New insights into the in vitro development and virulence of Culicinomyces spp. as fungal pathogens of Aedes aegypti

Authors: RODRIGUES, JUSCELINO - Federal University Of Goias; LUZ, CHRISTIAN - Federal University Of Goias; Humber, Richard

Submitted to: Journal of Invertebrate Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/29/2017
Publication Date: 3/31/2017
Citation: Rodrigues, J., Luz, C., Humber, R.A. 2017. New insights into the in vitro development and virulence of Culicinomyces spp. as fungal pathogens of Aedes aegypti. Journal of Invertebrate Pathology. doi:10.1016/j.jip.2017.03.012.

Interpretive Summary: Mosquitoes are important in the transmission of devastating diseases such as Zika, dengue, and yellow fever. This manuscript presents results of studies of temperature on the growth and sporulation of 11 isolates of two fungal pathogens (Culicinomyces species) of mosquito larvae, including Aedes aegypti. This manuscript also documents effects of serial repassage of isolates on the ability of these isolates to sporulate and kill Aedes aegypti larvae. Our results show that all isolates preferred rather moderate temperatures that are encountered in many sheltered tropical breeding sites but that these fungi cannot tolerate high temperatures characteristic of small bodies of water exposed to many daily hours of direct insolation. Many fungal pathogens affecting insects tend to lose many of their key properties (virulence, ability to sporulate, and more) over time, and it has been hypothesized that passing these fungi back through a natural host may restore some of these important properties. To test this hypothesis, a selection of the most productive isolates used in the temperature and growth studies were serial passaged through Aedes aegypti larvae, reisolated from dead larvae, grown out, and passed again through the mosquitoes, reisolated from cadavers, and passed a third time. Although the results of these repassages were inconsistent, there was some demonstrable improvement in the ability of these fungi to produce infective conidia, and in their virulence for Aedes aegypti. These experiments deal with possible improvements of Culicinomyces isolates for applied uses as biological control agents against field populations of mosquitoes. These data should encourage and facilitate further studies involving Culicinomyces species for the increasingly important non-pesticidal alternative control strategies used against disease-transmitting mosquito larvae throughout the world.

Technical Abstract: Culicinomyces spp. (Hypocreales: Cordycipitaceae) are facultative fungal pathogens affecting the larval stages from a range of mosquito species and are especially notable in their ability to infect hosts through the digestive tract after conidial ingestion. While Culicinomyces spp. were studied mainly in the 1980s, little is yet known about inter- and intraspecific variability of the in vitro development of these fungi at different temperatures, and nothing is known about the impact of serial host-passage on the development or virulence against Aedes aegypti larvae. The development of ten isolates of C. clavisporus (ARSEF 372, 582, 644, 706, 964, 1260, 2471, 2478, 2479 and 2480) and one of C. bisporalis (ARSEF 1479) was assessed on solid SDAY/4 and liquid SDY/4 at 15, 20, 25, 30 and 35ºC. Based on the results of these assays, three isolates were selected (ARSEF 644, 964 and 2479) for three serial host-passage/reisolation cycles, and comparison of the reisolates with the original stock isolates for their virulence, vegetative growth and conidiogenesis. The highest germination rates (= 95%) after 48 h incubation were obtained at 25 and 20ºC, and the lowest germination (= 12%) at 35ºC after the same time. The optimal temperature for radial growth was 25ºC (= 11.8 mm), followed by 20ºC for all isolates. ARSEF 706, 582 and 372 showed the greatest vegetative growth (= 20 mm). In general, there was little radial growth of colonies at 30ºC (= 2.5mm), and none at 35ºC. Isolates, especially ARSEF 964, 2479, and 644, generally produced the highest numbers of conidia at 25ºC (= 1.42 x 105 conidia/plate) after 15 days. After two host-passages, conidiogenesis increased significantly on SDAY/4 for ARSEF 2479 but not for ARSEF 644 or 964. All larvae exposed to these three isolates of C. clavisporus died within 7 days regardless of the concentration or host-passage; C. bisporalis was not tested in these experiments. The virulence of ARSEF 964 increased at lower concentrations (106–3 x 105 conidia/ml) after the first host-passage.