± 2  °C, relative humidity 70 ± 5% microscopy, were briefed at GMC about the study and with 12  h  light/12  h  dark photoperiod cycling. A pinch informed consent was obtained from volunteers prior of Cerelac powder (about 60  mg) and fish food (1:1) to blood collection. Approximately 5.5–6  ml of blood mixture was given to the growing larvae once a day till was drawn into acid dextrose vacutainer, and the vacu- they developed into pupae. The pupae were collected in tainer was immediately placed in a thermos flask main - plastic bowls containing 200 ml tap water and were kept tained at 37 °C and transported to the MESA insectary at inside a closed cage until the emergence of adults. Upon NIMR-Goa. Mohanty et al. Malar J (2018) 17:225 Page 3 of 10 Mosquito infection experiments laboratory-maintained mosquito populations have the Six to seven-day old adult females of wild and colonized same SD, paired t test was used to determine p value (two mosquito populations were used to compare their abil- tailed distribution, 95% confidence level). This was to ity to support P. vivax infections. Based on the avail- assess the significance of differences seen in oocyst infec - ability of the age-matched mosquitoes, 75–125 wild and tion rate, average oocyst load, sporozoite infection rate colonized mosquitoes were selected for any single experi- and sporozoite load (4+) between wild and laboratory ment. Each infection experiment used one patient’s blood populations. to infect both wild and colonized mosquitoes. Over 30 separate experiments were conducted, some for moni- Results toring the timing of infections and the rest to study the Establishment of Anopheles stephensi colony host-competence of wild versus laboratory-reared mos- Anopheles stephensi adapts well to laboratory conditions quitoes. The mosquitoes were securely kept in plastic and is easy to colonize. The colonized An . stephensi in cups covered by mesh netting. Since GMC blood collec- the MESA-ICEMR study site, maintained for 4 years and tion clinical site is just 7 km from the MESA NIMR-Goa currently in its 86th generation at the time of this writ- insectary, the freshly collected blood maintained at 37 °C ing, was derived from a single adult female caught from in a water flask could be transferred within 60–90  min the field. The mosquitoes were fed with human blood, to feed the wild and colonized mosquitoes. Blood feed- and the expected number of eggs, larvae, and pupae were ing was done by the standard membrane feeding assay obtained. Seasonality affected the amount of eggs pro - (MFA) as described in earlier studies [2, 7]. Briefly, 2  ml duced, and time required for emergence of adult mosqui- of blood was added to a 5 cm water- jacketed membrane toes, with slow growth in the months between December feeder positioned in the center of the plastic container and February. No alterations in lighting conditions or containing mosquitoes, and fitted to a circulating water temperature in the laboratory were made for the growth bath maintained at 37 °C. Mosquitoes were then allowed and propagation of mosquitoes. For infection experi- to feed for 90  min. After that, unfed mosquitoes were ments, wild versus laboratory-reared 6–7  days old mos- removed, and the plastic cup with fully-fed mosquitoes quitoes were used after starving them for 16–18 h. were kept in Percival incubators maintained at 27 °C ± 2 and 80% ± 2 relative humidity. Cotton pads soaked in Plasmodium vivax oocyst production in wild 10% glucose were provided for subsequent days until the versus laboratory‑colonized An. stephensi mosquitoes were dissected. Studying the development of oocysts in mosquitoes from different sources was expected to reveal potential dif - Mosquito dissections, microscopy and parasite counting ferences in ability to support development of different Equal number of wild and colonized mosquitoes were patient isolates of P. vivax. The oocyst load in 32 wild ver - dissected on days 7/8 post blood feeding for assessing sus colonized feeding experiments are shown in Table  1. oocyst load in the midgut. The oocysts were counted Oocyst infection rate ranged from 0 to 100% in both using a Carl Zeiss Axio Lab. A1 phase-contrast micro- wild and colonized mosquitoes, with mean (SD) 62.8% scope at 5× and 10× magnification. Sporozoite load was (35.2) and 53.8% (39.0) in wild and colonized mosquitoes, assessed on days 12 and 13, and imaging of dissected respectively. The 25 percentile, median and 75 percentile salivary glands was done using Carl Zeiss Axio Lab. A1 oocyst infection rate in wild (laboratory) mosquitoes was phase contrast microscope at 40×. Sporozoite load was 29.0% (10.8%), 76.1% (55.5%) and 91.2% (95%), respec- represented by gland index [4, 22, 23] and recorded as; tively. This means that 25% of wild samples have oocyst 1+ for (0–10 sporozoites), 2+ for (10–100 sporozoites), infection rate lower than 29.0, 25% of colonized samples 3+ for (101–1000 sporozoites), and 4+ for (> 1000 sporo- have oocyst infection rate lower than 10.8%. Similarly, zoites). Parasitaemia in the patient blood was counted in 50% of wild samples have oocyst infection rate lower than thin smears by two trained technicians independently. 76.1 and 50% of colonized samples have oocyst infec- For every smear, 100 fields were counted by the miller tion rate lower than 55.5%. Finally, 75% of wild samples reticule technique [24]. The ratio of large reticule to small have oocyst infection rate lower than 91.2 and 75% of reticule was 4:1 (ImageJ software), and the reticule factor colonized samples have  oocyst infection rate lower than was 25. 