MOSQUITO SAMPLING

To detect the presence of mosquito species all the developmental stages should be looked for: eggs, larvae, pupae and adults (Figure 7).

Figure 7. Examples of methods applied for mosquito sampling. a. Sampling eggs with an ovitrap built up for the survey on Ae. albopictus in Canton Ticino (photo A. Rose); b.

sampling of larvae with dipper (photo E. Flacio); c. adult trap placed on the field (EVS CO2-baited mosquito trap) (photo E. Flacio)

The developmental stage to be investigated depends on the straightforwardness and efficacy of the methodology adopted. In fact, for some species it is easier to detect the eggs, for others the larvae and by others the adult form. For example, by Aedes species that lay eggs in the ground like Ae. vexans, the eggs are difficult to sample. The soil close to water surface should be collected and then sieved (Silver 2008). In this case it is not evident to find the right place where eggs have been laid and then it is difficult after the sieving to have living eggs ready to hatch. Therefore it is easier to look after larvae or adults. Larvae of the genus Coquillettidia, stay under water attached to aquatic plants, therefore they are very difficult to detect. In this case it is worth to look after the adult form. Vice versa all forms of Ae. albopictus, a container breeding mosquito, can be sampled with different methodologies, but for an extensive survey system egg sampling with oviposition traps is the cheapest way, therefore this methodology has been privileged to others. Further every type of adult trap attract species in a different way (Lühken et al. 2014), therefore there is not a straight correlation in mosquito species densities with the use of a unique tool. Thus to have an overview on mosquito species in this work different sample methodologies were applied simultaneously.

a b c

Still waters in urban and natural ecosystems were investigated to collect juvenile forms of mosquitoes, traps for mosquito adults were placed, and the presence of eggs of Ae.

albopictus was checked.

3.2.1 EGGS

Ae. albopictus eggs were collected using oviposition traps (ovitraps) (Figure 7a). Ovitraps attract the female mosquito through the form of the container (black colour and the small size) and water. A support (the paddle) is added to allow the mosquito to lay its eggs, and eventually odours can be added. This support is then removed for analysis of the presence of the eggs, it is light coloured in order to facilitate the morphological detection of the eggs (black coloured) and should not have any repellent effect on the female mosquito. These ovitraps have been the main tool for the survey on Ae. albopictus. They are generally considered the easiest way to detect the presence of species belonging to the subgenus Stegomyia, i.e. Ae. aegypti (Linnaeus, 1862) and Ae. albopictus, (Romi 1996a, Silver 2008).

Ovitraps detect eggs even at low mosquito densities, are not expensive and are easy to handle even by unskilled staff. To be used in a large scale survey a compromise between attractiveness for the mosquito (colour, shape, odours, persistence of water), safeness, i.e.

not being breeding sites themselves, simplicity in handling and availability on the market has to be found.

We used black plastic containers of 1 l (Ramona Ø13/H12, Luwasa® Interhydro AG, Allmendingen, Switzerland) with an efflux hole on the top border (Figure 7a). A slat of steamed beechwood (200 x 26 x 6 mm) was placed as an oviposition support in an angular position in the plastic container, which was filled with tap water. Ten granules of VectoBac G® (Valent BioSciences, Libertyville, USA) were added in order to block the development of hatched larvae. The ovitraps were labelled with all the necessary information including phone number and the pledge not to remove it. Ovitraps were positioned close to or under vegetation or near buildings. An important factor was to select the most suitable place to position the ovitraps: it had to catch the mosquito at its arrival place, i.e. getting out from a vehicle, before finding several other breeding sites and/or it had to be placed where adults

could easily live, i.e. presence of hosts for blood meal, shadow, etc. The traps were controlled bi-weekly. They were rinsed and filled with fresh water and VectoBac G^® granules. The paddle was retrieved, labelled, wrapped into a plastic film, replaced and brought to the laboratory for examination of the presence of eggs.

