Michael G. Kaufman

Density-Dependent Development of Anopheles gambiae (Diptera: Culicidae) Larvae in Artificial Habitats

Abstract The growth and development of Anopheles gambiae Giles larvae were studied in artificial habitats in western Kenya. Larvae responded to increasing densities by extending their development time and by emerging as smaller adults, although survival was not significantly affected. Addition of nutrients in the form of cow dung collected near the study site had no impact on larval growth and development. Regression analysis showed that female development time increased by 0.020 d and female dry mass decreased by 0.74 μg with each additional larva. By fitting the data to the pupation window model, the estimated minimum dry mass to achieve pupation was 0.130 mg and the estimated minimum time to pupation was 5 d. The most likely food source for An. gambiae larvae was algal growth, which was significantly reduced by the presence of larvae. Bacterial densities were not significantly affected by the presence of larvae although total bacteria counts were lower at the higher densities indicating they may provide a secondary food source when algal resources are depleted. Similarly, the levels of nitrogen and phosphorus in the habitats were not significantly affected by the presence of larvae although there was evidence of decreasing nitrogen levels occurring with increasing larval densities suggesting that nitrogen may be a limiting resource in the larval environment. The data indicate that competition within the larval environment may indirectly regulate An. gambiae populations by reducing adult body size, which may in turn reduce adult survivorship and fecundity. The potential impact of density-dependent interactions among An. gambiae larvae on the transmission of Plasmodium falciparum is discussed.

Role of bacteria in mediating the oviposition responses of Aedes albopictus (Diptera: Culicidae).

Abstract The responses of Aedes albopictus to sources of oviposition attractants and stimulants were evaluated with a behavioral bioassay in which females attracted to odorants emanating from water were trapped on screens coated with an adhesive. Gravid mosquitoes were attracted to volatiles from larval-rearing water and soil-contaminated cotton towels. Bacteria were isolated from these substrates and from an organic infusion made with oak leaves. Through fatty acid-methyl ester analyses, six bacterial isolates from larval-rearing water, two isolates from soil-contaminated cotton towels, and three isolates from oak leaf infusion were identified to species. The response of gravid mosquitoes to these isolates was also evaluated in behavioral bioassays. Water containing Psychrobacter immobilis (from larval-rearing water), Sphingobacterium multivorum (from soil-contaminated cotton towels), and an undetermined Bacillus species (from oak leaf infusion) elicited significantly higher oviposition than control water without bacteria. Only volatiles collected from larval rearing water elicited significant electroantennogram responses in females.

The effect of an industrial effluent on an urban stream benthic community: Water quality vs. habitat quality

We studied the effect of an industrial effluent on the water quality, habitat quality, and benthic macroinvertebrates of an urban stream in southwestern Michigan (USA). The effluent affected water quality by raising in-stream temperatures 13-18 degree C during colder months and carrying high amounts of iron (> 20 x higher than ambient) that covered the streambed. The effluent also affected habitat conditions by increasing total stream discharge by 50-150%, causing a significant change in substrate and flow conditions. We used three methods to collect benthic macroinvertebrates in depositional and erosional habitats and to understand the relative importance of habitat quality and water quality alterations. Macroinvertebrate response variables included taxonomic richness, abundance, and proportional abundance of sensitive taxonomic groups. Results indicated that the effluent had a positive effect on macroinvertebrate communities by increasing the quantity of riffle habitat, but a negative effect on macroinvertebrate communities by reducing water quality. Results illustrated the need for careful consideration of habitat quality and water quality in restoration or remediation programs.

Influence of diet on the structure and function of the bacterial hindgut community of crickets

The effect of diets varying in carbohydrate and protein content on the structure and function of the hindgut microbiota of crickets was evaluated by determining bacterial densities, fermentation activity, and guanine plus cytosine (G + C) profiles of the DNA extracted from the microbial hindgut community. DNA isolated from the gut community was fractionated and quantified according to G + C content as a comprehensive, coarse‐level measure of the composition and structure of the community. The bacterial densities measured by direct counts were not significantly different among the four diets. The crickets were initially reared in the laboratory on cricket chow, which resulted in a hindgut community dominated by bacteria with a G + C content between 32% and 57%. Crickets shifted to an alfalfa diet showed a similar hindgut community G + C profile, although microbial populations with DNA between 35% and 45% G + C were more abundant in alfalfa‐ than chow‐fed crickets. The apparent complexity of the gut community was reduced in crickets fed beet‐pulp and protein‐based diets compared to those fed chow and alfalfa, and was dominated by populations with a low percentage G + C content. Hindgut communities in crickets fed pulp and protein diets also showed a decrease in hydrogen and carbon dioxide production, suggesting that these diets affected the biochemical activity of the hindgut community. The protein‐based diet resulted in a decrease in the rate of evolution of volatile fatty acids, while the ratio of butyrate production to acetate and propionate production was significantly higher in these crickets. Our results show the emergence of a new microbial community structure concomitant with changes in microbial biochemical activity due to shifts in the cricket’s dietary regime.

