Integrated control of Aedes albopictus in Southwest Germany supported by the Sterile Insect Technique.

BACKGROUND: The invasive species Aedes albopictus, commonly known as the Asian tiger mosquito, has undergone extreme range expansion by means of steady introductions as blind passengers in vehicles traveling from the Mediterranean to south-west Germany. The more than 25 established populations in the State of Baden-Württemberg, Palatine and Hesse (south-west Germany) have become a major nuisance and public health threat. Aedes albopictus deserves special attention as a vector of arboviruses, including dengue, chikungunya and Zika viruses. In Germany, Ae. albopictus control programs are implemented by local communities under the auspices of health departments and regulatory offices. METHODS: The control strategy comprised three pillars: (i) community participation (CP) based on the elimination of breeding sites or improved environmental sanitation, using fizzy tablets based on Bacillus thuringiensis israelensis (fizzy Bti tablets; Culinex® Tab plus); (ii) door-to-door (DtD) control by trained staff through the application of high doses of a water-dispersible Bti granular formulation (Vectobac® WG) aimed at achieving a long-lasting killing effect; and (iii) implementation of the sterile insect technique (SIT) to eliminate remaining Ae. albopictus populations. Prior to initiating large-scale city-wide treatments on a routine basis, the efficacy of the three elements was evaluated in laboratory and semi-field trials. Special emphasis was given to the mass release of sterile Ae. albopictus males. RESULTS: More than 60% of the local residents actively participated in the first pillar (CP) of the large-scale control program. The most effective element of the program was found to be the DtD intervention, including the application of Vectobac® WG (3000 ITU/mg) to potential breeding sites (10 g per rainwater container, maximum of 200 l = maximum of approx. 150,000 ITU/l, and 2.5 g per container < 50 l) with a persistence of at least 3 weeks. In Ludwigshafen, larval source management resulted in a Container Index for Ae. albopictus of < 1% in 2020 compared to 10.9% in 2019. The mean number of Aedes eggs per ovitrap per 2 weeks was 4.4 in Ludwigshafen, 18.2 in Metzgergrün (Freiburg) (SIT area) and 22.4 in the control area in Gartenstadt (Freiburg). The strong reduction of the Ae. albopictus population by Bti application was followed by weekly releases of 1013 (Ludwigshafen) and 2320 (Freiburg) sterile Ae. albopictus males per hectare from May until October, resulting in a high percentage of sterile eggs. In the trial areas of Ludwigshafen and Frieburg, egg sterility reached 84.7 ± 12.5% and 62.7 ± 25.8%, respectively; in comparison, the natural sterility in the control area was 14.6 ± 7.3%. The field results were in line with data obtained in cage tests under laboratory conditions where sterility rates were 87.5 ± 9.2% after wild females mated with sterile males; in comparison, the sterility of eggs laid by females mated with unirradiated males was only 3.3 ± 2.8%. The overall egg sterility of about 84% in Ludwigshafen indicates that our goal to almost eradicate the Ae. albopictus population could be achieved. The time for inspection and treatment of a single property ranged from 19 to 26 min depending on the experience of the team and costs 6-8 euros per property. CONCLUSIONS: It is shown that an integrated control program based on a strict monitoring scheme can be most effective when it comprises three components, namely CP, DtD intervention that includes long-lasting Bti-larviciding to strongly reduce Ae. albopictus populations and SIT to reduce the remaining Ae. albopictus population to a minimum or even to eradicate it. The combined use of Bti and SIT is the most effective and selective tool against Ae. albopictus, one of the most dangerous mosquito vector species.

Heterologous Production and Functional Characterization of Ageritin, a Novel Type of Ribotoxin Highly Expressed during Fruiting of the Edible Mushroom Agrocybe aegerita.

