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Keywords:

  • Corixidae;
  • Veliidae;
  • Aedes aegypti;
  • water jars;
  • PCR

ABSTRACT:

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Residents of Vietnam living in areas with water shortages and/or poor tap water maintain water storage containers, such as jars, in and around their domiciles in order to store water used in daily life. Although these water jars are known to be important breeding sources of the Aedes mosquito, use of chemical larvicides in such containers is legally prohibited in Vietnam. In this study, we identified the dominant mosquito insect predators in water jars in and around residences located in Tan Chanh, Long An, southern Vietnam. Of 3,646 Heteroptera collected from such jars, Corixidae (Micronecta spp.) and Veliidae (Microvelia spp.) were revealed to be the dominant predators. Polymerase chain reaction (PCR) analysis revealed that 40% of Micronecta and 12% of Veliidae had Aedes aegypti-positive reactions, indicating that these two dominant Heteroptera are important predators of Ae. aegypti. Our results suggest that aquatic Heteroptera may be an important mosquito control agent in addition to the currently used copepods.


INTRODUCTION

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Dengue fever virus is transmitted by vector mosquitoes, predominantly Aedes aegypti (L.) and Ae. albopictus (Skuse) (Gratz 2004, Phillips 2008). To date, reduction in the population density of vector mosquitoes has been the only treatment option for controlling the transmission of dengue virus in the human population, since there is no promising vaccine for the prevention of dengue (Farrar et al. 2007). The mosquitoes Ae. aegypti and Ae. albopictus feed on human blood and deposit their eggs in water collected in a variety of artificial containers found in and around the human habitat. Vector control activities, such as the use of larvivorous fish (e.g., Poecilia reticulate) and copepods (Mesocyclops), chemical larvicides, and adoption of preventive practices by local residents (e.g., the use of covers on water storage containers, frequent exchange of water in containers, or elimination of unnecessary containers) (Kay and Nam 2005, Morrison et al. 2008, Devine et al. 2009) have targeted the larvae or pupae of these mosquitoes in their attempts to reduce vector density.

In Vietnam, residents living in areas with poor tap water systems lack a reliable supply of water, especially in the dry season. To ensure that they are able to meet daily water needs, residents keep water storage containers in and around their living areas. Although water storage containers, such as jars, are regarded as important breeding sources of Aedes mosquitoes, it is legally prohibited in Vietnam to use chemical larvicides in such water storage containers. Thus, the use of lids, net covers, and predacious copepods have been adopted as the primary methods for controlling Aedes mosquitoes in Vietnam.

Various organisms have been considered as possible effective predators of mosquitoes (reviewed in Kumar and Hwang 2006, Mogi 2007, Quiroz-Martinez and Rodriguez-Castro 2007, Shaalan and Canyon 2009). Jenkins (1964) published a list of 220 species of invertebrate predators, of which only a few can be manipulated as biological controls. Of these few species, aquatic Heteroptera (Notonectidae, Belostomatidae, Nepidae, and Naucoridae) and semiaquatic Heteroptera (Veliidae and Gerridae), which inhabit rice fields and marshes, are ecologically important mosquito predators (reviewed in Mogi 2007, Quiroz-Martinez and Rodriguez-Castro 2007). Nam et al. (2000) revealed that in Vietnam, large domestic containers inhabited by Micronecta quadristigata (Corixidae) were positive for Ae. aegypti and Ae. albopictus less frequently than containers without such inhabitants. Thus, in addition to Mesocyclops spp. copepods, Heteroptera are believed to be effective as mosquito control agents in Vietnam (Nam et al. 2000). However, very few studies have investigated the biological communities of insect predators living in water jars.

Following the primary methods for detecting predation such as observation, excluding natural enemies, and the radionuclide technique (Service 1993), Service (1973, 1977) we used the serological method in order to confirm predation of Anopheles mosquito larvae in predators’ gut contents. Schielke et al. (2007) showed that Anopheles mosquito DNA can be detected after ingestion by members of the Odonata, Heteroptera. Therefore, the polymerase chain reaction (PCR)-based assay could be useful for detecting mosquito larval predators in natural breeding sites.

In the present study, we first examined the abundance of aquatic and semiaquatic Heteroptera inhabiting water storage jars near houses in southern Vietnam. We then carried out PCR analysis in order to determine whether the dominant Heteroptera insects do, in fact, feed on mosquito larvae.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Investigation of mosquito insect predator abundance in water jars

The survey was conducted within Tan Chanh District in Long An Province, Vietnam (10° 09′ N, 106° 42′ E), during March 4 to March 30, 2009. This period was within the dry season, and residents in this area usually had 5–18 jars placed around their houses. Jars were typically 0.60 to 0.88 m in height with a mouth diameter of 0.45 to 0.47 m and a maximum capacity of 200 to 260 liters.

