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Raw sewage as breeding site to Aedes (Stegomyia) aegypti (Diptera, culicidae)
Accepted Manuscript Title: Raw sewage as breeding site to Aedes (Stegomyia) aegypti (Diptera, Culicidae) Author: R.F. Chitolina F.A. Anjos T.S. Lima E.A. Castro M.C.V. Costa Ribeiro PII: DOI: Reference:
S0001-706X(16)30507-1 http://dx.doi.org/doi:10.1016/j.actatropica.2016.07.013 ACTROP 3991
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Acta Tropica
Received date: Revised date: Accepted date:
4-2-2016 28-6-2016 17-7-2016
Please cite this article as: Chitolina, R.F., Anjos, F.A., Lima, T.S., Castro, E.A., Costa Ribeiro, M.C.V., Raw sewage as breeding site to Aedes (Stegomyia) aegypti (Diptera, Culicidae).Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2016.07.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Raw sewage as breeding site to Aedes (Stegomyia) aegypti (Diptera,
Culicidae)
Chitolina, R. F.1, Anjos, F. A.1, Lima, T. S.1, Castro E. A.1, & Costa Ribeiro, M. C. V.1, *
1
Laboratório de Parasitologia Molecular, Departamento de Patologia Básica, Centro Politécnico, Universidade Federal do Paraná, Brasil.
*Corresponding author:
[email protected]
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Graphical abstract
Highlights 1. Aedes aegypti females can lay their eggs either in clean water or raw sewage. 2. Ae. aegypti females retain their eggs if there is not a suitable oviposition site 3. Raw sewage may be acting as breeding site for Ae. aegypti mosquitoes. 4. Control programs must re-think their action considering new breeding sites.
ABSTRACT
The selection of oviposition sites by females of Aedes (Stegomyia) aegypti is
a key factor for the larval survival and egg dispersion and has a direct influence
in vector control programs. In this study, we evaluated the aspects of
reproductive physiology of Ae. aegypti mosquitoes tested in the presence of raw
sewage. Ae. aegypti females were used in oviposition bioassays according to
two methodologies: (i) choice assay, in which three oviposition substrates were
offered in the same cage: treatment (raw sewage), positive control (distilled
water) and negative control (1% sodium hypochlorite) and; (ii) no choice assay,
in which only one substrate was available. The physicochemical and
microbiological analysis of the raw sewage used in this study indicated virtually
no levels of chlorine, low levels of dissolved oxygen and high levels of
nitrogenous compounds as well as the presence of Escherichia coli and total
fecal coliforms. After 72 hours of oviposition, the eggs were counted and there
was no statistically significant difference (p>0.05) in the oviposition rate
between raw sewage and positive control in both methodologies. In addition,
females were dissected to evaluate egg-retention and also there were no
appreciable differences in egg retention even when raw sewage was the only
substrate offered. The data also showed that egg hatching and larvae
development occurred normally in the raw sewage. Therefore, the present study
suggests that Ae. aegypti can adapt to new sites and lay eggs in polluted water,
such as the raw sewage. These findings are of particular importance for the
control and surveillance programs against Ae. aegypti in countries where the
conditions of poor infrastructure and lack of basic sanitation are still an issue.
KEY WORDS: Aedes aegypti, oviposition, raw sewage, skip-oviposition
behavior
1. INTRODUCTION
The selection of sites for oviposition by females of Aedes (Stegomyia)
aegypti (Linnaeus, 1762) is a key factor for the survival of their immature forms
(Bentley & Day 1989). The choice of oviposition sites is influenced by visual,
tactile and olfactory factors, the latter considered of primary importance usually
due to decomposition of organic matter, the presence of immature mosquitoes
and fauna-associated bacteria (Benzon & Apperson, 1988; Bentley & Day 1989;
Logan & Birkett, 2007;. Sharma et al 2008). In addition, physical factors like
temperature, humidity, presence of vegetation, and chemical factors such as
co-specifics and oviposition attractants also influence the choice of the breeding
site (Benzon & Apperson, 1988; Bentley & Day, 1989).
According to Varejão et al., (2005) Ae. aegypti breeds in clean water reservoirs, although it may be able to adapt to new situations imposed by the humans. Indeed, it was shown that the presence of fecal coliform bacteria was able to increase the attractiveness for oviposition (Navarro et al., 2003). The ability of the vector to develop in environments with high levels of pollution as well as in raw sewage is due to the high concentration of organic material, which contains nutrients such as proteins, carbohydrates and lipids (Beserra et al., 2010). According to the same authors, the choice of oviposition site is not only related to the degree of water pollution, but also to the environmental conditions necessary for the survival and development of immature mosquitoes, making them adaptable to lay eggs in sites considered unfavorable. For more than 40 years Ae. aegypti larvae, pupae and adults have been found in environments considered polluted, such as septic tanks, sewage disposal containers and raw sewage (Chan et al., 1971; Barrera et al., 2008;
Troyo et al., 2008; Burke at al., 2010, Banerjee et al., 2015). According to the
WHO, in 2015 more than 30% of the global population has no access to improved sanitation facilities (WHO/UNICEF, 2015) and in Francisco Beltrão municipality, Paraná state, Brazil, immatures forms of the Ae. aegypti were found in raw sewage during dengue epidemic (Personal communication, 2012).
Although several studies have reported the detection of this vector in raw sewage, only a few studies have addressed the attractiveness of oviposition of Ae. aegypti to collections of water with high levels of pollution such as raw sewage (Navarro et al., 2003; Beserra et al., 2010). Therefore, the aim of this study was to evaluate under laboratory conditions, aspects of the reproductive
physiology of Ae. aegypti in the presence of raw sewage.
2.
MATERIAL AND METHODS
2.1
Mosquitoes rearing
Ae. aegypti (Rockefeller strain) were reared and maintained under laboratory conditions (26°C±2°C, 12:12h photoperiod, 80% humidity). To
perform the oviposition bioassays 4-5-days old females were allowed to blood
feed on anesthetized mice (Mus musculus) (ketamine:xylazine 80–120:10–16
mg/kg) according to Ethics Committee for Animal Use (protocol number 714) of
the Federal University of Paraná.
2.2
Raw sewage samples
Raw sewage was collected at the Atuba Sewage Treatment Station -
SANEPAR in Curitiba, Paraná, and analyzed by microbiological, physical and
chemical tests according to the Standard Methods for the Examination of Waste
water (Standard Methods, APHA, 21st – 22nded) (Table 01) by Center for
Research and Food Processing (CEPPA) of the Federal University of Paraná.
In order to determine the best concentration of sewage, we performed
oviposition assays in the presence of different dilutions of raw sewage (25%, 50% and 100%). As no significant differences were found between the dilutions,
all further experiments were performed using raw sewage without dilution (see
below). 2.3
Sixty (60) mL of raw sewage without dilution, distilled water (positive control)
and 1% sodium hypochlorite (negative control) were allocated inside a
transparent recipient with a filter paper as an oviposition substrate, these were
Experimental solutions preparation
then placed inside a black vessel, known as attractive to the species. 2.4
Oviposition experimental design Fifteen fully engorged females with the same physiological age were allocated inside a cage with 30x30x30 cm. The solutions were disposed equidistantly in an equilateral triangle, replaced every 24 hours, and rotated in clockwise direction. Each experiment was repeated six times (n=6). To ensure that the females were in proper conditions for oviposition, a physiological control cage (vessels with distilled water only) was used (n=3). The solutions were placed inside the cages five days after the blood meal, in order to ensure that all the females would be able to lay eggs. After 72 hours, the number of eggs laid were quantified. The ovaries of all females were dissected in order to distinguish parous of non-parous females according to Detinova (1962) apud Consoli & Lourenço-de-Oliveira (1994) and to count the number of eggs retained inside the ovaries. The oviposition assay was performed using two distinct methodologies: (i) choice assay, in the same
cage all the vessels with solutions (treatment, positive and negative control)
were disposed together equidistantly inside the cage, allowing the females to
choose the best egg-laying sites, and the (ii) no choice assay, where only one
solution in each cage was available to the females to lay eggs. 2.5
Hatching and development assay In order to check the hatching rate of the eggs in the raw sewage, were put to hatch inside a tray containing one liter of raw sewage or aged water (control). The eggs were allowed to hatch for 2 days, and then the numbers of larvae were counted. The experiment was performed two times in triplicate. To check the development of the mosquitoes inside the raw sewage, ten
first instar larvae were placed inside a cup with 30 mL of raw sewage or aged water (control). To simulate the real conditions of growth, 15 mL of raw sewage
was replaced every other day in each cup containing the larvae. For the control 1 mL of food solution (2g/L) were provided every other day. Adult emergence was monitored every day until the emergence of the last adult mosquito. The development assay was repeated three times with five replicas in each.