Fungi produce various defense proteins against antagonists, including ribotoxins. These toxins cleave a single phosphodiester bond within the universally conserved sarcin-ricin loop of ribosomes and inhibit protein biosynthesis. Here, we report on the structure and function of ageritin, a previously reported ribotoxin from the edible mushroom Agrocybe aegerita The amino acid sequence of ageritin was derived from cDNA isolated from the dikaryon A. aegerita AAE-3 and lacks, according to in silico prediction, a signal peptide for classical secretion, predicting a cytoplasmic localization of the protein. The calculated molecular weight of the protein is slightly higher than the one reported for native ageritin. The A. aegerita ageritin-encoding gene, AaeAGT1, is highly induced during fruiting, and toxicity assays with AaeAGT1 heterologously expressed in Escherichia coli showed a strong toxicity against Aedes aegypti larvae yet not against nematodes. The activity of recombinant A. aegerita ageritin toward rabbit ribosomes was confirmed in vitro Mutagenesis studies revealed a correlation between in vivo and in vitro activities, indicating that entomotoxicity is mediated by ribonucleolytic cleavage. The strong larvicidal activity of ageritin makes this protein a promising candidate for novel biopesticide development.IMPORTANCE Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides.

Investigation of temperature conditions in Swiss urban and suburban microhabitats for the overwintering suitability of diapausing Aedes albopictus eggs.

BACKGROUND: In Switzerland, the invasive Asian tiger mosquito, Aedes albopictus, is firmly established in the Canton of Ticino, south of the Alps. According to a large-scale distribution model developed in 2013, suitable climatic conditions for the establishment of Ae. albopictus north of the Alps are found in Basel and Geneva while Zurich appears to be characterized by winters currently being too cold for survival of diapausing eggs. However, the spatial resolution of large-scale distribution models might not be sufficient to detect particular climatic conditions existing in urban settings, such as the presence of microclimatic temperatures, which may positively influence the probability of diapausing eggs to overwinter. In order to investigate this, microclimatic monitoring of potential diapausing sites (i.e. catch basins) and external controls was performed in January 2017 in Ticino and within the cities of Basel, Geneva and Zurich. RESULTS: Mean January temperatures in catch basins of Basel, Geneva and Zurich were always higher than the -1 °C temperature threshold previously set for survival probability of diapausing eggs, while mean January temperatures were below -1 °C in several catch basins south of the Alps, where Ae. albopictus eggs currently overwinter. The catch basin absolute January daily minimum temperatures both south and north of the Alps were in general higher than the external control temperatures. Absolute January daily minimum temperatures in catch basins in Basel, Geneva and Zurich were always above -10 °C, indicating that diapausing Ae. albopictus eggs could potentially survive winter nights in urban areas north of the Alps. CONCLUSIONS: The findings confirmed previous conclusions that urban catch basins can provide favourable conditions for overwintering of diapausing eggs compared to more cold-exposed sites. The results confirmed the presence of suitable winter conditions for the establishment of Ae. albopictus in the cities of Basel and Geneva. In addition, the microclimate-scale analysis added new information compared to the previous large-scale prevision model by showing that also the city of Zurich could provide winter conditions suitable for the establishment of Ae. albopictus. This illustrates the importance of the resolution of climate data in using models to predict Ae. albopictus distribution.

Strategies of a thirteen year surveillance programme on Aedes albopictus (Stegomyia albopicta) in southern Switzerland.

BACKGROUND: In Ticino, a canton located south of the Alps in Switzerland, a surveillance programme on Aedes albopictus (Stegomyia albopicta) started in 2000 seeing that the region was considered at high risk of introduction based on the rapid spread of this mosquito in neighbouring Italy. METHODS: The surveillance programme, which is still ongoing, was adapted continuously to handle preventive measures of arrival, dispersal and establishment of this invasive species. The monitoring was based on ovitraps supported by reports from the population. The integrated control measures included removal of breeding sites, larvicide applications with Bacillus thuringiensis israelensis or diflubenzuron and, in some circumstances, adulticide applications with permethrin. These measures involved citizens, municipalities and Civil Protection Units. RESULTS: Ae. albopictus was first observed in 2003 in Ticino. We describe the strategies adopted and their adaptations to the evolving problem since year 2000. The approach used allowed keeping the mosquito densities at tolerable levels and below the threshold of autochthonous Ae. albopictus borne disease transmission. During the surveillance period, new typologies of breeding sites for Ae. albopictus have been discovered. CONCLUSIONS: It was worth tackling the arrival of Ae. albopictus and adopting immediate control measures, followed by regular control measures after its establishment. Early intervention and prevention of the possible spread of the tiger mosquito over the territory avoided facing a crisis situation. This also reduced the difficulty of managing the situation and probably also reduced the overall cost if this had not been put in place.