For the entomological survey, a 0.1 mm gauze dip net (200 mm in diameter with a 1.2 m-long handle) was used to catch all insects in water jars. The water in the jars was sampled using a five-sweep netting technique, which involved 1 sweep around the periphery at the water surface (with the net held perpendicular to the surface), followed by 3–1/2 similar sweeps down through the water column, with the last full sweep at the container bottom. The final half sweep was brought up through the center of the column with the net held parallel to the water surface (see Knox et al. 2007). Insects thus trapped were transferred to the Pasteur Institute Laboratory in Ho Chi Minh City. Insects were then sorted on the basis of Polhemus (1996) and counted within each taxon.

Evaluation of prey found in the midgut of Heteroptera predators using PCR

PCR analysis was carried out in order to confirm (1) the digestive time duration in the midgut of dominant Heteroptera (Micronecta spp.) and (2) whether Corixidae (Micronecta spp.) and Veliidae (Microvelia spp.) feed on Aedes larvae, as Schielke et al. (2007) have reported. In this analysis, DNA was extracted using REDExtract-N-Amp Tissue PCR Kit (Sigma, St. Louis, MO). The extraction solution (20 μl) and tissue preparation solution (5 μl) were mixed, and each individual sample was homogenized in a 1.5-ml tube and incubated at room temperature for 10 min, followed by incubation at 95° C for 3 min. Neutralization solution (20 μl) was added to the sample and mixed by vortexing. The resultant mixture was used directly for the PCR. Although the total of 1,335 of Aedes larvae found in water jars in this area in August, October, and December 2008, and February 2009 were Aedes aegypti (100%, Tsunoda, unpublished data), Aedes aegypti and Ae. albopictus were found in the study area (Kawada et al. 2009). So, multiplex PCR was conducted by using two primers for both Ae. aegypti and Ae. albopictus (Higa et al., unpublished data). Internal transcribed spacer (ITS) regions, including 18S, 5.8S, and 16S of rDNA, were PCR-amplified using the forward primer: 18SFHIN, 5′-GTA AGC TTC CTT TGT ACA CAC CGC CCG T-3′ (Crabtree et al. 1995), and the newly designed specific reverse primers: aeg.r1 and alb.r1 (Higa et al. 2010). All PCRs were performed in a total volume of 8.65 μl that contained 1 μl of template DNA, 1 μl of 10× Ex Taq buffer, 0.8 μl of dNTP (2.5 mM each), 0.05 μl of TaKaRa Ex Taq polymerase (5 units/μl), and 0.5 μl of 0.4 picomole of each primer in 10 μl of reaction mixture (TaKaRa Ex Taq®, TaKaRa Bio Inc., Shiga, Japan). The PCR reaction mixture was heated to 96° C for 12 min and then put through 40 cycles of PCR amplification: 96° C for 30 sec, 52° C for 30 sec, and 72° C for 1.5 min, followed by 72° C for 4 min. The amplified DNA was loaded onto an agarose gel (2%) with the 100-bp ladder loading marker (Bio-Rad, Richmond, CA), stained with ethidium bromide solution (Wako Inc., Tokyo, Japan), and visualized on an ultraviolet (UV) transilluminator (TF-20C; Vilber Lourmat, Marne-la-Vallée, France).

Documentation of digestive time duration in the midgut of Micronecta

Because Micronecta was the most common insect among the insect predators, we confirmed a positive reaction to Ae. aegypti in the midgut of Micronecta at various times after feeding. To supply a single 4th instar Ae. aegypti larva, a single adult or final instar of Micronecta was placed into a 5 cm diameter plastic cup to which water had been added to a depth of 1 cm. After each individual Micronecta finished feeding on Ae. aegypti, it was preserved in 99% ethanol until analysis. The procedure of preserving individual Micronecta within 1 min (just after feeding) and at 1 h after feeding on an Ae. aegypti larva was replicated 15 times. As a preliminary test, we also carried out PCR analysis at 3, 6, 12, and 24 h after feeding (each experiment was replicated four times) using Micronecta spp. that had been previously collected from the study site in February, 2009. A stock culture of this species was established in the laboratory (F2–F3 generations).

Confirmation of Heteroptera midgut collected from water jars

PCR analysis was carried out in order to confirm whether the dominant Heteroptera, Corixidae (Micronecta spp.) and Veliidae (Microvelia spp.), had been collected from the water jars. The Micronecta and Microvelia inhabiting the mosquito-positive jars were collected in June, 2009 using a 0.1-mm gauze dip net and pipette. The collected specimens were immediately transferred to 99% ethanol and were subsequently transferred to fresh 99% ethanol 2 h after the first fixation for PCR analysis. Twenty-five individuals of both Micronecta and Microvelia were placed on clean paper for 30 min in order to remove the ethanol before PCR analysis.

RESULTS

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

Investigation of mosquito insect predator abundance in water jars

In this study, 3,646 aquatic and semiaquatic Heteroptera were collected from water jars, including Corixidae, Veliidae, Gerridae, and Pleidae. Of these Heteroptera, Corixidae (Micronecta spp.; 91.0%) and Veliidae (Microvelia spp.; 8.6%) were the dominant Heteroptera in the water jars. The number of individuals per jar was highest for Corixidae, followed by Veliidae (Table 1). The following analysis focused on these two species as mosquito predators.