95%. The oocyst load ranged from 0 to 215 in the wild and 0–210 in the colonized populations. The 25 per - centile, median and 75 percentile average oocyst load in Statistics wild (laboratory) was 0.5 (0.3), 3.8 (1.9) and 37.0 (23.5), Statistical analysis was performed using the GraphPad respectively. Statistically, there is significant difference Prism 7.02 software. Assuming that the wild and the between wild and colonized An. stephensi in the oocyst Mohanty et al. Malar J (2018) 17:225 Page 4 of 10 Table 1 Oocyte infection rate and load in wild and colonized Anopheles stephensi Experiment Gametocytaemia Wild mosquitoes Colonized mosquitoes no. No. dissected No. positive Oocyst range Average Oocyst No. dissected No. positive Oocyst range Average Oocyst oocyst no. infection rate oocyst no. infection (%) rate (%) 1 0.03 27 7 1–3 0.33 25.9 27 3 1–4 0.29 11.1 2 0.56 23 14 0–13 2.5 61 23 18 0–14 3.7 78.2 3 0.09 26 26 4–195 65.9 100 26 24 0–175 55.2 92.3 4 0.04 34 16 0–83 4.2 47 34 9 0–3 0.35 26.4 5 0.13 25 22 0–15 4.9 88 25 10 0–5 0.68 40 6 0.58 20 17 0–215 95.8 85 20 20 25–210 94.5 100 7 0.23 20 18 0–40 15.7 90 20 18 0–30 11.3 90 8 0.18 20 17 0–185 79.2 85 20 20 10–192 96.5 100 9 0.05 20 18 0–22 7.6 90 20 19 0–24 11.5 95 10 0.14 20 16 0–50 10.5 80 20 16 0–65 18 80 11 0.31 20 4 0–1 0.2 20 20 0 0–0 0 0 12 0.19 21 2 0–2 0.14 9.5 21 1 1 0.05 4.7 13 0.15 20 7 0–4 0.75 35 20 2 0–4 0.25 10 14 0.16 20 14 0–8 2.3 70 20 12 0–15 2.1 60 15 0.06 20 6 0–2 0.35 30 20 1 1 0.05 5 16 0.11 20 17 0–85 35.4 85 20 19 0–68 30 95 17 0.14 20 20 22–102 54.8 100 20 19 0–75 38.6 95 18 0.05 20 10 0–9 1.8 50 20 9 0–5 1.3 45 19 0.02 21 4 0–3 0.3 19 21 6 0–12 0.95 28.5 20 0.03 16 0 0 0 0 16 0 0 0 0 21 0.06 5 5 1–9 3.4 100 5 1 8 1.6 20 22 0.09 18 13 0–12 3 72.2 18 9 0–7 1.3 50 23 0.04 14 0 0 0 0 14 0 0 0 0 24 0.03 25 22 0–35 9.6 88 25 14 0–22 4.3 56 25 0.08 20 11 0-8 1.4 55 20 11 0–3 0.95 55 26 0.14 20 20 1–127 41.7 100 20 16 0–55 21.3 80 27 0.12 20 1 1 0.05 5 20 0 0 0 0 28 0.17 12 12 21–105 57.9 100 12 12 1–85 46.5 100 29 0.29 20 5 0–4 0.55 25 20 2 0–5 0.35 10 30 0.27 17 17 13–165 55.4 100 17 17 4–89 44.7 100 31 0.09 20 19 0–42 8.3 95 20 19 0–25 6.6 95 32 0.3 15 15 21–145 70.5 100 15 15 1–132 67.2 100 The data from experiments 1–15 in wild mosquitoes were used in Balabaskaran et al. [23] to study oocyst infection kinetics in wild mosquitoes Mohanty et al. Malar J (2018) 17:225 Page 5 of 10 Fig. 1 Oocyst infection rate and average oocyst load in laboratory and wild Anopheles stephensi. There is significant difference in a oocyst infection rate (paired t test, p = 0.01) and b no difference in average oocyst load (paired t test, p = 0.06) between wild and laboratory An. stephensi the sporozoite infection rate in wild and colonized mos- infection rate (paired t test, p = 0.01, mean of the differ - quitoes (Fig.  2a). Both wild and colonized mosquitoes ences = 9.0%) and no difference in the average oocyst had a wide range of sporozoite load ranging from 1+ to load (paired t test, p = 0.06, mean of the differences = 2.3) 4+. The number of mosquitoes with gland index of 1+, (Fig.  1a, b). These comparative feeding experiments 2+ and 3+ were similar in wild and colonized (Table  3), reveal a significant difference between wild and colonized and the two types of mosquitoes showed no significant mosquitoes in their susceptibility to oocyst stage infec- difference; paired t test p value is 0.67, 0.53, 0.89 for 1+, tion of P. vivax. 2+ and 3+ mosquitoes, respectively (Fig. 2b–d). Interest- ingly, in 12 of the 14 experiments where mosquitoes with 4+ gland index were seen (wild or colonized), the num- Sporozoite infection in wild and colonized Anopheles ber of mosquitoes with 4+ sporozoite load were higher stephensi in wild (68) than in colonized mosquitoes (30) (Table  3), Sporozoite infection rate and sporozoite load was and the difference is significant (paired t test, p = 0.002) expected to reveal possible variations in the infection (Fig.  2e). Overall, the sporozoite infection rate in wild kinetics of patient-derived isolates of P. vivax in wild ver- and colonized mosquitoes may be similar, however, the sus colonized An. stephensi and potential variations in parasites reach high sporozoite levels more efficiently in transmission potential of different P. vivax samples. In wild mosquitoes compared to colonized mosquitoes. 26 of the 32 experiments, there were surviving labora- Experiment numbers in Table  2 correspond to experi- tory and wild mosquitoes on days 12 and 13, and these ment numbers in Table  1. The data from experiments 3, were dissected to assess sporozoite infection rate and 5, 6–12, 14 and 15 in wild mosquitoes were used in Bala- gland index. The details of sporozoite infection rate and baskaran et al. [23] to study sporozoite infection kinetics load of individual experiments are given in Table  2. The in wild mosquitoes. sporozoite infection rate ranged from 0 to 100% in wild and colonized mosquitoes, and the mean (SD) was 60.7 Discussion (38.0)% and 55.3 (40.9)% in wild and colonized respec- Colonized mosquitoes are widely used around the world tively. The 25 percentile, median and 75 percentile oocyst to understand the developmental kinetics of Plasmo- infection rate in wild and (laboratory) was 23.5 (2.4), 80 dium isolates of that region [1–4, 8, 22, 25, 26]. Upon (74.4) and 90.9 (91.7), respectively. In experiments 14, colonization, mosquitoes tend to undergo genetic varia- 15 and 23 (Table  2), sporozoites were not seen in the tion over a period of many generations [14, 17, 18]. How colonized mosquitoes, whereas the infection rate in wild this genetic diversity associated with mosquito coloniza- mosquitos was 37.5, 25 and 18.75%, respectively. There tion affects Plasmodium development has not been clear. was no significant difference (paired t test, p = 0.18) in Mohanty et al. Malar J (2018) 17:225 Page 6 of 10 Table 2 Sporozoite infection rate and load in wild and colonized An. stephensi Experiment Gametocytaemia Wild mosquitoes Colonized mosquitoes no. No. No. Sporozoite Gland index No. No. Sporozoite Gland index dissected positive infection dissected positive infection rate (%) rate (%) 3 0.097 21 20 95.2 4+ (12), 3+ (8) 21 21 100 4+ (6), 3+ (15) 5 0.136 20 11 55 4+ (1), 3+ (7), 2+ (2), 20 4 20 3+ (4) 1+ (1) 6 0.585 12 11 91.6 4+ (2), 3+ (10) 12 12 100 4+ (3), 3+ (8), 2+ (1) 7 0.235 21 19 90.4 4+ (3), 3+ (15), 21 16 76.1 4+ (1), 3+ (10), 2+ (4), 1+ (1) 1+ (1) 8 0.18 20 17 85 4+ (7), 3+ (9), 2+ (1) 20 18 90 4+ (3), 3+ (9), 2+ (5), 1+ (1) 9 0.058 20 17 85 4+ (2), 3+ (7), 2+ (7), 20 20 100 3+ (10), 2+ (10) 1+ (1) 10 0.143 21 15 71.4 3+ (8), 2+ (7) 21 17 80.9 3+ (6), 2+ (8), 1+ (3) 11 0.3195 4 0 0 – 4 0 0 – 12 0.198 20 0 0 – 20 0 0 – 14 0.16 8 3 37.5 2+ (3) 8 0 0 – 15 0.06 20 5 25 3+ (1), 2+ (4) 20 0 0 – 16 0.115 18 18 100 4+ (12), 3+ (5), 18 17 94.4 4+ (5), 3+ (11), 2+ (1) 2+ (1) 17 0.145 22 20 90.9 4+ (11), 3+ (9) 22 21 95.4 4+ (5), 3+ (13), 2+ (3) 18 0.055 13 3 23 2+ (3) 13 11 84.6 3+ (1), 2+ (8), 1+ (2) 20 0.037 8 0 0 – 8 0 0 – 21 0.0625 24 19 79.1 4+ (1), 3+ (3), 2+ (6), 24 14 58.3 2+ (10), 1+ (4) 1+ (9) 22 0.095 22 20 90.9 4+ (1), 3+ (3), 2+ 22 16 72.7 4+ (1), 3+ (1), 2+ (7), (13), 1+ (3) 1+ (7) 23 0.040 16 3 18.7 2+ (2), 1+ (1) 16 0 0 0 25 0.088 20 16 80 3+ (4), 2+ (11), 1+ 20 10 50 3+ (1), 2+ (7), 1+ (2) (1), 26 0.141 13 12 92.3 4+ (1), 3+ (9), 2+ (1), 13 12 92.3 3+ (7), 2+ (2), 1+ (3) 1+ (1) 27 0.122 11 0 0 0 11 0 0 0 28 0.176 13 13 100 4+ (8), 3+ (5) 13 