2.6
Statistical analysis
The choice of the best raw sewage concentration was determined using a One Way ANOVA followed of a Tukey`s Multiple Comparisons Test (p<0.05). A t-test of Mann-Whitney was performed to check oviposition rate, hatching rate and the number of adult mosquitoes that raised from development assay. The time of development between the sewage and the control were analyzed by a Two Way ANOVA followed of a Bonferroni’s test.
To evaluate the oviposition behavior a One Way ANOVA followed of a Tukey`s Multiple Comparisons Test was used to compare the oviposition through the days and eggs retention in the no choice assay. In addition, to access the oviposition attractiveness of Ae. aegypti to raw sewage, the Oviposition Activity Index (OAI), given by Kramer & Mulla (1979), was calculated as follows: OAI = Nt – Ns /Nt + Ns Where: Nt = Number of eggs in the treatment Ns = Number of eggs of the control The OAI can range between +1 and -1. The positive values indicate that there are more eggs in the treatment compared to positive control, and negative
values indicate the reverse. According to Kramer and Mulla (1979) OAI ≥ +0.30 indicates the presence of substances that serve as oviposition attractants for
mosquitoes; conversely OAI≤ -0.30 indicates the presence of repellent substances.
3. RESULTS 3.1 Physical, chemical and microbiological analyses of raw sewage
The microbiological analysis of raw sewage showed high levels of
Escherichia coli and total fecal coliforms, up to 106 cells in all analysis. Physical
and chemical analysis indicated the presence of low levels of dissolved oxygen
(DO) associated with a high chemical oxygen demand (CDO), high levels of nitrogenated compounds and virtually no chlorine levels (Table 01).
3.2
Determination of raw sewage concentration.
First, we assayed the oviposition behavior of Ae. aegypti towards three different concentrations of raw sewage (25%, 50% and 100%). In the physiological control, an average number of 740.33 (± 121.14) eggs were obtained, showing that the females were able to lay eggs. In the experimental cages, it was obtained an average number of 381.67 (±118.07), 335.00 (±112.09) and 407.17 (± 328.67) eggs in the raw sewage solutions of 25%, 50% and 100%, respectively. As no significant differences were found (p= 0.8377) between the dilutions and in order to simulate natural environmental conditions, all further experiments using raw sewage were performed without dilution. 3.3
Oviposition assay
Following the determination of raw sewage concentration, an oviposition assay was performed to check whether or not raw sewage affects the oviposition rate of Ae. aegypti female under laboratory conditions. The oviposition assays were performed according to the methodologies of choice or no choice, as represented on figure 01. In the choice assay, no statistical difference (p= 0.3095) was found between the number of eggs laid in the raw sewage 526.50 (± 196.74) and the positive control 409.83 (± 137.19) (Fig. 02 A). As expected, no eggs were laid in
the negative control. In the physiological control an average of 1059.67 (± 255) eggs were observed. Similarly, in the no choice assay, no significant differences (p= 0.4848) were found between the number of eggs laid in the presence of only raw sewage (1237.83 ± 462.60) in comparison to the positive control (1003 ± 218.23) (Fig. 02 B). No eggs were laid in the negative control. These results suggested that, at least under laboratory conditions, raw sewage is a suitable 3.4
Dissection and evaluation of eggs retention
Since we did not observe any difference in the oviposition rate, was evaluated the egg retention ratio in the ovaries of Ae. aegypti females after both choice and no choice oviposition assays. In the choice assay it was found that in the experimental cages 273.50
(± 128.09) eggs were retained in the ovaries of Ae. aegypti females whereas 307.00 (± 126.58) eggs were retained in females of the physiological control group. No significant differences were found between the groups (p= 0.9048) (Fig 03 A). In the no choice assay the retention of eggs were compared between the three set of cages: i) raw sewage (470.67 ± 415.20 eggs retained); ii) positive control (318.50 ± 142.94 eggs retained); and iii) negative control (1106.33 ± 197.01 eggs retained). Statistical analysis revealed a significant difference between the groups compared (p=0.0004) with difference between the positive and the negative control (p<0.0001) and between raw sewage and the negative control (p< 0.001). However, no significant differences were found between the raw sewage solution and the positive control (Fig 03 B). These results reinforce the idea that raw sewage is a suitable place for oviposition.
3.5
Evaluation of oviposition behavior
We further evaluated the Oviposition Activity Indexes (OAI) as well as the oviposition behavior during the three days of experiment. The Oviposition Activity Indexes (OAI) values were +0.1246 and +0.1048 for the choice and no choice assay, respectively. In both assays (Choice and No Choice), we noted that females of the positive control group laid more eggs in the first day in comparison to day two and three in the choice assay (p= 0.0001) (Fig. 04 A) and compared to day three in the no choice assay (p= 0.0026) (Fig 04 B) respectively. Interestingly, the same daily pattern of oviposition was observed in the raw sewage group in the choice assay (p< 0.0001) (Fig. 04 C) and in the no choice assay (p= 0.0009) (Fig 04 D). These data suggested that Ae. aegypti females display the same oviposition behavior towards water and raw sewage.
3.6
Hatching and development of larvae in raw sewage
Since Ae. aegypti females do not discriminate between water and raw sewage as oviposition substrates under laboratory conditions, we further
evaluated whether raw sewage could support hatching and development of
larval stages. In the hatching assay we initially observed that eggs laid by female Ae. aegypti were able to hatch both in water and in sewage (p= 0.1797) (Fig 05 A). It was observed that the larvae that developed in water reached the adult stage approximately 2-3 days earlier than the larvae grown in the raw sewage (Fig 05 B). However, despite the difference in the larvae growth period, adult mosquitoes at the end of the development period were present in about same numbers in both conditions (p= 0.4256) (Fig 05 C). Altogether, these results demonstrate that raw sewage can provide all the conditions needed for mosquito eggs to hatch as well as for supporting the development of larvae into adult mosquitoes.
4.
DISCUSSION
In the present study we showed that under laboratory conditions Ae. aegypti females lay eggs in both clean water and raw sewage. Moreover, eggs and larvae are fully capable of hatch and develop in polluted water, respectively.
This data corroborates a few studies showing that Ae. aegypti
females can lay their eggs in contaminated water, such as the sewage, and these eggs are able to fully develop into adult mosquitoes (Navarro et al., 2003; Beserra et al., 2010). Furthermore, the positive values found for both methodologies (choice and no choice) in the OAI are an indicative that the raw sewage may be attractive to oviposition of females.
