# Effectiveness of native cyclopoid copepods in biological vector control related to their predatory behavior against the Asian tiger mosquito, Aedes albopictus | Parasites and Vectors

### Teaching material

#### Copepods of field origin

All copepods were collected from May to July 2021 from a pond in Philippsburg, Germany (49°14′44.6″N 8°26′45.0″E). Copepods were obtained from the shallow part of water sources, including aquatic vegetation, using a long-handled fine plankton net (mesh size 100 µm). Collected aquatic organisms were transferred to glass containers and transported to the laboratory in styrofoam boxes for identification and sorting of the respective species.

Sorting is done no later than 1 day after collection in laboratory conditions. Water samples were transferred to sorting containers (about 500 ml) and cyclopoid copepods with the largest relative body size, especially those carrying egg sacs, were selected and pipetted into plastic boxes filled with about 600 ml of fresh tap water (pH 7.4). , 20 °C, 521 µS; valid for all subsequent experiments). Selection of copepods based on body size ensured the use of typically larger cyclopoid females rather than smaller males or copepods. [36]. Copepods obtained from the field were stored at room temperature (24 ± 1 °C) and protected from direct insolation until use. Maximum capacity per box was kept below 150 people to ensure longevity. A spatula tip of fish food powder to the copepods (TetraMin Baby®, Tetra GmbH) per box, every 3-5 days. In addition, the residues were removed and fresh water was added when necessary. Copepods are stored for no more than 7 weeks.

#### Mosquito larvae

eggs Ae. albopictus Obtained from colonies managed by Institute of Dipterology.Gesellschaft zur Förderung der Stechmückenbekämpfung (GFS; Speyer, Germany) and Centro Agricoltura Ambiente “G. nicole” (CAA; Crevalcore, Italy). About 24 h before experiments, eggs were filled with fresh tap water at room temperature (24 ± 1 °C) to allow larval hatch. Egg rafts Cx. pipiens sl was collected from rain barrels in the IfD garden approximately 24 h before the experiment (Speyer, Germany). Egg rafts were kept in water from rearing sources at room temperature (24 ± 1 °C) until larvae hatched.

Larvae that were not used as newly hatched larvae were reared at room temperature (24 ± 1 °C) in glass containers covered with nets. Larvae were fed a spatula tip of fish food every other day until they reached the appropriate developmental stage for subsequent experiments.

### Experimental setting

Field-collected gravid or non-gravid female copepods of similar size and appearance were used in the experiments. After the experiments, the copepods were killed in alcohol and preserved in 70% ethanol and treated with a drop of glycerol for accurate species identification. [36]According to the fixation and preparation process described by Einsle [24].

### Laboratory experiments

#### Evaluation of predation efficiency of copepods against different larval instars Ae. albopictus

Copepod predation rates were assessed separately for the first three larval stages. Experiments on first-instar larvae were performed three times with five replicates for each control and treatment group (number of replicates per group: n = 15). The effectiveness of predation on the second and third developmental stages was tested only once (n = 5).

Ten clear plastic boxes (1.1 L) each filled with 500 mL of fresh tap water were used as experimental containers. Five larvae of the same mosquito species at the same corresponding developmental stage were transferred to each container. According to the procedures reported by Chansang et al. [37] and Ray et al. [31], one adult female copepod per box was introduced into five containers to create a 1:5 prey-to-prey ratio. Boxes with only larvae served as controls to calculate background prey mortality. A spatula tip of powdered fish feed was added to each box to provide nutrition. All laboratory experiments were performed at room temperature (24 ± 1 °C). To determine the number of larvae killed, the surviving larvae after 24 hours and 48 hours were counted. Larval remains (head capsules, fragments or dissected bodies) were removed, identified and counted under a binocular microscope (Motic SMZ 171-BP).®) as evidence of predatory behavior [29, 31, 36].

Predation rate was defined as the number of larvae killed per copepod per day [38]and predation efficiency was calculated using the Abbott formula [30, 39]:

$${text{Predation,efficiency}} = frac{Number,alive,in,control – Number,alive,in,treatment }{{Number,alive,,in,control }} left( {{1}00} right)$$

Following the same procedure described above, ad libitum feeding was performed on first-instar larvae. Ae. albopictus (n = 15), differing only in the amount of prey offered to copepods. Instead of five larvae, 24 first-instar larvae were exposed to one adult female copepod. This experiment was not performed for later developed larval stages.

#### Analysis of the efficiency of simultaneous predation against Ae. albopictus and Cx. pipiens sl larvae

One day before the experiment, five clear buckets (10 L), each filled with 8 L of fresh tap water, were inoculated with four adult female copepods for acclimation and starvation. Twenty-four hours later, 10 first instar larvae Ae. albopictus and 10 first instar larvae Cx. pipiens sl was added to each bucket, thus establishing a 1:5 ratio. Five buckets with the same number of larvae but no copepods served as controls. Larvae were fed two spatula-tip fish baits per bucket.