12 92.3 4+ (4), 3+ (6), 2+ (2) 29 0.295 21 1 4.7 1+ (1) 21 2 9.5 2+ (1), 1+ (1) 30 0.274 13 12 92.3 4+ (4), 3+ (5), 2+ (3) 13 10 76.9 4+ (2), 3+ (6), 2+ (1), 1+ (1) 31 0.092 20 18 90 4+ (3), 3+ (6), 2+ (8), 20 18 90 3+ (4), 2+ (11), 1+ (3), 1+ (1) 32 0.3 15 12 80 2+ (7), 1+ (5) 15 8 53.3 2+ (7), 1+ (1) Experiment numbers in Table 2 correspond to experiment numbers in Table 1. The data from experiments 3, 5, 6–12, 14 and 15 in wild mosquitoes were used in Balabaskaran et al. [23] to study sporozoite infection kinetics in wild mosquitoes Studies have mapped gene polymorphisms to altered passaged colonized mosquitoes to Plasmodium infection, levels of vector susceptibility to Plasmodium [5, 11, 27, especially P. vivax infection, has not been investigated so 28]. Anopheles gambiae from different geographical loca - far. tions in Africa show different infection intensity to infec - Mosquito infection experiments in wild Anophelines tion with Plasmodium falciparum [29], underscoring are complicated by logistics, availability of mosquitoes the importance of genetic differentiation. Even though year around, their ability to adapt to artificial feeding and varying infection intensities has been reported between new environment. The MESA-ICEMR mosquito infec - sympatric and allopatric vector-parasite combinations tion laboratory at NIMR-Goa is one of the very few sites [29] or between specific intercrosses of mosquito phe - in the world where feeding experiments can be done with notypes [5], the effect of genetic differentiation on highly wild An. stephensi and patient isolates of P. vivax. The Mohanty et al. Malar J (2018) 17:225 Page 7 of 10 Fig. 2 Sporozoite infection rate and 4+ sporozoite load in laboratory and wild Anopheles stephensi. a There is no significant difference in the sporozoite infection rate (paired t test, p = 0.18). b, c, d There is no significant difference in 1+, 2+ and 3+ sporozoite load; paired t test, p = 0.67, 0.53 and 0.89 respectively. e There is a significant difference in 4+ sporozoite load (paired t test, p = 0.002) between wild and laboratory An. stephensi. The values on Y axis were normalized and is reported as percent (%). Experiment number of each comparison experiment is plotted in the x axis Mohanty et al. Malar J (2018) 17:225 Page 8 of 10 Table 3 Distribution of  sporozoite load in  wild Overall, at the level of oocyst development, significant and colonized mosquitoes difference was found between the colonized and wild mosquitoes in their susceptibility to P. vivax. To the first Sporozoite load (gland Wild mosquitoes Colonized index) mosquitoes approximation, it should be possible to use freshly reared larvae of other mosquito species to understand parasite 1+ 25 29 infectivity. Although there was no significant difference 2+ 79 88 in sporozoite infection rate in the present study, a sig- 3+ 112 114 nificantly higher sporozoite load (4+) was found in wild 4+ 68 30 when compared to laboratory mosquitoes. These experi - ments illustrate why it may be important to exercise caution when studying parasite infection in long-term presence of highly passaged An. stephensi (66th–86th laboratory-reared colonies of An. stephensi, especially in generations) allowed to study the susceptibility of wild this region. It will be of interest to learn whether long- and colonized mosquitoes to patient derived isolates of term mosquito colonization alters Plasmodium suscep- P. vivax. In both the mosquito populations, there is sig- tibility in vectors from different geographical locations. nificant difference in oocyst infection rate and whereas Understanding the molecular mechanisms that modulate this was not in the average oocyst load. In three of the Plasmodium infection will also be a prime area of focus experiments, no sporozoites were found in the labora- in the future studies. tory when compared to wild mosquitoes. The average oocyst load ranged between 0 and 2.5 in these experi- ments, and is possible that some parasite strains may not Abbreviations An: Anopheles; GMC: Goa Medical College and Hospital; MESA: malaria develop to sporozoites as efficiently in wild as compared evolution in South Asia; ICEMR: International Center of Excellence for Malaria to laboratory, especially when the oocyst load is very low. Research; NIMR: National Institute of Malaria Research; NIAID: National Institute When the sporozoite load was compared between wild of Allergy and Infectious Diseases; NIH: US National Institutes of Health; US: United States of America; UW: University of Washington; DMID: Division of and laboratory, there was no significant difference in 1+, Microbiology and Infectious Disease. 2+ and 3+ load. However, in 12 of the 14 experiments where 4+ sporozoite load were seen, the number of mos- Authors’ contributions AKM, PBN, PKR, and AK designed the study. PKR, LC and AK administered the quitoes with 4+ load was significantly higher in wild (68) study. AKM, PBN, SB, SV, SP and MD carried out the experiments. PBN, AKM, than in colonized (30) mosquitoes. In An. gambiae, it has PKR, AK, ST and WZ analyzed the data. AKM, PBN, PKR, and AK wrote the been shown that genes involved in insecticide resistance, manuscript. All authors read and approved the final manuscript. immunity and olfaction are expressed higher in wild Author details mosquitoes when compared to colonized populations National Institute of Malaria Research, Field Unit, Campal, Goa 403001, India. [14]. Hence, it is important to learn here that, even under Departments of Chemistry and Global Health, University of Washington, Seattle, WA 98195, USA. Department of Epidemiology and Public Health, sterile rearing conditions lacking continual parasite Central University of Tamil Nadu, Thiruvarur, Tamil Nadu 610005, India. Goa infections, the immune system in colonized An. stephensi Medical College and Hospital, Bambolim, Goa 403202, India. Department was not weakened when challenged with P. vivax. It is of Biology, Stanford University, Stanford, CA 94305, USA. National Institute of Malaria Research (ICMR), Sector 8, Dwarka, New Delhi 110077, India. still possible that, in colonized mosquitoes, there could be a developmental delay that prevents the sporozoites to Acknowledgements populate the salivary gland on days 12 and 13. The authors thank all participating malaria patients at the Goa Medical College and Hospital who volunteered for this study, and Dr. Sachin Shinde, Special Mosquito gut microbiota is determined by the water Secretary (Health) and Administrator (GMC), for