Is Switzerland suitable for the invasion of Aedes albopictus [corrected]?

BACKGROUND: Over the last 30 years, the Asian tiger mosquito, Aedes albopictus, has rapidly spread around the world. The European distribution comprises the Mediterranean basin with a first appearance in Switzerland in 2003. Early identification of the most suitable areas in Switzerland allowing progressive invasion by this species is considered crucial to suggest adequate surveillance and control plans. METHODOLOGY/PRINCIPAL FINDINGS: We identified the most suitable areas for invasion and establishment of Ae. albopictus in Switzerland. The potential distribution areas linked to the current climatic suitability were assessed using remotely sensed land surface temperature data recorded by the MODIS satellite sensors. Suitable areas for adult survival and overwintering of diapausing eggs were also identified for future climatic conditions, considering two different climate change scenarios (A1B, A2) for the periods 2020-2049 and 2045-2074. At present, the areas around Lake Geneva in western Switzerland provide suitable climatic conditions for Ae. albopictus. In northern Switzerland, parts of the Rhine valley, around Lake Constance, as well as the surroundings of Lake Neuchâtel, appear to be suitable for the survival at least of adult Ae. albopictus. However, these areas are characterized by winters currently being too cold for survival and development of diapausing eggs. In southern Switzerland, Ae. albopictus is already well-established, especially in the Canton of Ticino. For the years 2020-2049, the predicted possible spread of the tiger mosquito does not differ significantly from its potential current distribution. However, important expansions are obtained if the period is extended to the years 2045-2074, when Ae. albopictus may invade large new areas. CONCLUSIONS/SIGNIFICANCE: Several parts of Switzerland provide suitable climatic conditions for invasion and establishment of Ae. albopictus. The current distribution and rapid spread in other European countries suggest that the tiger mosquito will colonize new areas in Switzerland in the near future.

Comparison between diflubenzuron and a Bacillus thuringiensis israelensis- and Lysinibacillus sphaericus-based formulation for the control of mosquito larvae in urban catch basins in Switzerland.

A field test was conducted to evaluate a commercial biolarvicide based on Bacillus thuringiensis var. israelensis and Lysinibacillus sphaericus to control mosquitoes breeding in catch basins in southern Switzerland. The efficacy and residual activity of the microbial mosquito larvicide applied at the recommended rate of 10 g per catch basin was compared to the currently used larvicide diflubenzuron. Both products provided a very good control activity (> 97% of reduction) of late instars (3rd and 4th instars) and pupae for 4 wk. However, only the microbial formulation controlled immature stages during the whole period of the trial, with > 98% of larval reduction. A single application of the microbial larvicide applied at 10 g per catch basin significantly reduced the number of immature mosquitoes for at least 70 days. The quantity of rainfall in the 48-h period before each sampling and the water temperature did not influence the efficacy of the treatments. Under the environmental conditions encountered in southern Switzerland, the larvicide tested may be a valid alternative to diflubenzuron to control mosquitoes in urban catch basins. The long-lasting control by the microbial larvicide further reduces the number of treatments required to keep the population of mosquitoes at low levels.

Dynamics of Bacillus thuringiensis var. israelensis and Lysinibacillus sphaericus spores in urban catch basins after simultaneous application against mosquito larvae.

