Hindawi BioMed Research International Volume 2018, Article ID 2472508, 10 pages https://doi.org/10.1155/2018/2472508 Research Article Second Blood Meal by Female Lutzomyia longipalpis: Enhancement by Oviposition and Its Effects on Digestion, Longevity, and Leishmania Infection C. S. Moraes,1,2 K. Aguiar-Martins,2 S. G. Costa,1,2 P. A. Bates ,2 R. J. Dillon,2,3 and F. A. Genta 1,3 1 Laboratory of Insect Biochemistry and Physiology, Oswaldo Cruz Institute, FIOCRUZ, 4365 Brasil Av., Leonidas Deane Building, Room 207, 21040-360 Manguinhos, RJ, Brazil 2Faculty of Health and Medicine, Division of Biomedical and Life Sciences, Lancaster University, Furness Building, Bailrigg, Lancaster LA1 4YG, UK 3National Institute of Science and Technology for Molecular Entomology, 373 Carlos Chagas Filho Av., Center for Health Science, Building D, Basement, Room 5, Cidade Universitaria,21941-590RiodeJaneiro,RJ,Brazil´ Correspondence should be addressed to F. A. Genta; [email protected] Received 9 October 2017; Revised 11 January 2018; Accepted 15 February 2018; Published 25 March 2018 Academic Editor: Marlene Benchimol Copyright © 2018 C. S. Moraes et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lutzomyia longipalpis is the main vector of visceral leishmaniasis (VL) in America. Physiological and molecular mechanisms of Leishmania infection in sand fies have been studied during the frst gonotrophic cycle. Tere are few studies about these interactions during the second gonotrophic cycle mainly because of the difculties maintaining sand fies through sequential feeds. Here we standardized conditions to perform the second blood feed efciently, and our results show that oviposition is an essential factor for the success of multiple feeds. We evaluated the impact of the second blood meal on longevity, protein digestion, trypsin activity, and Leishmania mexicana development within L. longipalpis gut. Mortality of blood-fed females increases afer second blood meal as compared to sugar-fed females. Trypsin activity was lower during the second gonotrophic cycle. However, no diference in protein intake was observed between blood meals. Tere was no diference in the population size of Leishmania in the gut afer both blood meals. In this work, we presented an optimized protocol for obtaining sufcient numbers of sand fy females fed on a second blood meal, and we described some physiological and parasitological aspects of the second gonotrophic cycle which might infuence the vectorial competence of sand fies. 1. Introduction of vertebrates as dogs, chickens, humans, and horses, among others [4]. Afer a saturating blood meal, the sand fy female Visceral leishmaniasis (VL) is a severe chronic disease caused starts its digestion, a process that takes an average of 3-4 days by protists belonging to the Leishmania donovani complex [1]. [5]. Female L. longipalpis are anautogenous insects, where egg Currently, the estimated incidence of VL is 0.2 to 0.4 million development occurs only afer a blood feed. Te oviposition cases per year with over 90% occurring in India, Bangladesh, in this species starts 3 days afer the blood meal and in general Sudan, South Sudan, Ethiopia, and Brazil [2]. lasts for 6 days [6, 7]. A second blood feed is necessary to start In the Americas, the phlebotomine sand fy Lutzomyia a new cycle of digestion and oviposition [8]. longipalpis isthemainvectorofVL[3].Bothmaleandfemale During an infective blood meal, sand fies ingest infected L. longipalpis adults, afer emergence from pupae, feed on macrophages that release amastigote forms of Leishmania sugars from plants and aphids in a regular basis, but only parasites in the midgut. Te amastigotes diferentiate in adult females are hematophagous, feeding on a wide range the sand fy midgut to procyclic promastigotes, replicative 2 BioMed Research International forms which can survive and develop in the presence of 2. Materials and Methods microbiota [9, 10] and digestive enzymes [11–17]. Afer 2–4 days, procyclic forms develop into nectomonad forms, which 2.1. Sand Flies Maintenance. L. longipalpis, originally from escape from the peritrophic matrix by the posterior opening Jacobina (Bahia, Brazil), were kept at Lancaster University of this structure, attach to the intestinal epithelium, and (United Kingdom) under standardized conditions of tem- 24 ± 2∘ migrate forward to the anterior thoracic midgut by chemo- perature ( C) and a photoperiod of 8 h of light/16 h taxis/osmotaxis [13, 14, 18–24]. Nectomonad forms transform of darkness. Adult insects (males and females) were fed into leptomonad forms, multiplicative forms responsible ad libitum with 70% (w/v) autoclaved sucrose during all for the