his support. The authors are source available in the breeding habitats, and has been most grateful for the administrative and scientific guidance provided by the shown to modulate the development of P. falciparum MESA-ICEMR Scientific Advisory Group, including the Government of India representatives Dr. Shiv Lal, Dr. P Joshi, Dr. Rashmi Arora, and Dr. Manju Rahi, [30–37]. Anti-Plasmodium effect of gut microbiota is and US NIH Programme Officer Dr. Malla Rao. This manuscript was approved suggested to be due to the effect of bacterial compounds by the publication committee of NIMR and bears Approval No. 057/2017. and/or mosquito immunity directed against the microbes Competing interests [31, 38]. It is possible that the altered gut biota in colo The authors declare that they have no competing interests. nized mosquitoes may elicit stronger basal immunity than in wild, and produce metabolites that may affect Availability of data and materials The datasets supporting the conclusions of this article are included within the oocyst maturation and sporozoite development. A recent article. study has implicated a specific Escherichia coli strain 444 in modulating P. falciparum infection in the mos- Availability of data and materials ST95 The datasets supporting the conclusions of this article are included within the quito midgut [38]. The observed differences in 4+ sporo - article. zoite load between wild and colonized mosquitoes may be further investigated in the future. Mohanty et al. Malar J (2018) 17:225 Page 9 of 10 Consent for publication 13. Muller P, Donnelly MJ, Ranson H. Transcription profiling of a recently Not applicable. colonised pyrethroid resistant Anopheles gambiae strain from Ghana. BMC Genomics. 2007;8:36. Ethics approval and consent to participate 14. Aguilar R, Simard F, Kamdem C, Shields T, Glass GE, Garver LS, et al. The human subjects protocol and consent forms for enrollment of Plasmo- Genome-wide analysis of transcriptomic divergence between laboratory dium positive individuals presenting to Goa Medical College and Hospital colony and field Anopheles gambiae mosquitoes of the M and S molecu- were approved by the institutional review boards of the Division of Microbiol- lar forms. Insect Mol Biol. 2010;19:695–705. ogy and Infectious Diseases at the US National Institute of Allergy and Infec- 15. Hoffmann AA, Hallas R, Sinclair C, Partridge L. 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Driv- An epidemiologically successful Escherichia coli sequence type modu- ing mosquito refractoriness to Plasmodium falciparum with engineered lates Plasmodium falciparum infection in the mosquito midgut. Infect symbiotic bacteria. Science. 2017;357:1399–402. Genet Evol. 2016;43:22–30. Ready to submit your research ? Choose BMC and benefit from: 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 Malaria Journal Springer Journals
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Abstract

Background: As much as 80% of global Plasmodium vivax infections occur in South Asia and there is a shortage of direct studies on infectivity of P. vivax in Anopheles stephensi, the most common urban mosquito carrying human malaria. In this quest, the possible effects of laboratory colonization of mosquitoes on infectivity and development of P. vivax is of interest given that colonized mosquitoes can be genetically less divergent than the field population from which they originated. Methods: Patient-derived P. vivax infected blood was fed to age-matched wild and colonized An. stephensi. Such a comparison requires coordinated availability of same-age wild and colonized mosquito populations. Here, P. vivax infection are studied in colonized An. stephensi in their 66th–86th generation and fresh field-caught An. stephensi. Wild mosquitoes were caught as larvae and pupae and allowed to develop into adult mosquitoes in the insectary. Parasite development to oocyst and sporozoite stages were assessed on days 7/8 and 12/13, respectively. Results: While there were batch to batch variations in infectivity of individual patient-derived P. vivax samples, both wild and colonized An. stephensi were roughly equally susceptible to oocyst stage Plasmodium infection. At the level of sporozoite development, significantly more mosquitoes with sporozoite load of 4 + were seen in wild than in colo- nized populations. Background experimental Plasmodium infections [2, 4–10]. However, Plasmodium-Anopheles interactions in malaria endemic it is also known that when colonized mosquitoes (and research sites are widely studied using colonized mos- other insects) are maintained in the laboratory for gen- quito populations [1–3]. Laboratory-adapted mosqui- erations, may not accurately reflect the genetic make-up toes offer significant advantages in logistics, ease of of a wild population due to founder effects, inbreeding, maintenance, flexibility of scaling up and reproducibil - genetic drift, and accumulation of traits that favour their ity of experimental infections. Many Anopheles species, survival in artificial breeding conditions [ 6, 11–14]. Colo- especially the Plasmodium vectors Anopheles gambiae, nized mosquito populations can also lose alleles that are Anopheles dirus, Anopheles albimanus, and Anoph- required in the wild. eles darlingi have been colonized and are used for Studies in Drosophila show that the laboratory bred populations have lower fitness and are less adaptable compared to the outbred population from which they *Correspondence: ashwani07@gmail.com originated [15, 16]. Transcriptome analysis of wild and Ajeet Kumar Mohanty and Praveen Balabaskaran Nina contributed laboratory An. gambiae indicate substantive divergence, equally to this work with elevated expression of genes involved in insecticide National Institute of Malaria Research, Field Unit, Campal, Goa 403001, India resistance, immunity and olfaction in wild mosquitoes, Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Mohanty et al. Malar J (2018) 17:225 Page 2 of 10 while metabolism and protein synthesis genes were emergence, the adult mosquitoes were fed 10% glucose expressed higher in colonized population [14]. In colo- soaked in a cotton pad. Adult mosquitoes were identi- nized An. gambiae, microsatellite DNA polymorphisms fied using standard keys by trained experts. On day 5 were lower compared to wild populations [17]. Simi- post-emergence, 10 female mosquitoes were starved larly, in An. albimanus colonized for 20 years, significant over-night (14–16 h) and then allowed to feed on human