New artificial hematophagy system with attractive polymeric biofilm for maintenance of Culex quinquefasciatus (Diptera: Culicidae) in the laboratory

doi:10.21203/rs.3.rs-3150477/v1

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Research Article

New artificial hematophagy system with attractive polymeric biofilm for maintenance of Culex quinquefasciatus (Diptera: Culicidae) in the laboratory

https://doi.org/10.21203/rs.3.rs-3150477/v1

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published 15 Mar, 2024

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Background: Maintaining mosquito colonies in the laboratory implies the need for a blood supply so that females can mature their oocytes and perform oviposition. In this study, a new artificial hematophagy system for colonization and maintenance of Cx quinquefasciatusin the laboratory, was developed and tested.

Methods: We developed an attractive polymeric biofilm with 25% L-lactic acid in its composition, to be used as a membrane in the artificial hematophagy system and compared the feeding rate of females with Parafilm - M®. We also evaluated the oviposition rate, larval survival, and adult emergence of females fed through the attractive biofilm.

Results: The average percentage of females of Cx. quinquefasciatusfed through the Attractive biofilm was 87%, while only 20% engorged with Parafilm-M® (p<0.0001). Feeding through the attractive biofilm developed by us was able to produce high rates of the evaluated biological parameters, the percentage of laying of females who performed artificial hematophagy through the biofilm was 90%, with an average of 158 eggs per raft. Of these eggs, 97% of the larvae hatched, of which 95% reached the pupal stage. The adult emergence rate corresponded to 93% of the pupae.

Conclusions: The attractive biofilm was superior to Parafilm-M® in terms of engorgement rate. In addition, feeding with the biofilm ensured significant rates of the evaluated biological parameters and there was no interference of the biofilm components in the biological parameters.

Mosquitoes

Blood feeding

Attractive membrane

Cx. quinquefasciatus

The mosquito Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is an important vector of pathogens such as the Wulchereria bancrofti and West Nile virus [1]. It is also a potential vector of the arbovirus responsible for Saint Louis encephalitis [2] and recently in Brazil, it was detected that specimens of this insect from urban environments were infected with ZIKV, suggesting participation in a new cycle of this arbovirus [3–4]. This mosquito species has a wide geographic distribution in urban and suburban areas from tropical to temperate zones. In addition, it exhibits opportunistic blood-feeding behavior in different hosts, and its ability to transmit zoonotic disease agents poses a risk for possible cross-breeding between wild bird arboviruses and spread to humans, especially in urban areas [5].

Culicidae females, in general, need to perform hematophagy for the maturation of their oocytes and subsequent egg development [6]. Thus, the blood supply of vertebrates is an essential step and represents the greatest challenge for the colonization and maintenance of these insects in the laboratory. Laboratory animals are often used as a source of blood, however, there are a number of restrictions and ethical limitations in this method, in addition to the high cost and the need for a specialized professional to maintain the animals in the vivarium [7]. The maintenance of mosquito species in the laboratory, as well as other arthropods of public health importance, is essential for studies on biology, behavior and the development of control techniques.

Artificial feeders have been developed and routinely used in procedures for maintaining mosquitoes in a laboratory environment [8]. These feeders are usually composed of one or more blood reservoirs, usually covered by thin membranes of animal tissue, Parafilm-M® or collagen [9] that facilitate the penetration of female mouthparts for food. To simulate the presence of a host and attract mosquitoes, heating methods are used [8]. In this context, in the present study, an artificial hematophagy system was developed and tested for the colonization and maintenance of Cx. quinquefasciatus in the laboratory, using an attractive polymeric biofilm as membrane, which contains L-lactic acid in its composition to increase the attraction and induction of the blood meal.

Colony establishment and maintenance

For establishment of the colony of Cx. quinquefasciatus in the laboratory, initially a field collection of Culex spp eggs was carried out, arranged in the form of a raft under the water, with the aid of an entomological shell and glass containers. The collection was carried out in the urban area of the city of Pelotas, Rio Grande do Sul, Brazil (31°46'28.4" S/ 52°21'07.0" W). Subsequently, the samples were sorted and the egg rafts were placed in trays containing dechlorinated water, kept at a temperature of 25ºC for the hatching of the larvae. These were identified by sampling each tray by dichotomous key [6–10]. After confirming the species, the larvae were fed wet chow for kittens (0.5 to 1 gram/tray) every two days and the water was changed once during the larval stage. After reaching the pupal stage, they were transferred to cages identified with the species, date and generation number (F.0 for specimens collected in the field) for colony control. The cages used were made of plastic material with screens on the top and sides, for air circulation, and the presence of a top opening with access to the feeding apparatus. After emergence of adults, 10% sugar solution was added ad libitum.