Evaluation was performed 5 days after larval introduction, when the larvae reached the second and third instar larval stages of development. To distinguish the species of the remaining larvae, they were collected from buckets and identified under a binocular microscope. The key feature for differentiation was the siphon, which differed in shape and length between the two species. [1].

#### assessment of suitability of use Bti and copepods in an integrated control strategy

Three different concentrations (1, 10, 50 ppm). Bti(3000 ITU/mg, VectoBac WG®Lot No. 320917PG30) was prepared by diluting the appropriate amount to 50 ppm. Bti stock solution (0.15 g Bti powder, 3.00 LH2O) with an appropriate amount of fresh tap water to reach a final experimental volume of 200 ml. One part per million was chosen as the lowest concentration because it represents a concentration of 1000 µg. Bti /L is widely used in mosquito control applications [14]. the effect of Bti tested in five Cx. pipiens sl first-instar larvae (as a positive control) or five adult female copepods. Negative controls consisted of 200 ml of tap water and five adult female copepods.

Analyzes for three groups (positive control, negative control and copepods Bti water) were performed in triplicate at three different concentrations, resulting in a total of 27 analyzes (n= 3). They were performed in transparent plastic bottles, to which a spatula tip of powdered fish feed was added, respectively. The results were recorded 24 hours and 48 hours after the injection of the organisms.

### Estimation of predation rate Ae. albopictuslarvae in half-field trials

Ten rain barrels (70 L) filled approximately three-quarters with tap water were set up in a semi-open, mosquito-free environment at room temperature (24 ± 1 °C) for 1 week prior to testing. running (laboratory premises of GFS, Speyer, Germany).

The day before the experiment, 10 adult female copepods were added to five of 10 rain barrels to account for acclimation and starvation. Twenty-four hours later, 50 newly hatched larvae Ae. albopictuswere included in each rain barrel, resulting in a predator-prey ratio of 1:5. The other five rain barrels served as controls containing larvae only. Ten spatula-tip fish baits were added to each barrel. Feeding was performed every other day throughout the duration of the experiment, gradually increasing the amount of food available with five additional spatula tips at each feeding.

Five days after establishment, the number of surviving predators was counted by removing the larvae from the barrels into sorting containers filled with water and digging them one at a time into another container filled with water. In the control condition, all counted larvae were killed to prevent further development. In contrast, only third- and fourth-instar larvae were destroyed from tubs containing copepods, as they were unlikely to be eaten later and, as in the control, further development had to be prevented. All first- or second-instar larvae were poured into the respective tubs with the copepods to account for the possibility that the copepods would still kill them during the remaining experimental period. After this procedure, another 2 days were counted. In addition, all copepods, copepodids, and larval fragments collected intentionally with a net were listed.

### Identification of species of copepods of field origin

For species identification, 12 preserved copepods from laboratory predation efficiency experiments were randomly selected, dissected, and identified under a microscope (Motic Panthera C2).® microscope; n= 12) according to the keys of Błędzki and Rybak [40] and Einsle [24]. For the identification of the final species, the fourth and fifth pairs of swimming legs (P4 and P5), furca, as well as the specific ecology, frequency of occurrence and body size of the different species were taken into account. Each of these characteristic body features was captured with the Canon EOS 90D® camera.

### Statistical analysis

Copepod predation rates in different experiments were analyzed using generalized linear (mixed) models. [GL(M)Ms]. The response variable, ie larval survival, is binomially distributed (alive or dead). Thus, generalized linear (mixed) models with binomial family using logit transition are applied.

Different treatment groups (i.e., with or without prey, low vs. high density of different prey species, or prey) were used as explanatory variables in GL(M)Ms as fixed factors. For logistical reasons, a series of five replicates per treatment had to be replicated three times in a row to obtain a total of 15 replicates in all experiments (except the half-field study). Thus, the variable “batch” (i.e., number of trial replicates) was used as a random factor in all GL(M)Ms (model 1). In addition, both prey species were kept in the same bucket for simultaneous predation analyses. Therefore, bucket number was also included as a random factor within batch in this analysis (model 2). The half-field experiment was analyzed using a generalized linear model (GLM) because it was conducted only once with five replications per treatment (model 3). Model hypotheses were evaluated using diagnostic plots.

All statistical analyzes were performed with the statistical and programming environment R (version 4.2.0). [41]. Installed using GL(M)Ms functions lme4package [42].

Differences between tested groups were considered significant P-value was ≤ 0.05, indicated by an asterisk in figures (*P< 0.05; **P< 0.01 or ***P< 0.001). In the Results section, data values ​​are expressed as mean ± standard deviation (SD).