genetic differentiation was found in the mitochondrial blood in a membrane feeder. A single fed female mos- gene cytb gene between laboratory and field populations. quito was kept inside the cage for oviposition, and its Even after just 21 generations of colonization, low to eggs were used for the continuous cyclic population of moderate genetic diversity was observed in An. darlingi pure An. stephensi colony. Larvae hatched from these [18]. The interaction of Plasmodium with Anopheles in eggs were reared in reverse-osmosis purified water under the wild must constantly evolve, and a successful vector- the same laboratory conditions described above. The parasite association must depend on the ability of para- colonized mosquitoes used in these experiments were in sites to continuously adapt to the changing ecosystem. their 66th–86th generation. How mosquito genetic differentiation affects the suscep - tibility of colonized mosquitoes to Plasmodium infec- Wild Anopheles stephensi larvae collection tion is poorly understood. One way to study the effect of and maintenance genetic diversity on vector-parasite interactions in wild Larvae and pupae of wild An. stephensi were collected and colonized mosquitoes would be to feed the same by dipping technique from the curing waters of natural patient-derived Plasmodium blood to age-matched wild breeding habitats in construction sites around the city of and laboratory mosquitoes, and track infection kinetics. Ponda in Goa, India. Along with larvae and pupae, the Anopheles stephensi is the major urban malaria vec- surrounding water was also transferred to plastic bowls, tor in India and is a dominant vector in Goa, where the and brought to MESA insectary at the NIMR Goa field MESA-ICEMR (Malaria Evolution in South Asia-Inter- station. In the laboratory, the third and fourth stage lar- national Center of Excellence for Malaria Research) vae were separated from the first and second, and were study site is located [19]. Anopheles stephensi was colo- allowed to develop separately. The field collected pupae nized from a single wild-caught female, and currently were kept in a separate 500  ml plastic bowl containing the colony is in its 86th generation. Infections in a high approximately 200  ml of tap water, and the bowl was passaged An. stephensi line were compared with wild An. kept in a cage for emergence. Temperature, humidity stephensi, after challenge with patient isolates of P. vivax. and feeding protocols were the same as described above To get age matched wild mosquitoes, field-caught larvae for the pure colony. The emerged wild adult mosquitoes and pupae of An. stephensi were allowed to develop into were species-verified using standard morphological keys adult mosquitoes and immediately used for comparison by trained experts [20, 21]. with colonized An. stephensi of the same age (day 6 or 7). The study directly compares the susceptibility of wild and Approvals for the study colonized An. stephensi when fed many different local All necessary approvals for collecting blood from malaria patient isolates of P. vivax from South Asia. patients and conducting the study were obtained from the Institutional Ethics Committee of Goa Medical Col- Methods lege and Hospital (GMC), the University of Washing- Establishment and maintenance of pure colony ton Institutional Review Board, NIH/NIAID Division of Anopheles stephensi of Microbiology and Infectious Disease (DMID), Health Anopheles stephensi larvae were collected by dipping Ministry Screening Committee (HMSC) of the Govern- technique from the curing waters, which are intended ment of India and by the Government of Goa Public for curing the cement slabs in the construction sites Health Department. in North Goa, India in January, 2012. The larvae were reared in plastic trays containing curing water col- Collection of Plasmodium vivax infected blood lected from the breeding habitat and set in the labora- Plasmodium vivax-positive patients, identified by tory at temperature 27 ± 2  °C, relative humidity 70 ± 5% microscopy, were briefed at GMC about the study and with 12  h  light/12  h  dark photoperiod cycling. A pinch informed consent was obtained from volunteers prior of Cerelac powder (about 60  mg) and fish food (1:1) to blood collection. Approximately 5.5–6  ml of blood mixture was given to the growing larvae once a day till was drawn into acid dextrose vacutainer, and the vacu- they developed into pupae. The pupae were collected in tainer was immediately placed in a thermos flask main - plastic bowls containing 200 ml tap water and were kept tained at 37 °C and transported to the MESA insectary at inside a closed cage until the emergence of adults. Upon NIMR-Goa. Mohanty et al. Malar J (2018) 17:225 Page 3 of 10 Mosquito infection experiments laboratory-maintained mosquito populations have the Six to seven-day old adult females of wild and colonized same SD, paired t test was used to determine p value (two mosquito populations were used to compare their abil- tailed distribution, 95% confidence level). This was to ity to support P. vivax infections. Based on the avail- assess the significance of differences seen in oocyst infec - ability of the age-matched mosquitoes, 75–125 wild and tion rate, average oocyst load, sporozoite infection rate colonized mosquitoes were selected for any single experi- and sporozoite load (4+) between wild and laboratory ment. Each infection experiment used one patient’s blood populations. to infect both wild and colonized mosquitoes. Over 30 separate experiments were conducted, some for moni- Results toring the timing of infections and the rest to study the Establishment of Anopheles stephensi colony host-competence of wild versus laboratory-reared mos- Anopheles stephensi adapts well to laboratory conditions quitoes. The mosquitoes were securely kept in plastic and is easy to colonize. The colonized An . stephensi in cups covered by mesh netting. Since GMC blood collec- the MESA-ICEMR study site, maintained for 4 years and tion clinical site is just 7 km from the MESA NIMR-Goa currently in its 86th generation at the time of this writ- insectary, the freshly collected blood maintained at 37 °C ing, was derived from a single adult female caught from in a water flask could be transferred within 60–90  min the field. The mosquitoes were fed with human blood, to feed the wild and colonized mosquitoes. Blood feed- and the expected number of eggs, larvae, and pupae were ing was done by the standard membrane feeding assay obtained. Seasonality