Besides corroborating the literature data, our findings showed that Ae.
aegypti can adapt to adverse conditions. As far as we know, studies aiming to check oviposition rate and behavior have only checked the oviposition rate comparing a treatment with a positive control. We not only analyzed the oviposition rate between raw sewage with a positive and negative controls but also the eggs retention, reinforcing our conclusion that raw sewage is a suitable place for oviposition. Moreover, in order to guarantee that all the females used in the assays were fully capable to lay eggs we performed the Detinova technique, which consists in check the tracheolar endings, where distended tracheolar endings characterizes a parous female. In both methods of the study (choice and no choice assay) all the females used in the bioassays were parous, proving that a blood meal was realized and they were able to lay eggs (data not show). Under natural environmental conditions, Ae. aegypti females choose the best place to lay their eggs, and oviposit a few eggs in several different sites, this is called skip oviposition behavior (Colton, et al., 2003; Abreu, et al., 2015). Moreover, Gomes et al., (2006) and Abreu et al., (2015) showed that under laboratory and semi-field condition observed that there is a pick of oviposition three days after blood meal. In this study, Ae. aegypti females were blood fed and deprived of oviposition for five days, which led the females to lay the great majority of eggs during the first day, in both positive control and raw sewage. We hypothesized that once the females were submitted to a stressful condition, maintained in a closed space with no access to an oviposition site, it would be expected that after this time the females would have a need to lay eggs. Thus, a higher number of eggs would be expected at day one, if a suitable place for
oviposition was available, what happened during our experiments. Furthermore,
unpublished data from our group showed that once a suitable place is not available (for example when a repellent substance is present) a higher amount of eggs is not observed in the first day, even after five days with no access to an oviposition site, but distributed homogeneously over the days of experiment. Therefore, we believe that raw sewage, under laboratory conditions, has no repellent properties against mosquitoes. Wong et al. (2012) showed that when ideal containers are eliminated in the environment, others recipients that are not the main breeding sites to oviposition are chosen by Ae. aegypti to lay their eggs. The raw sewage, therefore, needs to be carefully considered as an optional oviposition site. Not only this site is pointed as capable of increase the productivity of mosquitoes offspring (Chaves et al., 2009), but also has been considered as source of infestation (Chan et al., 1971; Barrera et al., 2008; Troyo et al., 2008; Burke et al., 2010, Banerjee et al., 2015). Therefore, in urban areas where basic sanitation conditions are not met, the open raw sewage may be acting as a natural environment for the development of mosquitoes. The factors that lead to this change in oviposition behavior are still unclear but several studies have shown that the presence of bacteria increase the oviposition site preference. Bacteria are responsible for increase the oviposition rate in organic infusions when compared to bacteria-free leaf infusions, suggesting a role for microbial activity in the production of odorants that assist the oviposition activity in mosquitoes (Ponnusamy et al., 2009; Ponnusamy et al., 2010).
In the physical, chemical and microbiological analysis of raw sewage, we
observed a high amount of bacteria and fecal coliforms that may be the
responsible for the positive oviposition response, and a low rate of dissolved
oxygen, which can be validated for the founding’s of Ponnusamy and
colleagues (2011). These authors suggest that, not only the bacteria are
responsible for the attractiveness of oviposition sites, but also for stimulating
hatch of Ae. aegypti eggs.
Besides that, other aspects in the raw sewage can contribute to the
oviposition such as the low amount of chlorine, known as harmful for the eggs
and larvae and the high levels of nitrogenous compounds found in our samples.
Darriet & Corbel (2008) found out that collection of water containing high
amounts of nitrogenous compounds, such as ammonium (NH+4), nitrite (NO−2)
and nitrate (NO−3) ions, were more attractive to gravid females. In addition,
Monteiro Marques et al., (2013) evaluated different public water supply for
human consumption and found that high concentrations of ammoniacal nitrogen in public water were responsible for the attractiveness of pregnant Ae. aegypti females. Associated to the fact that the females may lay their eggs in contaminated water, the fully development of these eggs is ensured by several factors such as the higher amount of nutrients available and the high water turbidity. The latter is particularly important because it provides a suitable site for Ae. aegypti development, taking into consideration that Ae. aegypti larvae are photofobics (Consoli & Lourenço-de-Oliveira (1994). Our data also revealed that the eggs can hatch properly in the sewage, however, the development from an immature stage to an adult phase was delayed by three days compared to
clean water. This observation does not corroborate results obtained by Beserra
et al. (2010), who reported a faster development of larvae in raw sewage.
However, this apparent discrepancy can be due to the difference in the raw
sewage composition used in both studies.
According to several authors, Ae. aegypti is the vector of many
arbovirosis such as dengue, chinkungunya and zika (Diallo et al., 1999;
Lourenço de Oliveira et al., 2002; Li et al., 2012; Musso et al., 2014). Therefore,
any environmental conditions that may favor the development of the vector,
such as the lack of infrastructure and minimal sanitary conditions might
contribute to Ae. aegypti development, increasing the risk for diseases
transmission.
4. CONCLUSIONS
The importance of our study is not only to understand the physiological factors associated with the choice of reservoir for oviposition, but also to clarify and highlight that Ae. aegypti females can adapt to new breeding sites. These observations must be taken into account in the development of future vector control actions in order to prevent the presence of the vector and consequently the transmission of viruses associated with it. In general, the control of Ae. aegypti is based on observations only in artificial breeding sites with clean water, however, our results indicate that Ae. aegypti can lay eggs not only in clean water but also in raw sewage. Still, more studies need to be performed, for example with field populations, in order to fully understand the oviposition conditions. Nevertheless, our work using a
laboratory strain suggests that the Ae. aegypti mosquitoes can adapt to the new
oviposition niches and, which may threat the vector control programs, once
aspects related to the adaptation of the species to new breeding sites have
being neglected.
5. ACKNOWLEDGMENTS
We would like to thank to the SANEPAR in the person of Angélica de
Lima de Araujo, who helped and allowed the collection of sewage material. To
the Laboratório de Entomologia Prof. Ângelo Moreira da Costa Lima in the
person of Dra. Maria Aparecida Cassilha Zawadneak. To the Laboratório de
Fisiologia e Controle de Artrópodes Vetores – IBEX/Rio de Janeiro, Brazil which
provided the Rockeffeler strain and to Dr. Emerson Soares Bernardes for the
critical reading of the manuscript.
6. FUNDING
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 7. REFERENCES
Abreu, F. V. S. D., Morais, M. M., Ribeiro, S. P., & Eiras, Á. E. 2015. Influence of breeding site availability on the oviposition behaviour of Aedes aegypti. Mem do Inst Oswaldo Cruz, 110 (5): 669-676.
Banerjee, S., Mohan, S., Saha, N., Mohanty, S. P., Saha, G. K., & Aditya, G.
2015. Pupal productivity & nutrient reserves of Aedes mosquitoes breeding in
sewage drains & other habitats of Kolkata, India: implications for habitat
expansion & vector management. Indian J Med Res, 142: 87-94.
Barrera, R., Amador, M., Diaz, A., Smith, J.,Munoz-Jordan, J.L., Rosario, Y.
2008. Unusual productivity of Aedes aegypti in septic tanks and its implications
for dengue control. Med Vet Entomol, 22: 62-69.
Bentley, M. D., Day, J. F. 1989. Chemical ecology and behavioral aspects of
mosquito oviposition. Ann Rev Entom, 34: 401–421.
Benson, G. L., Apperson, C. S. 1988. Reexamination of chemically mediated
oviposition behavior in Aedes aegypti (L.)(Diptera: Culicidae). J Med Entomol, 25 (3): 158-164.
Beserra, E. B., Fernandes, C. R. M., Souza, J. T., Freitas, E. M., Santos, K. D. 2010. The Effect of Water Quality in the Life Cycle and in the Attraction for the Egg Oviposition of Aedes aegypti (L.) (Diptera: Culicidae). Neotrop Entom, 39
(6):1016-1023.
Burke,R., Barrera, R., Lewis, M., Kluchinsky, T., Claborn, D. 2010. Septic tanks as larval habitats for the mosquitoes Aedes aegypti and Culex quinquefasciatus in Playa-Playita, Puerto Rico. Med Vet Entom, 24:117–123.
Chan, Y. C., Ho, B. C., Chan, K. L. 1971. Aedes aegypti (L.) and Aedes albopictus (Skuse) in Singapore City. Bull World Health Organ, 44 (5): 651– 657.
Chaves, L.F., Keogh, C.L., Vazquez-Prokopec, G.M., Kitron, U.D. 2009.
Combined sewage overflow enhances oviposition of Culex quinquefasciatus (Diptera: Culicidae) in urban areas. J Med Entomol, 46 (2): 220-226.
Colton, Y. M., Chadee, D. D., Severson, D. W. 2003. Natural skip oviposition of the mosquito Aedes aegypti indicated by codominant genetic markers. Med Vet Entomol, 17: 195-204.
Consoli, R. A. G. B., De Oliveira, R. L. Principais mosquitos de importância
sanitária no Brasil. SciELO-Editora FIOCRUZ, 1994.