Table 1.  Aquatic Heteroptera individuals in jars, Tan Chanh District, Long An Province, southern Vietnam.
FamilyGenusTotal no. surveyed jarsNo. positive jarsPositive %No. individuals per jar ± S.E.
CorixidaeMicronecta24710241.311.89 ± 2.59
VeliidaeMicrovelia2475120.61.13 ± 0.19
PleidaeUnknown24720.80.02 ± 0.01
GerridaeUnknown24720.80.02 ± 0.02

Evaluation of prey found in the midgut of Heteroptera predators using PCR

We could find a positive reaction to mosquito DNA from the midgut of Micronecta (Corixidae) just after feeding (87%, 13/15 = no. of positive individuals/total individuals) and 1 h after 1 h feeding (13%, 2/15). No positive reaction from the midgut of Micronecta was found at 3, 6, 12, and 24 h after feeding (Figure 1). Therefore, the PCR method confirmed the presence of mosquitoes in the midgut of Micronecta just after feeding. Our PCR analysis further revealed that 40% of Micronecta and 12% of Microvelia (Veliidae) showed an Ae. aegypti-positive reaction (10/25 and 3/25, Figure 2).

image

Figure 1. Examination of the Aedes gene in the midgut of Micronecta by polymerase chain reaction (PCR) amplification. Lanes 1 and 2: just after feeding; lanes 3 and 4: 1 h after feeding; lanes 5 and 6: 3 h after feeding; lanes 7 and 8: 6 h after feeding; and lane M represents the 100-bp ladder loading marker.

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image

Figure 2. Examination of the Aedes gene in the midgut of Micronecta and Microvelia by polymerase chain reaction (PCR) amplification. Lanes 1 and 2: Aedes aegypti; lanes 3 and 4: Ae. albopictus; lanes 5 and 6: Micronecta; lanes 7 and 8: Micronecta and Microvelia feeding on Ae. aegypti; and lane M represents the 100-bp ladder loading marker.

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DISCUSSION

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

We did not find Odonata nymphs, Coleoptera (Dytiscidae and Hydrophylidae), or predatory mosquitoes (Toxorhynchites spp. and Lutzia spp.) in the water jars. Therefore, we focused on Heteropteran insects. Among the Heteroptera, we focused on Notonectidae (backswimmer) as the most effective mosquito predator (reviewed in Mogi 2007, Quiroz-Martinez and Rodriguez-Castro 2007). However, even though we confirmed the presence of Notonectidae in the water jars, their abundances were very low. Thus, it was necessary to consider other Heteropteran mosquito predators such as Belostomatidae (Saha et al. 2009), Naucoridae (McCoull et al. 1998), Nepidae (Ohba and Nakasuji 2006), Veliidae (Miura and Takahashi 1998), and Gerridae (Spence and Andersen 1994). Further examination revealed that Corixidae (Micronecta spp.) and Veliidae (Microvelia spp.) were the dominant Heteroptera in the water jars (Table 1).

We found a positive reaction to mosquito DNA from the midgut of Micronecta (Corixidae). Therefore, the PCR method confirmed the presence of mosquitoes in the midgut of Micronecta just after feeding. Our PCR analysis further revealed that 40% of Micronecta and 12% of Microvelia (Veliidae) showed an Ae. aegypti-positive reaction (10/25 and 3/25; Figure 2). Nam et al. (2000) suggested that insect predators such as Micronecta (Corixidae) living in artificial containers could suppress Aedes larva, but their study did not show direct evidence that Micronecta preyed upon Ae. aegypti larvae. This study, then, is the first to offer direct evidence that Micronecta and Microvelia feed on Ae. aegypti in water jars. In addition, under laboratory conditions, we observed that Micronecta and Microvelia attacked Ae. aegypti larvae and pupae (Ohba, unpublished data), supporting the concept that these two dominant Heteroptera are important predators of Ae. aegypti in water jars. Thus, our results suggest that these two insect predators, in addition to copepods, are important mosquito control agents in water jars (Kay and Nam 2005, Morrison et al. 2008). Unlike copepods, however, these heteropteran insects can distribute themselves widely by flight, which is an important advantage for heteropteran insect predators.

In the present study, we did not confirm whether Gerridae or Pleidae in water jars feed on Aedes larvae. Therefore, revealing the predatory ability of each Heteropteran insect is an important subject for future study. Moreover, it will be necessary to collect heteropteran insects in water jars during the rainy season in order to reveal their abundance, life cycles, and the relationship between aquatic Heteroptera and copepods such as intraguild predation and competition.

Acknowledgments

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED

We thank N. Minakawa and T. Tsunoda of Nagasaki University for their important suggestions during this study. We are also grateful to the entomological staff of the Ho Chi Minh Pasteur Institute. This study was supported in part by KAKENHI 19256002, the Japan Society for the Promotion of Science (JSPS), and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to M.T.

REFERENCES CITED

  1. Top of page
  2. ABSTRACT:
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. REFERENCES CITED
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