affected the amount of eggs pro - (MFA) as described in earlier studies [2, 7]. Briefly, 2  ml duced, and time required for emergence of adult mosqui- of blood was added to a 5 cm water- jacketed membrane toes, with slow growth in the months between December feeder positioned in the center of the plastic container and February. No alterations in lighting conditions or containing mosquitoes, and fitted to a circulating water temperature in the laboratory were made for the growth bath maintained at 37 °C. Mosquitoes were then allowed and propagation of mosquitoes. For infection experi- to feed for 90  min. After that, unfed mosquitoes were ments, wild versus laboratory-reared 6–7  days old mos- removed, and the plastic cup with fully-fed mosquitoes quitoes were used after starving them for 16–18 h. were kept in Percival incubators maintained at 27 °C ± 2 and 80% ± 2 relative humidity. Cotton pads soaked in Plasmodium vivax oocyst production in wild 10% glucose were provided for subsequent days until the versus laboratory‑colonized An. stephensi mosquitoes were dissected. Studying the development of oocysts in mosquitoes from different sources was expected to reveal potential dif - Mosquito dissections, microscopy and parasite counting ferences in ability to support development of different Equal number of wild and colonized mosquitoes were patient isolates of P. vivax. The oocyst load in 32 wild ver - dissected on days 7/8 post blood feeding for assessing sus colonized feeding experiments are shown in Table  1. oocyst load in the midgut. The oocysts were counted Oocyst infection rate ranged from 0 to 100% in both using a Carl Zeiss Axio Lab. A1 phase-contrast micro- wild and colonized mosquitoes, with mean (SD) 62.8% scope at 5× and 10× magnification. Sporozoite load was (35.2) and 53.8% (39.0) in wild and colonized mosquitoes, assessed on days 12 and 13, and imaging of dissected respectively. The 25 percentile, median and 75 percentile salivary glands was done using Carl Zeiss Axio Lab. A1 oocyst infection rate in wild (laboratory) mosquitoes was phase contrast microscope at 40×. Sporozoite load was 29.0% (10.8%), 76.1% (55.5%) and 91.2% (95%), respec- represented by gland index [4, 22, 23] and recorded as; tively. This means that 25% of wild samples have oocyst 1+ for (0–10 sporozoites), 2+ for (10–100 sporozoites), infection rate lower than 29.0, 25% of colonized samples 3+ for (101–1000 sporozoites), and 4+ for (> 1000 sporo- have oocyst infection rate lower than 10.8%. Similarly, zoites). Parasitaemia in the patient blood was counted in 50% of wild samples have oocyst infection rate lower than thin smears by two trained technicians independently. 76.1 and 50% of colonized samples have oocyst infec- For every smear, 100 fields were counted by the miller tion rate lower than 55.5%. Finally, 75% of wild samples reticule technique [24]. The ratio of large reticule to small have oocyst infection rate lower than 91.2 and 75% of reticule was 4:1 (ImageJ software), and the reticule factor colonized samples have  oocyst infection rate lower than was 25. 95%. The oocyst load ranged from 0 to 215 in the wild and 0–210 in the colonized populations. The 25 per - centile, median and 75 percentile average oocyst load in Statistics wild (laboratory) was 0.5 (0.3), 3.8 (1.9) and 37.0 (23.5), Statistical analysis was performed using the GraphPad respectively. Statistically, there is significant difference Prism 7.02 software. Assuming that the wild and the between wild and colonized An. stephensi in the oocyst Mohanty et al. Malar J (2018) 17:225 Page 4 of 10 Table 1 Oocyte infection rate and load in wild and colonized Anopheles stephensi Experiment Gametocytaemia Wild mosquitoes Colonized mosquitoes no. No. dissected No. positive Oocyst range Average Oocyst No. dissected No. positive Oocyst range Average Oocyst oocyst no. infection rate oocyst no. infection (%) rate (%) 1 0.03 27 7 1–3 0.33 25.9 27 3 1–4 0.29 11.1 2 0.56 23 14 0–13 2.5 61 23 18 0–14 3.7 78.2 3 0.09 26 26 4–195 65.9 100 26 24 0–175 55.2 92.3 4 0.04 34 16 0–83 4.2 47 34 9 0–3 0.35 26.4 5 0.13 25 22 0–15 4.9 88 25 10 0–5 0.68 40 6 0.58 20 17 0–215 95.8 85 20 20 25–210 94.5 100 7 0.23 20 18 0–40 15.7 90 20 18 0–30 11.3 90 8 0.18 20 17 0–185 79.2 85 20 20 10–192 96.5 100 9 0.05 20 18 0–22 7.6 90 20 19 0–24 11.5 95 10 0.14 20 16 0–50 10.5 80 20 16 0–65 18 80 11 0.31 20 4 0–1 0.2 20 20 0 0–0 0 0 12 0.19 21 2 0–2 0.14 9.5 21 1 1 0.05 4.7 13 0.15 20 7 0–4 0.75 35 20 2 0–4 0.25 10 14 0.16 20 14 0–8 2.3 70 20 12 0–15 2.1 60 15 0.06 20 6 0–2 0.35 30 20 1 1 0.05 5 16 0.11 20 17 0–85 35.4 85 20 19 0–68 30 95 17 0.14 20 20 22–102 54.8 100 20 19 0–75 38.6 95 18 0.05 20 10 0–9 1.8 50 20 9 0–5 1.3 45 19 0.02 21 4 0–3 0.3 19 21 6 0–12 0.95 28.5 20 0.03 16 0 0 0 0 16 0 0 0 0 21 0.06 5 5 1–9 3.4 100 5 1 8 1.6 20 22 0.09 18 13 0–12 3 72.2 18 9 0–7 1.3 50 23 0.04 14 0 0 0 0 14 0 0 0 0 24 0.03 25 22 0–35 9.6 88 25 14 0–22 4.3 56 25 0.08 20 11 0-8 1.4 55 20 11 0–3 0.95 55 26 0.14 20 20 1–127 41.7 100 20 16 0–55 21.3 80 27 0.12 20 1 1 0.05 5 20 0 0 0 0 28 0.17 12 12 21–105 57.9 100 12 12 1–85 46.5 100 29 0.29 20 5 0–4 0.55 25 20 2 0–5 0.35 10 30 0.27 17 17 13–165 55.4 100 17 17 4–89 44.7 100 31 0.09 20 19 0–42 8.3 95 20 19 0–25 6.6 95 32 0.3 15 15 21–145 70.5 100 15 15 1–132 67.2 100 The data from experiments 1–15 in wild mosquitoes were used in Balabaskaran et al. [23] to study oocyst infection kinetics in wild mosquitoes Mohanty et al. Malar J (2018) 17:225 Page 5 of 10 Fig. 1 Oocyst infection rate and average oocyst load in laboratory and wild Anopheles stephensi. There is significant difference in a oocyst infection rate (paired t test, p = 0.01) and b no difference in average oocyst load (paired t test, p = 0.06) between wild and laboratory An. stephensi the sporozoite infection rate in wild and colonized mos- infection rate (paired t test, p = 0.01, mean of the differ - quitoes (Fig.  2a). Both wild and colonized mosquitoes ences = 9.0%) and no difference in the average oocyst had a wide range of sporozoite load ranging from 1+ to load (paired t test, p = 0.06, mean of the differences = 2.3) 4+. The number of mosquitoes with gland index of 1+, (Fig.  1a, b). These comparative feeding experiments 2+ and 3+ were similar in wild and colonized (Table  3), reveal a significant difference between wild and colonized and the two types of mosquitoes showed no significant mosquitoes in their susceptibility to oocyst stage infec- difference; paired t test p value is 0.67, 0.53, 0.89 for 1+, tion of P. vivax. 