Darriet, F., & Corbel, V.
2008. Propriétés
attractives et
modifications
physicochimiques des eaux de gîtes colonisées par des larves de Aedes aegypti (Diptera: Culicidae). Comptes Rendus Biologies, 331 (8): 617-622.
Diallo, M., Thonnon, J., Traore-Lamizana, M., & Fontenille, D. 1999. Vectors of Chikungunya virus in Senegal: current data and transmission cycles. Am J Trop Med Hyg, 60 (2): 281-286.
Gomes, A. D. S., Sciavico, C. J., Eiras, Á. E. 2006. Periodicity of oviposition of females of Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) in laboratory and field. Rev Bras Med Trop, 39: 327-332.
Kramer, L.W., Mulla, S.M. 1979. Oviposition attractants and repellents of mosquitoes: oviposition responses of Culex mosquito to organic infusions. Environ Entomol, 8: 1111-1117.
Li, M. I., Wong, P. S., Ng, L. C., & Tan, C. H. 2012. Oral susceptibility of
Singapore Aedes (Stegomyia) aegypti (Linnaeus) to Zika virus. PLoS Negl Trop Dis, 6 (8): e1792.
Logan, J. G.,Birkett, M. A. 2007. Semiochemicals for biting fly control: their identification and explotation. Pest Manag Sci, 63: 647-657.
Lourenço-de-Oliveira, R., Honório, N. A., Castro, M. G., Schatzmayr, H. G., Miagostovich, M. P., Alves, J. C., ... & Nogueira, R. M. 2002. Dengue virus type 3 isolation from Aedes aegypti in the municipality of Nova Iguaçu, State of Rio de Janeiro. Mem I Oswaldo Cruz, 97 (6): 799-800.
Monteiro-Marques, G. R. A., Chaves, L. S. M., Serpa, L. L. N., Arduino. M. B., Chaves, F. J. M. 2013.Public drinking water supply and egg laying by Aedes aegypti. Rev Saúde Públi, 47 (3): 579-587.
Musso, D., Nilles, E. J., & Cao‐Lormeau, V. M. 2014. Rapid spread of emerging Zika virus in the Pacific area. Clin Microbiol Infec, 20 (10): O595-O596.
Navarro, D. M. A. F., Oliveira, P. E. S., Potting, R. P. J., Brito, A. C., Fital, S. J. F., Sant'Ana, A. E. G. 2003. The potential attractant or repellent effects of different water types on oviposition in Aedes aegypti L. (Dipt.,Culicidae). J Appl Entomol, 127: 46-50.
Ponnusamy, L., Wesson, D. M., Arellano, C., Schal, C., Apperson, C. S. 2009. Species
composition
of
bacterial
communities
influences
attraction
mosquitoes to experimental plant infusions. Microb Ecol, 59: 158–173.
of
Ponnusamy, L., Xu, N., Böröczky, K., Wesson, D. M., Ayyash, L. A., Schal, C.,
Apperson, C. S. 2010.Oviposition responses of the mosquitoes Aedes aegypti and Aedes albopictus to experimental plant infusions in laboratory bioassays. J Chem Ecol, 36: 709–719.
Ponnusamy L., Böröczky,K., Wesson, D.M., Schal, C., Apperson,C. S. 2011. Bacteria stimulate hatching of yellow fever mosquito eggs. PLoS ONE, 6 (9): e24409.
Sharma, K. R., Seenivasagan, T., Rao, A. N., Ganesan, K., Agarwal, O. P.,
Malhotra, R. C., Prakash, S. 2008. Oviposition responses of Aedes aegypt iand Aedes albopictus to certain fatty acid esters. Parasitol Res, 103: 1065-1073.
STANDARD methods for the examination of water and wastewater. 2012. 22nd ed. Washington: APHA; AWWA; WEF.
STANDARD methods for the examination of water and wastewater. 2005. 21st ed. Washington: APHA; AWWA; WEF.
Troyo, A., Calderón-Arguedas, O., Fuller, D.O., Solano, M.E., Avendaño, A., Arheart, K.L., Chadee, D.D., Beier, J,C. 2008. Seasonal profiles of Aedes aegypti (Diptera: Culicidae) larval habitats in an urban area of Costa Rica with a history of mosquito control. J Vector Ecol, 33 (1): 76-88.
Varejão, J.B.M., Santos, C.B., Rezende, H.R., Bevilacqua, L.C., Falqueto, A. 2005. Aedes (Stegomyia) aegypti (Linnaeus, 1762) breeding sites in native bromeliads in Vitória City, ES. Ver Soc Brasil Med Trop, 38: 238-240.
Wong, J., Morrison, A.C., Stoddard, S.T., Astete, H., Chu, Y.Y., Baseer, I.,
Scott, T.W. 2012. Linking oviposition site choice to offspring fitness in Aedes aegypti: consequences for targeted larval control of dengue vectors. PLoS Negl Trop Dis, 6 (5): e1632.
WHO/UNICEF, Progress on sanitation and drinking water: 2015 update and MDG assessment. World Health Organization (WHO), 2015.
Figure 01. Schematic representation of oviposition assay methodologies. A) Choice assay. B) No choice assay. Exp. Cages: Experimental Cages; Phys. Cages: Physiological Cages.
Figure 02: Oviposition assay. A) Comparisons of the oviposition rate of the raw
sewage and positive control, according to the methodology of choice assay. Statistic performed by Mann Whitney T-test (p<0.05). B) Comparisons of the oviposition rate of the raw sewage and positive control, according to the methodology of no choice assay. Statistic performed by Mann Whitney T-test
(p<0.05). Bars indicate mean number of eggs with standard deviation
Figure 03: Comparison of eggs retention inside the ovaries A) Eggs retention
(choice assay). Statistic performed by Mann Whitney T-test (p<0.05). B) Eggs retention (no choice assay). Statistic performed by One-Way ANOVA followed by Tukey’s Multiple Comparisons Test (p<0.05). Bars indicate mean number of eggs with standard deviation. Exp. Cage = Experimental cage; Phys. Cage =
Physiological Cage.
Figure 04: Oviposition behavior of Ae. aegypti females throughout the days. A)
Positive control (distilled water) in the choice assay. B) Positive control (distilled water) in the no choice assay. C) Raw sewage in the choice assay. D) Raw sewage in the no choice assay. Statistic performed by One-Way ANOVA followed by Tukey’s Multiple Comparisons Test (p<0.05). Bars indicate mean
number of eggs with standard deviation.
Figure 05: Hatching and development assay. A) Percentage of hatching of eggs
from Ae. aegypti in the raw sewage or in aged water (control). Statistic performed by Mann Whitney T-test (p<0.05). Bars indicate mean percentage of hatching
with standard
deviation.
B) Cumulative
percentage
of adult’s
appearance throughout the development experiment. Statistic performed by Two-Way ANOVA followed by Bonferroni’s test (p<0.05). Spots represents mean percentage of adults in each day. C) Comparison between the numbers of adults that reached the adult stage after the development experiment. Statistic performed by Mann Whitney T-test (p<0.05). Bars indicate mean number of adults with standard deviation. Cumulative percentage of adult’s appearance throughout the development experiment. Statistic performed by Two-Way ANOVA followed by Bonferroni’s test (p<0.05). Spots represents
mean percentage of adults in each day.
Table 01: Microbiological and physicochemical
parameters
of
raw
sewage used to assess the development and pattern oviposition of Ae. aegypti (Rockefeller strain). Total coliforms (NMP/100ml)
3.2 x 107
Total of Escherichia coli (NMP/100ml)
8.1x 106
Total alkalinity (mg CaCO3/L) Total residual chlorine (mg Cl2/L)
213.3 ± 52.6 <0.10
Chemical oxygen demand (mg O2/L)
348.4 ±119.8
Total Kjeldahl nitrogen (mg NH3-N/L)
51.7 ± 10
Ammoniacal nitrogen (mg NH3-N/L)
38 ±5.8
Oxygen consumed in acid medium (mg O2/L)
9.7 ± 2
Dissolved oxygen (mg O2/L) Temperature (ºC)
Turbidity (NTU) Ph The values represent six samples of raw sewage.