In all stages, the environment was maintained under controlled conditions of light/dark photoperiod of 12 h, relative humidity (RH) 60–70%, temperature 25°C ± 2°C and a screened exhaust system.

Preparation of the polymeric biofilm

The biofilm was prepared by the casting method with some modifications [11]. Initially, an aqueous solution of gelatin 1.3% (w/v) was prepared, which was kept under magnetic stirring at room temperature for 1 h. In parallel, an aqueous solution of poly(vinyl alcohol) (PVA) 1.3% (w/v) was also prepared and kept under magnetic stirring for 1 h at 80°C. Then, the two solutions were mixed and homogenized for 30 min at room temperature. Soon after, an amount of glycerol equivalent to 40% of the mass of the polymers was added to the mixture, to act as a plasticizer. To make the biofilm attractive to blood-sucking insects, L-lactic acid (25% in relation to the total mass of the system) was added to the filmogenic solution. Finally, the solution resulting from this process was poured into a Petri dish and transferred to an oven at 37°C until the complete evaporation of the solvent and biofilm formation. The attractive polymeric biofilm is part of the artificial hematophagy system that we prepared and produced, and this is published with the INPI under patent number (BR 102021015280-0 A2).

Analysis of the mechanical properties of the biofilm

The mechanical properties of the prepared biofilm (rupture strength, maximum elasticity and Young's modulus) were determined from stress tests as described by Voss et al. [12]. The tests were carried out in a texturometer equipment (Stable Microsystems TA.XT2 texturometer, United Kingdom) using rectangular specimens (26mm long, 76mm wide and 0.18mm thick). The analysis parameters followed the ASTM D882-12 standard and the tests were performed with 4 replicates (n = 4) using samples before and after exposure to the EDTA solution (1% w/v).

Artificial hematophagy system

The artificial hematophagy system was based on the method described by Rutledge et al. [13], composed of a plastic cup with a small external cavity at its bottom, to house the blood and covered with biofilm or Parafilm - M® forming the feeding surface. For the use of biofilm (Fig. 1a) it was necessary to immerse it in 1% ethylenediaminetetraacetic acid (EDTA) solution for 1 minute (Fig. 1b) to make the biofilm impermeable and hydrophobic, so that it could not absorb blood and was a physical barrier between the food and the insect. Blood was collected from bovine animals housed at the Veterinary Hospital of the Federal University of Pelotas (HUV/UFPel) by puncturing the jugular vein in a BD Vacutainer® tube with EDTA K2. Approximately 2 mL of the blood sample was transferred to the external cavity of the cup, using a pipette. Then the biofilm was spread over the cavity, staying in contact with the blood, and fixed to the outer side of the cup with double-sided tape (Fig. 1c). Water at 37°C was added to the inner cavity of the cup to keep the blood at an ideal temperature for hematophagy (Fig. 1d). For validation of this system, the apparatus was also made with the method using Parafilm - M®, which was rubbed on human skin for adhesion of natural odor binders [14].

Essay

For the artificial hematophagy test, 2 treatments were performed, sorted into the following groups: A (artificial hematophagy system with polymeric biofilm with 25% L25 lactic acid) and B (artificial hematophagy system with Parafilm-M®). The test was performed in triplicate, in each treatment 60 females were used, divided into three cages (n = 20) with an average age of 5 days. They came from a cage containing males. Feeding lasted 1 h in a dark environment with controlled temperature (25ºC). Three days after hematophagy, a dark container with dechlorinated water was placed inside each cage so that the females could lay eggs.

Evaluation of biological parameters

The engorgement rate was evaluated by the number of female mosquitoes with a distended and dark abdomen after the blood meal. The oviposition rate was evaluated by counting the number of rafts and dividing it by the number of engorged females. To evaluate the other biological parameters, 5 rafts were randomly selected from each cage of the biofilm treatment (n = 15). The average number of eggs produced per female was obtained by counting eggs per raft with the aid of a stereomicroscope. Subsequently, these eggs were transferred to trays with dechlorinated water for the eggs to hatch. The larvae were fed daily with macerated wet kitten food. After 48 h, the larvae were quantified, as well as the pupae from these offspring. Subsequently, these pupae were transferred to cages to assess the adult emergence rate.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 9.0 for Windows, Graph Pad Software (San Diego, CA, USA). One-way analysis of variance (ANOVA) followed by Tukey's test was used to determine whether there were statistically significant differences (p < 0.05) between the artificial blood feeding methods.

