The Potential of a New Beauveria bassiana Isolate for Mosquito Larval Control.

The African malaria mosquito, Anopheles gambiae Giles (Diptera: Culicidae), and the Asian tiger mosquito, Aedes albopictus Skuse (Diptera: Culicidae) are of public health concern due to their ability to transmit disease-causing parasites and pathogens. Current mosquito control strategies to prevent vector-borne diseases rely mainly on the use of chemicals. However, insecticide resistance in mosquito populations necessitates alternative control measures, including biologicals such as entomopathogenic fungi. Here we report the impact of a new Beauveria bassiana (Balsamo) Vuillemin (Hyprocreales: Cordycipitaeceae) isolate, isolated from field-collected Ae. albopictus larvae on mosquito survival and development. Larval infection bioassays using three B. bassiana conidial concentrations were performed on the second and third larval instars of An. gambiae and Ae. albopictus mosquitoes. Larvae were monitored daily for survival and development to pupae and adults. Our results show that B. bassiana MHK was more effective in killing An. gambiae than Ae. albopictus larvae. We further observed delays in development to pupae and adults in both mosquito species exposed the varying concentrations of B. bassiana as compared to the water control. In addition, larval exposure to B. bassiana reduced adult male and female survival in both mosquito species, further contributing to mosquito population control. Thus, this study identifies a new B. bassiana isolate as a possible biological control agent of two mosquito species of public health concern, increasing the arsenal for integrated mosquito control.

Patterns of fungal community assembly across two Culex mosquito species.

In the aquatic environment, mosquito larvae encounter bacteria and fungi that assemble into bacterial and fungal communities. The composition and impact of mosquito-associated bacterial community has been reported across larvae of various mosquito species. However, knowledge on the composition of mosquito-associated fungal communities and the drivers of their assembly remain largely unclear, particularly across mosquito species. In this study, we used high throughput sequencing of the fungal Internal transcribed spacer 2 (ITS2) metabarcode marker to identify fungal operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) associated with field-collected Culex restuans and Culex pipiens larvae and their breeding water. Our analyses identified diverse fungal communities across larval breeding sites collected on a fine geographic scale. Our data show that the larval breeding site is the major determinant of fungal community assembly in these mosquito species. We also identified distinct fungal communities in guts and carcasses within each species. However, these tissue-specific patterns were less evident in Cx. restuans than in Cx. pipiens larvae. The broad ecological patterns of fungal community assembly in mosquito larvae did not vary between OTU and ASV analyses. Together, this study provides the first insight into the fungal community composition and diversity in field collected Cx. restuans and Cx. pipiens larvae using OTUs and ASVs. While these findings largely recapitulate our previous analyses in Aedes albopictus larvae, we report minor differences in tissue-specific fungal community assembly in Cx. restuans larvae. Our results suggest that while the fungal community assembly in mosquito larvae may be generalized across mosquito species, variation in larval feeding behavior may impact fungal community assembly in the guts of mosquito larvae.

Host-Environment Interplay Shapes Fungal Diversity in Mosquitoes.

Mosquito larvae encounter diverse assemblages of bacteria (i.e., "microbiota") and fungi in the aquatic environments that they develop in. However, while a number of studies have addressed the diversity and function of microbiota in mosquito life history, relatively little is known about mosquito-fungus interactions outside several key fungal entomopathogens. In this study, we used high-throughput sequencing of internal transcribed spacer 2 (ITS2) metabarcode markers to provide the first simultaneous characterization of the fungal communities in field-collected Aedes albopictus larvae and their associated aquatic environments. Our results reveal unprecedented variation in fungal communities among adjacent but discrete larval breeding habitats. Our results also reveal a distinct fungal community assembly in the mosquito gut versus other tissues, with gut-associated fungal communities being most similar to those present in the environment where larvae feed. Altogether, our results identify the environment as the dominant factor shaping the fungal community associated with mosquito larvae, with no evidence of environmental filtering by the gut. These results also identify mosquito feeding behavior and fungal mode of nutrition as potential drivers of tissue-specific fungal community assembly after environmental acquisition. IMPORTANCE The Asian tiger mosquito, Aedes albopictus, is the dominant mosquito species in the United States and an important vector of arboviruses of major public health concern. One aspect of mosquito control to curb mosquito-borne diseases has been the use of biological control agents such as fungal entomopathogens. Recent studies also demonstrate the impact of mosquito-associated microbial communities on various mosquito traits, including vector competence. However, while much research attention has been dedicated to understanding the diversity and function of mosquito-associated bacterial communities, relatively little is known about mosquito-associated fungal communities. A better understanding of the factors that drive fungal community diversity and assembly in mosquitoes will be essential for future efforts to target mosquito-associated bacteria and fungi for mosquito and mosquito-borne disease control.

Real-Time PCR Protocol for Phytoplasma Detection and Quantification.

Phytoplasmas are mollicutes restricted to plant phloem tissue and are normally present at very low concentrations. Real-time polymerase chain reaction (qPCR) offers several advantages over conventional PCR. It is a fast, sensitive, and reliable detection technique amenable to high throughput. Two fluorescent chemistries are available, intercalating dyes or hybridization probes. Intercalating dyes are relatively less expensive than TaqMan® hybridization probes but the latter chemistry is the most commonly used for phytoplasma detection. qPCR may be designed for universal detection of phytoplasma, group or subgroup specific detection, or for simultaneous detection of up to three or four phytoplasmas (multiplexing). qPCR may be used for relative or absolute quantification in host plants and in insect vectors. Therefore, qPCR plays an important role in phytoplasma detection as well as in host-pathogen interaction and in epidemiological studies. This chapter outlines the protocols followed in qPCR assay for phytoplasma detection and quantification, focusing mainly on the use of TaqMan® probes.

Specific PCR and real-time PCR assays for detection and quantitation of 'Candidatus Phytoplasma phoenicium'.

Almond witches' broom (AlmWB) is a fast-spreading lethal disease of almond, peach and nectarine associated with 'Candidatus Phytoplasma phoenicium'. The development of PCR and quantitative real-time PCR (qPCR) assays for the sensitive and specific detection of the phytoplasma is of prime importance for early detection of 'Ca. P. phoenicium' and for epidemiological studies. The developed qPCR assay herein uses a TaqMan(®) probe labeled with Black Hole Quencher Plus. The specificity of the PCR and that of the qPCR detection protocols were tested on 17 phytoplasma isolates belonging to 11 phytoplasma 16S rRNA groups, on samples of almond, peach, nectarine, native plants and insects infected or uninfected with the phytoplasma. The developed assays showed high specificity against 'Ca. P. phoenicium' and no cross-reactivity against any other phytoplasma, plant or insect tested. The sensitivity of the developed PCR and qPCR assays was similar to the conventional nested PCR protocol using universal primers. The qPCR assay was further validated by quantitating AlmWB phytoplasma in different hosts, plant parts and potential insect vectors. The highest titers of 'Ca. P. phoenicium' were detected in the phloem tissues of stems and roots of almond and nectarine trees, where they averaged from 10(5) to 10(6) genomic units per nanogram of host DNA (GU/ng of DNA). The newly developed PCR and qPCR protocols are reliable, specific and sensitive methods that are easily applicable to high-throughput diagnosis of AlmWB in plants and insects and can be used for surveys of potential vectors and alternative hosts.