4.35 ± 2.9 24.1 ±2 31.14 ±16.3 7.09 ±0.12
S0001-706X(16)30507-1 http://dx.doi.org/doi:10.1016/j.actatropica.2016.07.013 ACTROP 3991
To appear in:
Acta Tropica
Received date: Revised date: Accepted date:
4-2-2016 28-6-2016 17-7-2016
Please cite this article as: Chitolina, R.F., Anjos, F.A., Lima, T.S., Castro, E.A., Costa Ribeiro, M.C.V., Raw sewage as breeding site to Aedes (Stegomyia) aegypti (Diptera, Culicidae).Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2016.07.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Raw sewage as breeding site to Aedes (Stegomyia) aegypti (Diptera,
Culicidae)
Chitolina, R. F.1, Anjos, F. A.1, Lima, T. S.1, Castro E. A.1, & Costa Ribeiro, M. C. V.1, *
1
Laboratório de Parasitologia Molecular, Departamento de Patologia Básica, Centro Politécnico, Universidade Federal do Paraná, Brasil.
*Corresponding author:
[email protected]
Tel:
+55
41
3361
170.
address:
Graphical abstract
Highlights 1. Aedes aegypti females can lay their eggs either in clean water or raw sewage. 2. Ae. aegypti females retain their eggs if there is not a suitable oviposition site 3. Raw sewage may be acting as breeding site for Ae. aegypti mosquitoes. 4. Control programs must re-think their action considering new breeding sites.
ABSTRACT
The selection of oviposition sites by females of Aedes (Stegomyia) aegypti is
a key factor for the larval survival and egg dispersion and has a direct influence
in vector control programs. In this study, we evaluated the aspects of
reproductive physiology of Ae. aegypti mosquitoes tested in the presence of raw
sewage. Ae. aegypti females were used in oviposition bioassays according to
two methodologies: (i) choice assay, in which three oviposition substrates were
offered in the same cage: treatment (raw sewage), positive control (distilled
water) and negative control (1% sodium hypochlorite) and; (ii) no choice assay,
in which only one substrate was available. The physicochemical and
microbiological analysis of the raw sewage used in this study indicated virtually
no levels of chlorine, low levels of dissolved oxygen and high levels of
nitrogenous compounds as well as the presence of Escherichia coli and total
fecal coliforms. After 72 hours of oviposition, the eggs were counted and there
was no statistically significant difference (p>0.05) in the oviposition rate
between raw sewage and positive control in both methodologies. In addition,
females were dissected to evaluate egg-retention and also there were no
appreciable differences in egg retention even when raw sewage was the only
substrate offered. The data also showed that egg hatching and larvae
development occurred normally in the raw sewage. Therefore, the present study
suggests that Ae. aegypti can adapt to new sites and lay eggs in polluted water,
such as the raw sewage. These findings are of particular importance for the
control and surveillance programs against Ae. aegypti in countries where the
conditions of poor infrastructure and lack of basic sanitation are still an issue.
KEY WORDS: Aedes aegypti, oviposition, raw sewage, skip-oviposition
behavior
1. INTRODUCTION
The selection of sites for oviposition by females of Aedes (Stegomyia)
aegypti (Linnaeus, 1762) is a key factor for the survival of their immature forms
(Bentley & Day 1989). The choice of oviposition sites is influenced by visual,
tactile and olfactory factors, the latter considered of primary importance usually
due to decomposition of organic matter, the presence of immature mosquitoes
and fauna-associated bacteria (Benzon & Apperson, 1988; Bentley & Day 1989;
Logan & Birkett, 2007;. Sharma et al 2008). In addition, physical factors like
temperature, humidity, presence of vegetation, and chemical factors such as
co-specifics and oviposition attractants also influence the choice of the breeding
site (Benzon & Apperson, 1988; Bentley & Day, 1989).
According to Varejão et al., (2005) Ae. aegypti breeds in clean water reservoirs, although it may be able to adapt to new situations imposed by the humans. Indeed, it was shown that the presence of fecal coliform bacteria was able to increase the attractiveness for oviposition (Navarro et al., 2003). The ability of the vector to develop in environments with high levels of pollution as well as in raw sewage is due to the high concentration of organic material, which contains nutrients such as proteins, carbohydrates and lipids (Beserra et al., 2010). According to the same authors, the choice of oviposition site is not only related to the degree of water pollution, but also to the environmental conditions necessary for the survival and development of immature mosquitoes, making them adaptable to lay eggs in sites considered unfavorable. For more than 40 years Ae. aegypti larvae, pupae and adults have been found in environments considered polluted, such as septic tanks, sewage disposal containers and raw sewage (Chan et al., 1971; Barrera et al., 2008;
Troyo et al., 2008; Burke at al., 2010, Banerjee et al., 2015). According to the
WHO, in 2015 more than 30% of the global population has no access to improved sanitation facilities (WHO/UNICEF, 2015) and in Francisco Beltrão municipality, Paraná state, Brazil, immatures forms of the Ae. aegypti were found in raw sewage during dengue epidemic (Personal communication, 2012).
Although several studies have reported the detection of this vector in raw sewage, only a few studies have addressed the attractiveness of oviposition of Ae. aegypti to collections of water with high levels of pollution such as raw sewage (Navarro et al., 2003; Beserra et al., 2010). Therefore, the aim of this study was to evaluate under laboratory conditions, aspects of the reproductive
physiology of Ae. aegypti in the presence of raw sewage.
2.
MATERIAL AND METHODS
2.1
Mosquitoes rearing
Ae. aegypti (Rockefeller strain) were reared and maintained under laboratory conditions (26°C±2°C, 12:12h photoperiod, 80% humidity). To
perform the oviposition bioassays 4-5-days old females were allowed to blood
feed on anesthetized mice (Mus musculus) (ketamine:xylazine 80–120:10–16
mg/kg) according to Ethics Committee for Animal Use (protocol number 714) of
the Federal University of Paraná.
2.2
Raw sewage samples
Raw sewage was collected at the Atuba Sewage Treatment Station -
SANEPAR in Curitiba, Paraná, and analyzed by microbiological, physical and
chemical tests according to the Standard Methods for the Examination of Waste
water (Standard Methods, APHA, 21st – 22nded) (Table 01) by Center for
Research and Food Processing (CEPPA) of the Federal University of Paraná.
In order to determine the best concentration of sewage, we performed
oviposition assays in the presence of different dilutions of raw sewage (25%, 50% and 100%). As no significant differences were found between the dilutions,
all further experiments were performed using raw sewage without dilution (see
below). 2.3
Sixty (60) mL of raw sewage without dilution, distilled water (positive control)
and 1% sodium hypochlorite (negative control) were allocated inside a
transparent recipient with a filter paper as an oviposition substrate, these were
Experimental solutions preparation
then placed inside a black vessel, known as attractive to the species. 2.4
Oviposition experimental design Fifteen fully engorged females with the same physiological age were allocated inside a cage with 30x30x30 cm. The solutions were disposed equidistantly in an equilateral triangle, replaced every 24 hours, and rotated in clockwise direction. Each experiment was repeated six times (n=6). To ensure that the females were in proper conditions for oviposition, a physiological control cage (vessels with distilled water only) was used (n=3). The solutions were placed inside the cages five days after the blood meal, in order to ensure that all the females would be able to lay eggs. After 72 hours, the number of eggs laid were quantified. The ovaries of all females were dissected in order to distinguish parous of non-parous females according to Detinova (1962) apud Consoli & Lourenço-de-Oliveira (1994) and to count the number of eggs retained inside the ovaries. The oviposition assay was performed using two distinct methodologies: (i) choice assay, in the same
cage all the vessels with solutions (treatment, positive and negative control)
were disposed together equidistantly inside the cage, allowing the females to
choose the best egg-laying sites, and the (ii) no choice assay, where only one
solution in each cage was available to the females to lay eggs. 2.5
Hatching and development assay In order to check the hatching rate of the eggs in the raw sewage, were put to hatch inside a tray containing one liter of raw sewage or aged water (control). The eggs were allowed to hatch for 2 days, and then the numbers of larvae were counted. The experiment was performed two times in triplicate. To check the development of the mosquitoes inside the raw sewage, ten
first instar larvae were placed inside a cup with 30 mL of raw sewage or aged water (control). To simulate the real conditions of growth, 15 mL of raw sewage
was replaced every other day in each cup containing the larvae. For the control 1 mL of food solution (2g/L) were provided every other day. Adult emergence was monitored every day until the emergence of the last adult mosquito. The development assay was repeated three times with five replicas in each.