The polymeric biofilm showed translucency, and when stretched the thickness ranged from 0.10 to 0.12 mm, demonstrating good flexibility and stability. After being immersed in 1% EDTA solution (w/v) and extended again, it was noted that the average thickness reduced to 0.05 to 0.07 mm. However, no visual changes were observed in the biofilm. Comparatively, the biofilm with EDTA showed good mechanical properties, where the maximum rupture stress and Young's modulus were slightly higher compared to the sample without EDTA (Table 1). Regarding elasticity, no statistically significant changes were observed for both samples (with or without EDTA). We noticed that the EDTA biofilm was also more hydrophobic, which can be an advantage in terms of handling and practicality of application.

Table 1

Mechanical properties determined from the tensile test for samples of biofilm before and after exposure to 1% EDTA solution (w/v).
Sample	Breaking force (N)	Maximum elongation (%)		Young's modulus (kPa)
Biofilm without EDTA	1,1 ± 0,1	63,42 ± 8,03	3,84 ± 0,16
Biofilm with EDTA	2,2 ± 0,8	70,32 ± 12,34	6,38 ± 0,27

The average percentage of females of Cx. quinquefasciatus artificially fed with a system using attractive biofilm was 87%, while only 20% engorged with Parafilm-M®. There was a statistically significant difference between the different membranes used (p < 0.0001), and the data are shown in Fig. 2. In group A, the egg production rate, its viability in relation to hatching and development of larvae, pupae and adults were evaluated. The laying rate of females that performed artificial hematophagy through the biofilm was 90% with an average of 158 eggs per raft. The percentage of larvae that hatched was 97%, of which 95% reached the pupal stage. The adult emergence rate corresponded to 93% of the pupae.

Maintenance of mosquito colonies in the laboratory requires a blood supply so that females can mature their oocytes and develop eggs [6]. Due to current bioethical parameters, the direct use of animals is being replaced by artificial blood feeding. Large-scale mosquito breeding laboratories use Hemotek®, a specific equipment for artificial blood feeding (Hemotek Ltd., Blackburn, UK) [15]. This method has a high cost of execution, in addition to requiring infrastructure and inspection [16]. The present study is innovative because it describes the development and applicability of an attractive membrane to hematophagous insects, capable of attracting and inducing the artificial blood meal of Cx. quinquefasciatus under laboratory conditions with lower cost.

Different membranes have been used in artificial feeding procedures in an attempt to simulate the skin of vertebrate animals. Some studies have indicated a predilection of mosquitoes for natural membranes, but there are records that other synthetic membranes, such as intestinal collagen and Parafilm-M®, have been successful with some Culicidae species [13, 17–18]. The polymeric biofilm developed in this study is composed of materials that guarantee its functionality and application. The polymers used were chosen according to their characteristics. Gelatin, for example, is a collagen-derived biopolymer and has relevant properties such as non-toxicity, biodegradability and biocompatibility [19]. PVA, in turn, is a synthetic, biodegradable and low-toxic polymer, which has excellent filmogenic properties [20]. L-lactic acid is one of the main volatiles secreted by the eccrine sweat glands of vertebrates, and plays an important role in attracting mosquitoes. This was the first substance isolated and identified that had an attractive effect for mosquitoes in laboratory behavior tests [21]. In double-choice olfactometer studies, Culex quinquefasciatus was attracted to L-lactic acid [22–23].

Upon exposure of the biofilm to EDTA, the biofilm maintains the mechanical properties observed for the film without EDTA, however, a slight increase in stiffness was observed. Probably, the interaction of the anticoagulant with the polymer matrix of the film explains this behavior. In addition, the biofilm with EDTA preserves a high elasticity, which facilitates its handling during application. This feature allows it to be stretched under the blood forming a physical barrier between this food and the insect. Finally, it was noted that during the use of the biofilm with EDTA there was no absorption of the blood contained in the reservoir. It is suggested that this occurred due to the increase in hydrophobicity on the surface of the same, caused by the presence of the substance.

Another advantage of this system is the fact that the biofilm is produced from biodegradable polymers, which allows its degradation to be rapid, minimizing damage to the environment. Therefore, after its use, it can be autoclaved and discarded.