2+ and 3+ mosquitoes, respectively (Fig. 2b–d). Interest- ingly, in 12 of the 14 experiments where mosquitoes with 4+ gland index were seen (wild or colonized), the num- Sporozoite infection in wild and colonized Anopheles ber of mosquitoes with 4+ sporozoite load were higher stephensi in wild (68) than in colonized mosquitoes (30) (Table  3), Sporozoite infection rate and sporozoite load was and the difference is significant (paired t test, p = 0.002) expected to reveal possible variations in the infection (Fig.  2e). Overall, the sporozoite infection rate in wild kinetics of patient-derived isolates of P. vivax in wild ver- and colonized mosquitoes may be similar, however, the sus colonized An. stephensi and potential variations in parasites reach high sporozoite levels more efficiently in transmission potential of different P. vivax samples. In wild mosquitoes compared to colonized mosquitoes. 26 of the 32 experiments, there were surviving labora- Experiment numbers in Table  2 correspond to experi- tory and wild mosquitoes on days 12 and 13, and these ment numbers in Table  1. The data from experiments 3, were dissected to assess sporozoite infection rate and 5, 6–12, 14 and 15 in wild mosquitoes were used in Bala- gland index. The details of sporozoite infection rate and baskaran et al. [23] to study sporozoite infection kinetics load of individual experiments are given in Table  2. The in wild mosquitoes. sporozoite infection rate ranged from 0 to 100% in wild and colonized mosquitoes, and the mean (SD) was 60.7 Discussion (38.0)% and 55.3 (40.9)% in wild and colonized respec- Colonized mosquitoes are widely used around the world tively. The 25 percentile, median and 75 percentile oocyst to understand the developmental kinetics of Plasmo- infection rate in wild and (laboratory) was 23.5 (2.4), 80 dium isolates of that region [1–4, 8, 22, 25, 26]. Upon (74.4) and 90.9 (91.7), respectively. In experiments 14, colonization, mosquitoes tend to undergo genetic varia- 15 and 23 (Table  2), sporozoites were not seen in the tion over a period of many generations [14, 17, 18]. How colonized mosquitoes, whereas the infection rate in wild this genetic diversity associated with mosquito coloniza- mosquitos was 37.5, 25 and 18.75%, respectively. There tion affects Plasmodium development has not been clear. was no significant difference (paired t test, p = 0.18) in Mohanty et al. Malar J (2018) 17:225 Page 6 of 10 Table 2 Sporozoite infection rate and load in wild and colonized An. stephensi Experiment Gametocytaemia Wild mosquitoes Colonized mosquitoes no. No. No. Sporozoite Gland index No. No. Sporozoite Gland index dissected positive infection dissected positive infection rate (%) rate (%) 3 0.097 21 20 95.2 4+ (12), 3+ (8) 21 21 100 4+ (6), 3+ (15) 5 0.136 20 11 55 4+ (1), 3+ (7), 2+ (2), 20 4 20 3+ (4) 1+ (1) 6 0.585 12 11 91.6 4+ (2), 3+ (10) 12 12 100 4+ (3), 3+ (8), 2+ (1) 7 0.235 21 19 90.4 4+ (3), 3+ (15), 21 16 76.1 4+ (1), 3+ (10), 2+ (4), 1+ (1) 1+ (1) 8 0.18 20 17 85 4+ (7), 3+ (9), 2+ (1) 20 18 90 4+ (3), 3+ (9), 2+ (5), 1+ (1) 9 0.058 20 17 85 4+ (2), 3+ (7), 2+ (7), 20 20 100 3+ (10), 2+ (10) 1+ (1) 10 0.143 21 15 71.4 3+ (8), 2+ (7) 21 17 80.9 3+ (6), 2+ (8), 1+ (3) 11 0.3195 4 0 0 – 4 0 0 – 12 0.198 20 0 0 – 20 0 0 – 14 0.16 8 3 37.5 2+ (3) 8 0 0 – 15 0.06 20 5 25 3+ (1), 2+ (4) 20 0 0 – 16 0.115 18 18 100 4+ (12), 3+ (5), 18 17 94.4 4+ (5), 3+ (11), 2+ (1) 2+ (1) 17 0.145 22 20 90.9 4+ (11), 3+ (9) 22 21 95.4 4+ (5), 3+ (13), 2+ (3) 18 0.055 13 3 23 2+ (3) 13 11 84.6 3+ (1), 2+ (8), 1+ (2) 20 0.037 8 0 0 – 8 0 0 – 21 0.0625 24 19 79.1 4+ (1), 3+ (3), 2+ (6), 24 14 58.3 2+ (10), 1+ (4) 1+ (9) 22 0.095 22 20 90.9 4+ (1), 3+ (3), 2+ 22 16 72.7 4+ (1), 3+ (1), 2+ (7), (13), 1+ (3) 1+ (7) 23 0.040 16 3 18.7 2+ (2), 1+ (1) 16 0 0 0 25 0.088 20 16 80 3+ (4), 2+ (11), 1+ 20 10 50 3+ (1), 2+ (7), 1+ (2) (1), 26 0.141 13 12 92.3 4+ (1), 3+ (9), 2+ (1), 13 12 92.3 3+ (7), 2+ (2), 1+ (3) 1+ (1) 27 0.122 11 0 0 0 11 0 0 0 28 0.176 13 13 100 4+ (8), 3+ (5) 13 12 92.3 4+ (4), 3+ (6), 2+ (2) 29 0.295 21 1 4.7 1+ (1) 21 2 9.5 2+ (1), 1+ (1) 30 0.274 13 12 92.3 4+ (4), 3+ (5), 2+ (3) 13 10 76.9 4+ (2), 3+ (6), 2+ (1), 1+ (1) 31 0.092 20 18 90 4+ (3), 3+ (6), 2+ (8), 20 18 90 3+ (4), 2+ (11), 1+ (3), 1+ (1) 32 0.3 15 12 80 2+ (7), 1+ (5) 15 8 53.3 2+ (7), 1+ (1) Experiment numbers in Table 2 correspond to experiment numbers in Table 1. The data from experiments 3, 5, 6–12, 14 and 15 in wild mosquitoes were used in Balabaskaran et al. [23] to study sporozoite infection kinetics in wild mosquitoes Studies have mapped gene polymorphisms to altered passaged colonized mosquitoes to Plasmodium infection, levels of vector susceptibility to Plasmodium [5, 11, 27, especially P. vivax infection, has not been investigated so 28]. Anopheles gambiae from different geographical loca - far. tions in Africa show different infection intensity to infec - Mosquito infection experiments in wild Anophelines tion with Plasmodium falciparum [29], underscoring are complicated by logistics, availability of mosquitoes the importance of genetic differentiation. Even though year around, their ability to adapt to artificial feeding and varying infection intensities has been reported between new environment. The MESA-ICEMR mosquito infec - sympatric and allopatric vector-parasite combinations tion laboratory at NIMR-Goa is one of the very few sites [29] or between specific intercrosses of mosquito phe - in the world where feeding experiments can be done with notypes [5], the effect of genetic differentiation on highly wild An. stephensi and patient isolates of P. vivax. The Mohanty et al. Malar J (2018) 17:225 Page 7 of 10 Fig. 2 Sporozoite infection rate and 4+ sporozoite load in laboratory and wild Anopheles stephensi. a There is no significant difference in the sporozoite infection rate (paired t test, p = 0.18). b, c, d There is no significant difference in 1+, 2+ and 3+ sporozoite load; paired t test, p = 0.67, 0.53 and 0.89 respectively. e There is a significant difference in 4+ sporozoite load (paired t test, p = 0.002) between wild and laboratory An. stephensi. The values on Y axis were normalized and is reported as percent (%). Experiment number of each comparison experiment is plotted in the x axis Mohanty et al. Malar J (2018) 17:225 Page 8 of 10 Table 3 Distribution of  sporozoite load in  wild Overall, at the level of oocyst development, significant and colonized mosquitoes difference was found between the colonized and wild mosquitoes in their susceptibility to P. vivax. To