2.6
Statistical analysis
The choice of the best raw sewage concentration was determined using a One Way ANOVA followed of a Tukey`s Multiple Comparisons Test (p<0.05). A t-test of Mann-Whitney was performed to check oviposition rate, hatching rate and the number of adult mosquitoes that raised from development assay. The time of development between the sewage and the control were analyzed by a Two Way ANOVA followed of a Bonferroni’s test.
To evaluate the oviposition behavior a One Way ANOVA followed of a Tukey`s Multiple Comparisons Test was used to compare the oviposition through the days and eggs retention in the no choice assay. In addition, to access the oviposition attractiveness of Ae. aegypti to raw sewage, the Oviposition Activity Index (OAI), given by Kramer & Mulla (1979), was calculated as follows: OAI = Nt – Ns /Nt + Ns Where: Nt = Number of eggs in the treatment Ns = Number of eggs of the control The OAI can range between +1 and -1. The positive values indicate that there are more eggs in the treatment compared to positive control, and negative
values indicate the reverse. According to Kramer and Mulla (1979) OAI ≥ +0.30 indicates the presence of substances that serve as oviposition attractants for
mosquitoes; conversely OAI≤ -0.30 indicates the presence of repellent substances.
3. RESULTS 3.1 Physical, chemical and microbiological analyses of raw sewage
The microbiological analysis of raw sewage showed high levels of
Escherichia coli and total fecal coliforms, up to 106 cells in all analysis. Physical
and chemical analysis indicated the presence of low levels of dissolved oxygen
(DO) associated with a high chemical oxygen demand (CDO), high levels of nitrogenated compounds and virtually no chlorine levels (Table 01).
3.2
Determination of raw sewage concentration.
First, we assayed the oviposition behavior of Ae. aegypti towards three different concentrations of raw sewage (25%, 50% and 100%). In the physiological control, an average number of 740.33 (± 121.14) eggs were obtained, showing that the females were able to lay eggs. In the experimental cages, it was obtained an average number of 381.67 (±118.07), 335.00 (±112.09) and 407.17 (± 328.67) eggs in the raw sewage solutions of 25%, 50% and 100%, respectively. As no significant differences were found (p= 0.8377) between the dilutions and in order to simulate natural environmental conditions, all further experiments using raw sewage were performed without dilution. 3.3
Oviposition assay
Following the determination of raw sewage concentration, an oviposition assay was performed to check whether or not raw sewage affects the oviposition rate of Ae. aegypti female under laboratory conditions. The oviposition assays were performed according to the methodologies of choice or no choice, as represented on figure 01. In the choice assay, no statistical difference (p= 0.3095) was found between the number of eggs laid in the raw sewage 526.50 (± 196.74) and the positive control 409.83 (± 137.19) (Fig. 02 A). As expected, no eggs were laid in
the negative control. In the physiological control an average of 1059.67 (± 255) eggs were observed. Similarly, in the no choice assay, no significant differences (p= 0.4848) were found between the number of eggs laid in the presence of only raw sewage (1237.83 ± 462.60) in comparison to the positive control (1003 ± 218.23) (Fig. 02 B). No eggs were laid in the negative control. These results suggested that, at least under laboratory conditions, raw sewage is a suitable 3.4
Dissection and evaluation of eggs retention
Since we did not observe any difference in the oviposition rate, was evaluated the egg retention ratio in the ovaries of Ae. aegypti females after both choice and no choice oviposition assays. In the choice assay it was found that in the experimental cages 273.50
(± 128.09) eggs were retained in the ovaries of Ae. aegypti females whereas 307.00 (± 126.58) eggs were retained in females of the physiological control group. No significant differences were found between the groups (p= 0.9048) (Fig 03 A). In the no choice assay the retention of eggs were compared between the three set of cages: i) raw sewage (470.67 ± 415.20 eggs retained); ii) positive control (318.50 ± 142.94 eggs retained); and iii) negative control (1106.33 ± 197.01 eggs retained). Statistical analysis revealed a significant difference between the groups compared (p=0.0004) with difference between the positive and the negative control (p<0.0001) and between raw sewage and the negative control (p< 0.001). However, no significant differences were found between the raw sewage solution and the positive control (Fig 03 B). These results reinforce the idea that raw sewage is a suitable place for oviposition.
3.5
Evaluation of oviposition behavior
We further evaluated the Oviposition Activity Indexes (OAI) as well as the oviposition behavior during the three days of experiment. The Oviposition Activity Indexes (OAI) values were +0.1246 and +0.1048 for the choice and no choice assay, respectively. In both assays (Choice and No Choice), we noted that females of the positive control group laid more eggs in the first day in comparison to day two and three in the choice assay (p= 0.0001) (Fig. 04 A) and compared to day three in the no choice assay (p= 0.0026) (Fig 04 B) respectively. Interestingly, the same daily pattern of oviposition was observed in the raw sewage group in the choice assay (p< 0.0001) (Fig. 04 C) and in the no choice assay (p= 0.0009) (Fig 04 D). These data suggested that Ae. aegypti females display the same oviposition behavior towards water and raw sewage.
3.6
Hatching and development of larvae in raw sewage
Since Ae. aegypti females do not discriminate between water and raw sewage as oviposition substrates under laboratory conditions, we further
evaluated whether raw sewage could support hatching and development of
larval stages. In the hatching assay we initially observed that eggs laid by female Ae. aegypti were able to hatch both in water and in sewage (p= 0.1797) (Fig 05 A). It was observed that the larvae that developed in water reached the adult stage approximately 2-3 days earlier than the larvae grown in the raw sewage (Fig 05 B). However, despite the difference in the larvae growth period, adult mosquitoes at the end of the development period were present in about same numbers in both conditions (p= 0.4256) (Fig 05 C). Altogether, these results demonstrate that raw sewage can provide all the conditions needed for mosquito eggs to hatch as well as for supporting the development of larvae into adult mosquitoes.
4.
DISCUSSION
In the present study we showed that under laboratory conditions Ae. aegypti females lay eggs in both clean water and raw sewage. Moreover, eggs and larvae are fully capable of hatch and develop in polluted water, respectively.
This data corroborates a few studies showing that Ae. aegypti
females can lay their eggs in contaminated water, such as the sewage, and these eggs are able to fully develop into adult mosquitoes (Navarro et al., 2003; Beserra et al., 2010). Furthermore, the positive values found for both methodologies (choice and no choice) in the OAI are an indicative that the raw sewage may be attractive to oviposition of females.