Several factors may influence the feeding of Culicidae, such as tactile, visual, olfactory stimuli, temperature of the feeding surface, blood temperature, among others [24]. The way commonly used to attract females to artificial devices is by simulating the presence of the host by heat, by heating the blood confined in the reservoir, mainly through water circulation systems [16, 25, 26–27]. Devices that do not have water circulation keep the blood warm by supplying internal compartments with heated liquid [14], thermal bag [28], ceramic with electrical connection for heating [29] or electronic equipment with a metal plate [30]. Based on dual-choice olfactometer studies, Cx. Quinquefasciatus was attracted to L-lactic acid [22–23]. Thus, the biofilm we produce contains this chemical compound in its composition that works as an attractant to mosquitoes, simulating the presence of a host. In this way, it is suggested that the biofilm we develop can attract and influence females of Cx. quinquefasciatus to perform the blood meal, ensuring high rates of engorgement in the laboratory. (Fig. 2). Much higher number than those found in the literature, Moutailler et al. [31] studying the transmission of the Rift Valley fever virus, artificially fed Cx. quinquefasciatus using Parafilm-M® membrane, and obtained only 1% of engorged females. In experiments performed with Cx. quinquefasciatus through the use of devices with circulating water for heating the blood, there was a variation in the average feeding rate between 45.0 and 48.0% [26].

Novak et al. [32], comparing different types of membranes for artificial feeding of mosquitoes, observed that females of Cx. quinquefasciatus are more reluctant to feed through artificial membranes than directly on live animals. Dias et al., [33] evaluated the efficiency of different membranes, namely collagen, latex and Parafilm - M® in the feeding of this culid and obtained percentages of fed females of 65%, 60% and 80% respectively, but the rate The fecundity rate was only 55 to 80 eggs, with Parafilm-M® the one with the highest fecundity rate. Dias et al. [26] evaluated blood sources of artificial feeding for Cx. quinquefasciatus, and observed that citrated rabbit blood presented the best result with an average of 44 eggs compared to citrated sheep blood (average of 39 eggs), defibrinated sheep blood (average of 30 eggs) and defibrinated rabbit blood (average of of 36 eggs). The average direct feeding in guinea pigs was slightly higher (55 eggs). In our work, the laying rate of Cx. quinquefasciatus fed using biofilm was 90% with an average of 158 eggs per raft with anticoagulated bovine blood, therefore, in addition to ensuring high feeding rates, the biofilm also does not interfere with fecundity, allowing large egg production by females.

In addition to satisfactory fecundity using the biofilm, this method also ensured high hatchability rates reaching 97%, larvae developed without major losses, and 95% reached the pupal stage. The adult emergence rate corresponded to 93%, therefore, no interference of biofilm components was observed in any developmental stage of the cycle of Cx quinquefasciatus. Laboratories that produce mosquitoes on a large scale need updated protocols, with alternatives for improving colonies, such as speed in the insect feeding process, less loss of adult mosquitoes, greater egg productivity after blood meal, and consequently, greater production of mosquitoes [ 25]. Our artificial hematophagy process with attractive polymeric biofilm is able to guarantee high production rates Cx. quinquefasciatus of fertile eggs with a very expressive percentage reaching the adult stage. Currently our colony of Cx. quinquefasciatus is in the F.9 generation, the insects are acclimatized and the artificial hematophagy process optimizes the maintenance of the biological cycle in the laboratory.

The females of Cx. quinquefasciatus artificially fed with a system using the attractive polymeric biofilm, prepared in this study, showed a higher rate of engorgement than Parafilm-M®. The polymeric biofilm has mechanical properties suitable for its application and its hydrophobic character ensured its stability during the test period. In addition, it was produced from biodegradable polymers, which allows its degradation to be fast, minimizing damage to the environment. It can be concluded that there is no interference of biofilm components in any stage of development of the cycle and in the biological parameters evaluated such as fecundity, egg hatching rate, development of larvae into pupae and adult emergence rate. This study contributes significantly to research involving the maintenance of the biological cycle of Cx. quinquefascitus in the laboratory. The artificial hematophagy system with attractive polymeric biofilm is effective, practical and can be used for other species of hematophagous insects. Thus, it will also be able to contribute to research on behavior, inter and intraspecific interactions, environmental control and vectorial capacity.

ZIKV: Zika virus; INPI: National Institute of Industrial Property; ASTM: American Society for Testing and Materials; EDTA: Ethylenediamine tetraacetic acid; PVA: Polyvinyl acetate.

Acknowledgment

The present work was carried out with the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Financing Code 001.

Ethics approval and consent to participate

The Committee of Ethics in the use of Animals of the Federal University of Pelotas, under the protocol 23110. 009653/2021-45 approved this research.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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Journal Publication

published 15 Mar, 2024

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Editorial decision: Major revision
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You are reading this latest preprint version

New artificial hematophagy system with attractive polymeric biofilm for maintenance of Culex quinquefasciatus (Diptera: Culicidae) in the laboratory

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Methods

Colony establishment and maintenance

Preparation of the polymeric biofilm

Analysis of the mechanical properties of the biofilm

Artificial hematophagy system

Essay

Evaluation of biological parameters

Statistical analysis

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1