the first Sporozoite load (gland Wild mosquitoes Colonized index) mosquitoes approximation, it should be possible to use freshly reared larvae of other mosquito species to understand parasite 1+ 25 29 infectivity. Although there was no significant difference 2+ 79 88 in sporozoite infection rate in the present study, a sig- 3+ 112 114 nificantly higher sporozoite load (4+) was found in wild 4+ 68 30 when compared to laboratory mosquitoes. These experi - ments illustrate why it may be important to exercise caution when studying parasite infection in long-term presence of highly passaged An. stephensi (66th–86th laboratory-reared colonies of An. stephensi, especially in generations) allowed to study the susceptibility of wild this region. It will be of interest to learn whether long- and colonized mosquitoes to patient derived isolates of term mosquito colonization alters Plasmodium suscep- P. vivax. In both the mosquito populations, there is sig- tibility in vectors from different geographical locations. nificant difference in oocyst infection rate and whereas Understanding the molecular mechanisms that modulate this was not in the average oocyst load. In three of the Plasmodium infection will also be a prime area of focus experiments, no sporozoites were found in the labora- in the future studies. tory when compared to wild mosquitoes. The average oocyst load ranged between 0 and 2.5 in these experi- ments, and is possible that some parasite strains may not Abbreviations An: Anopheles; GMC: Goa Medical College and Hospital; MESA: malaria develop to sporozoites as efficiently in wild as compared evolution in South Asia; ICEMR: International Center of Excellence for Malaria to laboratory, especially when the oocyst load is very low. Research; NIMR: National Institute of Malaria Research; NIAID: National Institute When the sporozoite load was compared between wild of Allergy and Infectious Diseases; NIH: US National Institutes of Health; US: United States of America; UW: University of Washington; DMID: Division of and laboratory, there was no significant difference in 1+, Microbiology and Infectious Disease. 2+ and 3+ load. However, in 12 of the 14 experiments where 4+ sporozoite load were seen, the number of mos- Authors’ contributions AKM, PBN, PKR, and AK designed the study. PKR, LC and AK administered the quitoes with 4+ load was significantly higher in wild (68) study. AKM, PBN, SB, SV, SP and MD carried out the experiments. PBN, AKM, than in colonized (30) mosquitoes. In An. gambiae, it has PKR, AK, ST and WZ analyzed the data. AKM, PBN, PKR, and AK wrote the been shown that genes involved in insecticide resistance, manuscript. All authors read and approved the final manuscript. immunity and olfaction are expressed higher in wild Author details mosquitoes when compared to colonized populations National Institute of Malaria Research, Field Unit, Campal, Goa 403001, India. [14]. Hence, it is important to learn here that, even under Departments of Chemistry and Global Health, University of Washington, Seattle, WA 98195, USA. Department of Epidemiology and Public Health, sterile rearing conditions lacking continual parasite Central University of Tamil Nadu, Thiruvarur, Tamil Nadu 610005, India. Goa infections, the immune system in colonized An. stephensi Medical College and Hospital, Bambolim, Goa 403202, India. Department was not weakened when challenged with P. vivax. It is of Biology, Stanford University, Stanford, CA 94305, USA. National Institute of Malaria Research (ICMR), Sector 8, Dwarka, New Delhi 110077, India. still possible that, in colonized mosquitoes, there could be a developmental delay that prevents the sporozoites to Acknowledgements populate the salivary gland on days 12 and 13. The authors thank all participating malaria patients at the Goa Medical College and Hospital who volunteered for this study, and Dr. Sachin Shinde, Special Mosquito gut microbiota is determined by the water Secretary (Health) and Administrator (GMC), for his support. The authors are source available in the breeding habitats, and has been most grateful for the administrative and scientific guidance provided by the shown to modulate the development of P. falciparum MESA-ICEMR Scientific Advisory Group, including the Government of India representatives Dr. Shiv Lal, Dr. P Joshi, Dr. Rashmi Arora, and Dr. Manju Rahi, [30–37]. Anti-Plasmodium effect of gut microbiota is and US NIH Programme Officer Dr. Malla Rao. This manuscript was approved suggested to be due to the effect of bacterial compounds by the publication committee of NIMR and bears Approval No. 057/2017. and/or mosquito immunity directed against the microbes Competing interests [31, 38]. It is possible that the altered gut biota in colo The authors declare that they have no competing interests. nized mosquitoes may elicit stronger basal immunity than in wild, and produce metabolites that may affect Availability of data and materials The datasets supporting the conclusions of this article are included within the oocyst maturation and sporozoite development. A recent article. study has implicated a specific Escherichia coli strain 444 in modulating P. falciparum infection in the mos- Availability of data and materials ST95 The datasets supporting the conclusions of this article are included within the quito midgut [38]. The observed differences in 4+ sporo - article. zoite load between wild and colonized mosquitoes may be further investigated in the future. Mohanty et al. Malar J (2018) 17:225 Page 9 of 10 Consent for publication 13. Muller P, Donnelly MJ, Ranson H. Transcription profiling of a recently Not applicable. colonised pyrethroid resistant Anopheles gambiae strain from Ghana. BMC Genomics. 2007;8:36. Ethics approval and consent to participate 14. Aguilar R, Simard F, Kamdem C, Shields T, Glass GE, Garver LS, et al. The human subjects protocol and consent forms for enrollment of Plasmo- Genome-wide analysis of transcriptomic divergence between laboratory dium positive individuals presenting to Goa Medical College and Hospital colony and field Anopheles gambiae mosquitoes of the M and S molecu- were approved by the institutional review boards of the Division of Microbiol- lar forms. Insect Mol Biol. 2010;19:695–705. ogy and Infectious Diseases at the US National Institute of Allergy and Infec- 15. Hoffmann AA, Hallas R, Sinclair C, Partridge L. 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Malaria JournalSpringer Journals

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