Bacillus thuringiensis var. israelensis (Bti) and Lysinibacillus sphaericus (Lsph) are extensively used in mosquito control programs. These biocides are the active ingredients of a commercial larvicide. Quantitative data on the fate of both Bti and Lsph applied together for the control of mosquitoes in urban drainage structures such as catch basins are lacking. We evaluated the dynamics and persistence of Bti and Lsph spores released through their concomitant application in urban catch basins in southern Switzerland. Detection and quantification of spores over time in water and sludge samples from catch basins were carried out using quantitative real-time PCR targeting both cry4A and cry4B toxin genes for Bti and the binA gene for Lsph. After treatment, Bti and Lsph spores attained concentrations of 3.76 (± 0.08) and 4.13 (± 0.09) log ml(-1) in water, then decreased progressively over time, reaching baseline values. For both Bti and Lsph, spore levels in the order of 10(5) g(-1) were observed in the bottom sludge two days after the treatment and remained constant for the whole test period (275 days). Indigenous Lsph strains were isolated from previously untreated catch basins. A selection of those was genotyped using pulsed field gel electrophoresis of SmaI-digested chromosomal DNA, revealing that a subset of isolates were members of the clonal population of strain 2362. No safety issues related to the use of this biopesticide in the environment have been observed during this study, because no significant increase in the number of spores was seen during the long observation period. The isolation of native Lysinibacillus sphaericus strains belonging to the same clonal population as strain 2362 from catch basins never treated with Lsph-based products indicates that the use of a combination of Bti and Lsph for the control of mosquitoes does not introduce non-indigenous microorganisms in this area.

Distribution of Bacillus thuringiensis subsp. israelensis in Soil of a Swiss Wetland reserve after 22 years of mosquito control.

Recurrent treatments with Bacillus thuringiensis subsp. israelensis are required to control the floodwater mosquito Aedes vexans that breeds in large numbers in the wetlands of the Bolle di Magadino Reserve in Canton Ticino, Switzerland. Interventions have been carried out since 1988. In the present study, the spatial distribution of resting B. thuringiensis subsp. israelensis spores in the soil was measured. The B. thuringiensis subsp. israelensis concentration was determined in soil samples collected along six transects covering different elevations within the periodically flooded zones. A total of 258 samples were processed and analyzed by quantitative PCR that targeted an identical fragment of 159 bp for the B. thuringiensis subsp. israelensis cry4Aa and cry4Ba genes. B. thuringiensis subsp. israelensis spores were found to persist in soils of the wetland reserve at concentrations of up to 6.8 log per gram of soil. Continuous accumulation due to regular treatments could be excluded, as the decrease in spores amounted to 95.8% (95% confidence interval, 93.9 to 97.7%). The distribution of spores was correlated to the number of B. thuringiensis subsp. israelensis treatments, the elevation of the sampling point, and the duration of the flooding periods. The number of B. thuringiensis subsp. israelensis treatments was the major factor influencing the distribution of spores in the different topographic zones (P < 0.0001). These findings indicated that B. thuringiensis subsp. israelensis spores are rather immobile after their introduction into the environment.

Biotoxicity assays for fruiting body lectins and other cytoplasmic proteins.

Recent studies suggest that a specific class of fungal lectins, commonly referred to as fruiting body lectins, play a role as effector molecules in the defense of fungi against predators and parasites. Hallmarks of these fungal lectins are their specific expression in reproductive structures, fruiting bodies, and/or sclerotia and their synthesis on free ribosomes in the cytoplasm. Fruiting body lectins are released upon damage of the fungal cell and bind to specific carbohydrate structures of predators and parasites, which leads to deterrence, inhibition of growth, and development or even killing of these organisms. Here, we describe assays to assess the toxicity of such lectins and other cytoplasmic proteins toward three different model organisms: the insect Aedes aegypti, the nematode Caenorhabditis elegans, and the amoeba Acanthamoeba castellanii. All three assays are based on heterologous expression of the examined proteins in the cytoplasm of Escherichia coli and feeding of these recombinant bacteria to omnivorous and bacterivorous organisms.