Besides corroborating the literature data, our findings showed that Ae.
aegypti can adapt to adverse conditions. As far as we know, studies aiming to check oviposition rate and behavior have only checked the oviposition rate comparing a treatment with a positive control. We not only analyzed the oviposition rate between raw sewage with a positive and negative controls but also the eggs retention, reinforcing our conclusion that raw sewage is a suitable place for oviposition. Moreover, in order to guarantee that all the females used in the assays were fully capable to lay eggs we performed the Detinova technique, which consists in check the tracheolar endings, where distended tracheolar endings characterizes a parous female. In both methods of the study (choice and no choice assay) all the females used in the bioassays were parous, proving that a blood meal was realized and they were able to lay eggs (data not show). Under natural environmental conditions, Ae. aegypti females choose the best place to lay their eggs, and oviposit a few eggs in several different sites, this is called skip oviposition behavior (Colton, et al., 2003; Abreu, et al., 2015). Moreover, Gomes et al., (2006) and Abreu et al., (2015) showed that under laboratory and semi-field condition observed that there is a pick of oviposition three days after blood meal. In this study, Ae. aegypti females were blood fed and deprived of oviposition for five days, which led the females to lay the great majority of eggs during the first day, in both positive control and raw sewage. We hypothesized that once the females were submitted to a stressful condition, maintained in a closed space with no access to an oviposition site, it would be expected that after this time the females would have a need to lay eggs. Thus, a higher number of eggs would be expected at day one, if a suitable place for
oviposition was available, what happened during our experiments. Furthermore,
unpublished data from our group showed that once a suitable place is not available (for example when a repellent substance is present) a higher amount of eggs is not observed in the first day, even after five days with no access to an oviposition site, but distributed homogeneously over the days of experiment. Therefore, we believe that raw sewage, under laboratory conditions, has no repellent properties against mosquitoes. Wong et al. (2012) showed that when ideal containers are eliminated in the environment, others recipients that are not the main breeding sites to oviposition are chosen by Ae. aegypti to lay their eggs. The raw sewage, therefore, needs to be carefully considered as an optional oviposition site. Not only this site is pointed as capable of increase the productivity of mosquitoes offspring (Chaves et al., 2009), but also has been considered as source of infestation (Chan et al., 1971; Barrera et al., 2008; Troyo et al., 2008; Burke et al., 2010, Banerjee et al., 2015). Therefore, in urban areas where basic sanitation conditions are not met, the open raw sewage may be acting as a natural environment for the development of mosquitoes. The factors that lead to this change in oviposition behavior are still unclear but several studies have shown that the presence of bacteria increase the oviposition site preference. Bacteria are responsible for increase the oviposition rate in organic infusions when compared to bacteria-free leaf infusions, suggesting a role for microbial activity in the production of odorants that assist the oviposition activity in mosquitoes (Ponnusamy et al., 2009; Ponnusamy et al., 2010).
In the physical, chemical and microbiological analysis of raw sewage, we
observed a high amount of bacteria and fecal coliforms that may be the
responsible for the positive oviposition response, and a low rate of dissolved
oxygen, which can be validated for the founding’s of Ponnusamy and
colleagues (2011). These authors suggest that, not only the bacteria are
responsible for the attractiveness of oviposition sites, but also for stimulating
hatch of Ae. aegypti eggs.
Besides that, other aspects in the raw sewage can contribute to the
oviposition such as the low amount of chlorine, known as harmful for the eggs
and larvae and the high levels of nitrogenous compounds found in our samples.
Darriet & Corbel (2008) found out that collection of water containing high
amounts of nitrogenous compounds, such as ammonium (NH+4), nitrite (NO−2)
and nitrate (NO−3) ions, were more attractive to gravid females. In addition,
Monteiro Marques et al., (2013) evaluated different public water supply for
human consumption and found that high concentrations of ammoniacal nitrogen in public water were responsible for the attractiveness of pregnant Ae. aegypti females. Associated to the fact that the females may lay their eggs in contaminated water, the fully development of these eggs is ensured by several factors such as the higher amount of nutrients available and the high water turbidity. The latter is particularly important because it provides a suitable site for Ae. aegypti development, taking into consideration that Ae. aegypti larvae are photofobics (Consoli & Lourenço-de-Oliveira (1994). Our data also revealed that the eggs can hatch properly in the sewage, however, the development from an immature stage to an adult phase was delayed by three days compared to
clean water. This observation does not corroborate results obtained by Beserra
et al. (2010), who reported a faster development of larvae in raw sewage.
However, this apparent discrepancy can be due to the difference in the raw
sewage composition used in both studies.
According to several authors, Ae. aegypti is the vector of many
arbovirosis such as dengue, chinkungunya and zika (Diallo et al., 1999;
Lourenço de Oliveira et al., 2002; Li et al., 2012; Musso et al., 2014). Therefore,
any environmental conditions that may favor the development of the vector,
such as the lack of infrastructure and minimal sanitary conditions might
contribute to Ae. aegypti development, increasing the risk for diseases
transmission.
4. CONCLUSIONS
The importance of our study is not only to understand the physiological factors associated with the choice of reservoir for oviposition, but also to clarify and highlight that Ae. aegypti females can adapt to new breeding sites. These observations must be taken into account in the development of future vector control actions in order to prevent the presence of the vector and consequently the transmission of viruses associated with it. In general, the control of Ae. aegypti is based on observations only in artificial breeding sites with clean water, however, our results indicate that Ae. aegypti can lay eggs not only in clean water but also in raw sewage. Still, more studies need to be performed, for example with field populations, in order to fully understand the oviposition conditions. Nevertheless, our work using a
laboratory strain suggests that the Ae. aegypti mosquitoes can adapt to the new
oviposition niches and, which may threat the vector control programs, once
aspects related to the adaptation of the species to new breeding sites have
being neglected.
5. ACKNOWLEDGMENTS
We would like to thank to the SANEPAR in the person of Angélica de
Lima de Araujo, who helped and allowed the collection of sewage material. To
the Laboratório de Entomologia Prof. Ângelo Moreira da Costa Lima in the
person of Dra. Maria Aparecida Cassilha Zawadneak. To the Laboratório de
Fisiologia e Controle de Artrópodes Vetores – IBEX/Rio de Janeiro, Brazil which
provided the Rockeffeler strain and to Dr. Emerson Soares Bernardes for the
critical reading of the manuscript.
6. FUNDING
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. 7. REFERENCES
Abreu, F. V. S. D., Morais, M. M., Ribeiro, S. P., & Eiras, Á. E. 2015. Influence of breeding site availability on the oviposition behaviour of Aedes aegypti. Mem do Inst Oswaldo Cruz, 110 (5): 669-676.
Banerjee, S., Mohan, S., Saha, N., Mohanty, S. P., Saha, G. K., & Aditya, G.
2015. Pupal productivity & nutrient reserves of Aedes mosquitoes breeding in
sewage drains & other habitats of Kolkata, India: implications for habitat
expansion & vector management. Indian J Med Res, 142: 87-94.
Barrera, R., Amador, M., Diaz, A., Smith, J.,Munoz-Jordan, J.L., Rosario, Y.
2008. Unusual productivity of Aedes aegypti in septic tanks and its implications
for dengue control. Med Vet Entomol, 22: 62-69.
Bentley, M. D., Day, J. F. 1989. Chemical ecology and behavioral aspects of
mosquito oviposition. Ann Rev Entom, 34: 401–421.
Benson, G. L., Apperson, C. S. 1988. Reexamination of chemically mediated
oviposition behavior in Aedes aegypti (L.)(Diptera: Culicidae). J Med Entomol, 25 (3): 158-164.
Beserra, E. B., Fernandes, C. R. M., Souza, J. T., Freitas, E. M., Santos, K. D. 2010. The Effect of Water Quality in the Life Cycle and in the Attraction for the Egg Oviposition of Aedes aegypti (L.) (Diptera: Culicidae). Neotrop Entom, 39
(6):1016-1023.
Burke,R., Barrera, R., Lewis, M., Kluchinsky, T., Claborn, D. 2010. Septic tanks as larval habitats for the mosquitoes Aedes aegypti and Culex quinquefasciatus in Playa-Playita, Puerto Rico. Med Vet Entom, 24:117–123.
Chan, Y. C., Ho, B. C., Chan, K. L. 1971. Aedes aegypti (L.) and Aedes albopictus (Skuse) in Singapore City. Bull World Health Organ, 44 (5): 651– 657.
Chaves, L.F., Keogh, C.L., Vazquez-Prokopec, G.M., Kitron, U.D. 2009.
Combined sewage overflow enhances oviposition of Culex quinquefasciatus (Diptera: Culicidae) in urban areas. J Med Entomol, 46 (2): 220-226.
Colton, Y. M., Chadee, D. D., Severson, D. W. 2003. Natural skip oviposition of the mosquito Aedes aegypti indicated by codominant genetic markers. Med Vet Entomol, 17: 195-204.
Consoli, R. A. G. B., De Oliveira, R. L. Principais mosquitos de importância
sanitária no Brasil. SciELO-Editora FIOCRUZ, 1994.
Darriet, F., & Corbel, V.