3.2.2 JUVENILE STAGES

Juvenile stages can be sampled in water either directly with a Pasteur pipette with the tip cut off or using a standard pint dipper (model 1132, BioQuip Products, Rancho Dominguez, USA), consisting of a white (to contrast the larvae or pupae colour, which is brown or black) plastic container of 11 cm in diameter with a capacity of 350 ml (Figure 7b). It is important to sample without creating shadows on the water surface not to scare larvae and make them sink to the bottom. For every sampling site, water collection was repeated at least 3 times, each time in a different part of the pound in order to cover a total water surface of 30 m². In case of catch basins the sampling was also repeated three times but at intervals of 3-5 minutes from each tray in order to permit the larvae to reach the water surface again after sinking.

For sampling of Ae. albopictus larvae in deep containers, such as catch basins or drums, a fish net for aquariums can be used because larvae can remain at bottom for several minutes. Afterwards, the fish net has to be cleaned out in a recipient with water.

In case larvae had not reached the fourth stadium they have to be transported to the laboratory in cold conditions so they do not stress and die during the transport. The larvae are concentrated in a plastic container with a cap using a sifter or a pipette and the recipient was kept in thermic bag containing frozen freezer packs. Once in the laboratory larvae are kept at 28^oC, and under 12 hours/ daylight in plastic trays filled up with tap water, feed with pulverized (using a mortar) fish food (TetraMin^® Fish Food Flakes), in the presence of an aquarium aerator not to create mouldy water surface. Once larvae reached the fourth stadium, they were stored in 70% ethanol. Pupae were kept in the laboratory at room temperature until they emerge as adults under mosquito cages.

3.2.3 ADULTS

Traps for adult sampling have all the same principle: attracting the mosquito and sucking it out to a collector recipient. Light can be used as attractant, but it is not very specific.

Mosquitoes, like other haematophagous insects, detect their prey following different chemical stimuli, like carbon dioxide, released with breathing, or body odours. Carbon dioxide can be added to traps for adults with a bottle or in form of dry ice whereas there are different products that simulate body odours.

For adult sampling, we used heavy duty EVS CO2-baited mosquito traps (model 2801A, BioQuip Products) placed with the mosquito entrance at 1.5 m above ground level and dry ice (CO2) was used as attractant (Figure 7c).

In some cases we used Mosquito Magnet® Liberty plus with either Octenol or Lurex™ or Lurex3™ as an attractant (www.mosquitomagnet.com). Traps were placed in the afternoon and removed the following day no later than midday. To catch Ae. albopictus adults, BG-sentinel traps (Biogents® AG, Regensburg, Germany) equipped with BG-Lure (Biogents® AG) and CO2 as attractants were placed on the ground.

In this case traps were preferably placed in the morning and removed the following day at the same time. For instant catches an insect net or aspirator (Flashlight Aspirator, 2809C, www.bioquip.com) was used. To estimate nuisance Human Landing Collections were performed by estimating the number of females landing for 15 minutes (Silver 2008).

Adults were killed by exposure to dry ice and stored at -20°C or dry for conservation purposes.

3.2.4 PERIOD OF SAMPLING

The period of sampling for the tiger mosquitoes was adapted to their known activity periods according to reports from Italy (Romi 1996a). The extensive sampling started generally at the beginning of May till the end of September, sometimes from April till the end of October and some more restricted samplings continued in the cold season from December till May in order to see if this species could be active during the cold season.

The sampling period for the other species was chosen when mosquito and human activities matched i.e. in the warm season. Samplings were conducted between mid-April and end of October returning at least twice in the same sampling sites each season.

3.2.5 COMMUNITY PARTICIPATION

Because the trapping system for Ae. albopictus was insufficient to cover the whole territory, community participation was requested: residents were asked to report the occurrence of this species. The residents could report the presence of tiger mosquito in sending samples to the laboratory. Samples had to be sent in a small box to avoid disruption in shifting and origin, data of collection as well as telephone number had to be indicated. All notifications were followed by an answer of the expert of the GLZ to the residents.