parasites’ second replicative cycle and secretion of experiments. promastigote secretory gel (PSG) [25]. Haptomonad forms Unless specifed, sand fies received the frst blood meal colonizethestomodealvalvebyattachmenttothecuticular four days afer emergence of adults. Second blood feeding lining, blocking this structure physically, together with lep- was done seven days afer frst blood feeding (females were tomonads and metacyclics that are embedded in the PSG [13, 11 days old). We chose these time points because in our conditions these are the minimal times to have successful frst 14]. Metacyclics are the infective forms for the mammalian andsecondbloodmeals.Longertimeswouldimpairthetime vertebrateanddevelopintheforegutorbehindthestomodeal course analysis of blood digestion and infection due to high valve from leptomonad forms [25]. Teir transmission to the mortality rates (see below). vertebrate host occurs through a bite during a subsequent Sheep blood, with Alsever’s anticoagulant (Cat. No. blood feed [14]. SB068), was purchased from TCS Biosciences (Buckingham, Trypsinisakeyenzymeresponsibleforprimarydigestion United Kingdom) and used in all experiments. Blood feeds of blood proteins in female sand fies [16]. Leishmania were carried out via artifcial apparatus (Hemotek, Discovery ∘ parasites can modulate the activity of this enzyme during Workshops), with chicken skin membranes held at 37 Cfor1 infectioninthesandfyvector[11,12,15–17,26].Despitethese hour. studies, nothing is known about the regulation of digestion in infected fies during the second gonadotrophic cycle. 2.2. Percentage of Fed Females, Oviposition, and Counting of Elnaiem et al. [27] described the development of Leish- Developed Oocytes afer Uninfected Blood Feeding. Females mania chagasi parasites within L. longipalpis gut during the (� = 125)andmales(�=25) were added to cages with second gonadotrophic cycle. It was shown that the second dimensions of 17 × 17 × 19 cm (height × width × length, resp.) blood feed is essential for the relatively rapid appearance for the frst blood feed. Afer blood feeding, fed females were of metacyclic forms and its fast migration to the vector equally divided between 2 small cages (dimensions 11 × 11 × proboscis, perhaps leading to an increase of vectorial com- 14 cm, height × width × length, resp.), one of them containing petence in infected sand fies undergoing the second cycle. a plastic oviposition pot inside of it (on the bottom of the Similarly, Vivenes et al. [28] showed the rise of migration and cage).Tepot(4.6cmdiameter× 4.3 cm height) included a colonization of Leishmania mexicana in Lutzomyia evansi 1 cm thick layer of plaster of Paris in the base, moistened with esophagus afer a second blood meal. deionized water. Tese cages were maintained as described in One crucial limitation for studying multiple gonotrophic sand fy maintenance section and afer seven days, 25 females cycles in sand fies is the difculty in obtaining engorged from each group were ofered a second blood feed. Te females afer a second blood feed. Rearing large colonies percentage of fed fies was estimated afer visual inspection of sand fies in the laboratory is problematic due to high of blood engorgement. Immediately afer the second blood mortality afer the frst blood feed in contrast to breeding feed, all females (engorged and nonengorged) were dissected the commonly available mosquito species that can be easily in PBS or 0.9% w/v saline, and the number of oocytes within maintained through multiple gonotrophic cycles. Previous thebodywascounted.Becausesandfiesareanautogenous workhasevaluatedthepercentageofblood-fedfemalesof insects, we assume that the frst blood meal does not interfere L. longipalpis afer frst and second blood feeds and, in in the oocyte counting afer the second blood feed. Females general, the number of fed females was much lower afer with small amounts of blood in the gut (partial blood feeding) the second blood feed than the frst feed [8]. Stamper et were not counted as fed. al. [29] observed that Phlebotomus duboscqi sand fies that had laid eggs previously accepted a second blood meal more 2.3. Longevity of Uninfected Females afer First and Second efciently compared to insects that retained eggs. Tis result Blood Meal. In an initial series of experiments, we compared suggested that oviposition was an essential prerequisite for sugar-fed and blood-fed females (frst blood meal). For both a successful second blood feed. Considering the critical role groups, we used 4-day-old females (30–50 insects per group) of the multiple blood feedings for Leishmania transmission, in 17 × 17 × 19 cm cages. we aimed to investigate conditions that would