2008. Propriétés
attractives et
modifications
physicochimiques des eaux de gîtes colonisées par des larves de Aedes aegypti (Diptera: Culicidae). Comptes Rendus Biologies, 331 (8): 617-622.
Diallo, M., Thonnon, J., Traore-Lamizana, M., & Fontenille, D. 1999. Vectors of Chikungunya virus in Senegal: current data and transmission cycles. Am J Trop Med Hyg, 60 (2): 281-286.
Gomes, A. D. S., Sciavico, C. J., Eiras, Á. E. 2006. Periodicity of oviposition of females of Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) in laboratory and field. Rev Bras Med Trop, 39: 327-332.
Kramer, L.W., Mulla, S.M. 1979. Oviposition attractants and repellents of mosquitoes: oviposition responses of Culex mosquito to organic infusions. Environ Entomol, 8: 1111-1117.
Li, M. I., Wong, P. S., Ng, L. C., & Tan, C. H. 2012. Oral susceptibility of
Singapore Aedes (Stegomyia) aegypti (Linnaeus) to Zika virus. PLoS Negl Trop Dis, 6 (8): e1792.
Logan, J. G.,Birkett, M. A. 2007. Semiochemicals for biting fly control: their identification and explotation. Pest Manag Sci, 63: 647-657.
Lourenço-de-Oliveira, R., Honório, N. A., Castro, M. G., Schatzmayr, H. G., Miagostovich, M. P., Alves, J. C., ... & Nogueira, R. M. 2002. Dengue virus type 3 isolation from Aedes aegypti in the municipality of Nova Iguaçu, State of Rio de Janeiro. Mem I Oswaldo Cruz, 97 (6): 799-800.
Monteiro-Marques, G. R. A., Chaves, L. S. M., Serpa, L. L. N., Arduino. M. B., Chaves, F. J. M. 2013.Public drinking water supply and egg laying by Aedes aegypti. Rev Saúde Públi, 47 (3): 579-587.
Musso, D., Nilles, E. J., & Cao‐Lormeau, V. M. 2014. Rapid spread of emerging Zika virus in the Pacific area. Clin Microbiol Infec, 20 (10): O595-O596.
Navarro, D. M. A. F., Oliveira, P. E. S., Potting, R. P. J., Brito, A. C., Fital, S. J. F., Sant'Ana, A. E. G. 2003. The potential attractant or repellent effects of different water types on oviposition in Aedes aegypti L. (Dipt.,Culicidae). J Appl Entomol, 127: 46-50.
Ponnusamy, L., Wesson, D. M., Arellano, C., Schal, C., Apperson, C. S. 2009. Species
composition
of
bacterial
communities
influences
attraction
mosquitoes to experimental plant infusions. Microb Ecol, 59: 158–173.
of
Ponnusamy, L., Xu, N., Böröczky, K., Wesson, D. M., Ayyash, L. A., Schal, C.,
Apperson, C. S. 2010.Oviposition responses of the mosquitoes Aedes aegypti and Aedes albopictus to experimental plant infusions in laboratory bioassays. J Chem Ecol, 36: 709–719.
Ponnusamy L., Böröczky,K., Wesson, D.M., Schal, C., Apperson,C. S. 2011. Bacteria stimulate hatching of yellow fever mosquito eggs. PLoS ONE, 6 (9): e24409.
Sharma, K. R., Seenivasagan, T., Rao, A. N., Ganesan, K., Agarwal, O. P.,
Malhotra, R. C., Prakash, S. 2008. Oviposition responses of Aedes aegypt iand Aedes albopictus to certain fatty acid esters. Parasitol Res, 103: 1065-1073.
STANDARD methods for the examination of water and wastewater. 2012. 22nd ed. Washington: APHA; AWWA; WEF.
STANDARD methods for the examination of water and wastewater. 2005. 21st ed. Washington: APHA; AWWA; WEF.
Troyo, A., Calderón-Arguedas, O., Fuller, D.O., Solano, M.E., Avendaño, A., Arheart, K.L., Chadee, D.D., Beier, J,C. 2008. Seasonal profiles of Aedes aegypti (Diptera: Culicidae) larval habitats in an urban area of Costa Rica with a history of mosquito control. J Vector Ecol, 33 (1): 76-88.
Varejão, J.B.M., Santos, C.B., Rezende, H.R., Bevilacqua, L.C., Falqueto, A. 2005. Aedes (Stegomyia) aegypti (Linnaeus, 1762) breeding sites in native bromeliads in Vitória City, ES. Ver Soc Brasil Med Trop, 38: 238-240.
Wong, J., Morrison, A.C., Stoddard, S.T., Astete, H., Chu, Y.Y., Baseer, I.,
Scott, T.W. 2012. Linking oviposition site choice to offspring fitness in Aedes aegypti: consequences for targeted larval control of dengue vectors. PLoS Negl Trop Dis, 6 (5): e1632.
WHO/UNICEF, Progress on sanitation and drinking water: 2015 update and MDG assessment. World Health Organization (WHO), 2015.
Figure 01. Schematic representation of oviposition assay methodologies. A) Choice assay. B) No choice assay. Exp. Cages: Experimental Cages; Phys. Cages: Physiological Cages.
Figure 02: Oviposition assay. A) Comparisons of the oviposition rate of the raw
sewage and positive control, according to the methodology of choice assay. Statistic performed by Mann Whitney T-test (p<0.05). B) Comparisons of the oviposition rate of the raw sewage and positive control, according to the methodology of no choice assay. Statistic performed by Mann Whitney T-test
(p<0.05). Bars indicate mean number of eggs with standard deviation
Figure 03: Comparison of eggs retention inside the ovaries A) Eggs retention
(choice assay). Statistic performed by Mann Whitney T-test (p<0.05). B) Eggs retention (no choice assay). Statistic performed by One-Way ANOVA followed by Tukey’s Multiple Comparisons Test (p<0.05). Bars indicate mean number of eggs with standard deviation. Exp. Cage = Experimental cage; Phys. Cage =
Physiological Cage.
Figure 04: Oviposition behavior of Ae. aegypti females throughout the days. A)
Positive control (distilled water) in the choice assay. B) Positive control (distilled water) in the no choice assay. C) Raw sewage in the choice assay. D) Raw sewage in the no choice assay. Statistic performed by One-Way ANOVA followed by Tukey’s Multiple Comparisons Test (p<0.05). Bars indicate mean
number of eggs with standard deviation.
Figure 05: Hatching and development assay. A) Percentage of hatching of eggs
from Ae. aegypti in the raw sewage or in aged water (control). Statistic performed by Mann Whitney T-test (p<0.05). Bars indicate mean percentage of hatching
with standard
deviation.
B) Cumulative
percentage
of adult’s
appearance throughout the development experiment. Statistic performed by Two-Way ANOVA followed by Bonferroni’s test (p<0.05). Spots represents mean percentage of adults in each day. C) Comparison between the numbers of adults that reached the adult stage after the development experiment. Statistic performed by Mann Whitney T-test (p<0.05). Bars indicate mean number of adults with standard deviation. Cumulative percentage of adult’s appearance throughout the development experiment. Statistic performed by Two-Way ANOVA followed by Bonferroni’s test (p<0.05). Spots represents
mean percentage of adults in each day.
Table 01: Microbiological and physicochemical
parameters
of
raw
sewage used to assess the development and pattern oviposition of Ae. aegypti (Rockefeller strain). Total coliforms (NMP/100ml)
3.2 x 107
Total of Escherichia coli (NMP/100ml)
8.1x 106
Total alkalinity (mg CaCO3/L) Total residual chlorine (mg Cl2/L)
213.3 ± 52.6 <0.10
Chemical oxygen demand (mg O2/L)
348.4 ±119.8
Total Kjeldahl nitrogen (mg NH3-N/L)
51.7 ± 10
Ammoniacal nitrogen (mg NH3-N/L)
38 ±5.8
Oxygen consumed in acid medium (mg O2/L)
9.7 ± 2
Dissolved oxygen (mg O2/L) Temperature (ºC)
Turbidity (NTU) Ph The values represent six samples of raw sewage.
4.35 ± 2.9 24.1 ±2 31.14 ±16.3 7.09 ±0.12