Invasion Biology of Aedes japonicus japonicus (Diptera: Culicidae)

Aedes japonicus japonicus (Theobald) (Diptera: Culicidae) has recently expanded beyond its native range of Japan and Korea into large parts of North America and Central Europe. Population genetic studies begun immediately after the species was detected in North America revealed genetically distinct introductions that subsequently merged, likely contributing to the successful expansion. Interactions, particularly in the larval stage, with other known disease vectors give this invasive subspecies the potential to influence local disease dynamics. Its successful invasion likely does not involve superior direct competitive abilities, but it is associated with the use of diverse larval habitats and a cold tolerance that allows an expanded seasonal activity range in temperate climates. We predict a continued but slower expansion of Ae. j. japonicus in North America and a continued rapid expansion into other areas as this mosquito will eventually be considered a permanent resident of much of North America, Europe, Asia, and parts of Hawaii.

The contribution of hindgut bacteria to dietary carbohydrate utilization by crickets (Orthoptera: Gryllidae)

Abstract 1. 1. Growth rates of germfree house crickets fluctuated during development more than their conventional counterparts in response to an alternating diet regime. 2. 2. Gut bacteria increased the digestive efficiency of soluble plant polysaccharides for crickets on each diet treatment and further, dampened the effect of diet change on the digestion of that pool of carbohydrates. 3. 3. Comparisons of carbohydrase activity in different gut segments and in germfree and conventional crickets, showed that bacteria in the anterior hindgut supply most of the conventional insects degradative capabilities against a heterogenous class of soluble plant storage/structural polysaccharides. 4. 4. Gut bacteria in crickets aid the insects ability to utilize a wider range of dietary carbohydrates by responding to changes in dietary sources with induced production of enzymes and/or changes in population levels.

Bacterial and Fungal Biomass Responses to Feeding by Larval Aedes triseriatus (Diptera: Culicidae)

Abstract We investigated the effect of different densities (0, 20, or 40) of developing larval Aedes triseriatus (Say) on bacterial abundance, bacterial productivity, and leaf fungal biomass in a microcosm experiment. Larvae in the low-density treatment developed normally, but larvae at the high density were significantly slower to develop. Both bacterial abundance (direct microscopic counts) and bacterial productivity (3H-leucine incorporation rates) on leaf material were significantly lower in the presence of larvae. Bacterial abundance in the water column did not change significantly with treatment, but bacterial productivity varied with time and declined significantly at both larval densities. Bacteria on the walls and bottom of the containers also were less abundant and significantly less productive in the presence of larvae. Aside from presence/absence effects, there was no clear evidence that larval impacts were density-dependent. Leaf-associated fungal biomass, as measured by ergosterol levels, varied with time but was not significantly affected by any treatment, suggesting most fungal tissue was incorporated in the leaf matrix and unavailable to larvae. Based upon estimated biomass accrual and respiration of larvae, it appears that bacterial biomass and production were insufficient to account for carbon demands of growing larvae. Because fungal biomass and leaf mass likely contributed little to gross larval demands, other carbon sources (e.g., protozoa and extracellular microbial components) were probably used by larvae. Although apparently insufficient for all larval carbon demands, bacterial and leaf fungal biomass may be adequate for other larval nutritional needs (i.e., nitrogen and essential lipids).

Importance of Algal Biomass to Growth and Development of Anopheles gambiae Larvae

Abstract We conducted experiments to investigate the importance of algal food resources for larval growth and adult emergence of Anopheles gambiae Giles s.s. in simulated larval habitats in Kenya, and in greenhouse and laboratory microcosms in the United States. In the first experiment, we used shading to reduce algal biomass, and because algal production and larval development might be a function of underlying soil nutrients, we crossed sun–shade treatments with soils of two distinct types collected near larval habitats. Shading reduced pupation rates and total adult biomass of An. gambiae by ≈50%. Soil type had no significant effect on mosquito production, but it did significantly affect concentrations of phosphorus and chlorophyll a in the surface microlayer. In a subsequent experiment conducted in the greenhouse to reduce temperature differences found between the shaded and sunlit treatments, <1% of larvae in the shaded treatments reached the pupal stage. There was a marked reduction of chlorophyll a levels as a function of shading and larval density. In a third experiment, larvae receiving material harvested from sunlit surface microlayers performed as well as those receiving liver powder, whereas those receiving surface microlayer from shaded habitats suffered >90% mortality and failed to pupate. In a fourth experiment, glucose was added to shaded microcosms to stimulate bacterial activity in the absence of algae. Bacterial growth rates were 2 to 3 times higher, and larval development was enhanced in glucose-amended treatments. However, pupation rates and adult weights in glucose-amended shaded microcosms were still poor compared with those in nonamended sunlit microcosms. Overall, these results demonstrate the importance of algal biomass in the surface microlayers of larval habitats to development and adult production of An. gambiae.

Mutational Analysis of the ompA Promoter from Flavobacterium johnsoniae

Sequences that mediate the initiation of transcription in Flavobacterium species are not well known. The majority of identified Flavobacterium promoter elements show homology to those of other members of the phylum Bacteroidetes, but not of proteobacteria, and they function poorly in Escherichia coli. In order to analyze the Flavobacterium promoter structure systematically, we investigated the -33 consensus element, -7 consensus element, and spacer length of the Flavobacterium ompA promoter by measuring the effects of site-directed mutations on promoter activity. The nonconserved sequences in the spacer region and in regions close to the consensus motifs were randomized in order to determine their importance for promoter activity. Most of the base substitutions in these regions caused large decreases in promoter activity. The optimal -33/-7 motifs (TTTG/TANNTTTG) were identical to Bacteroides fragilis sigma(ABfr) consensus -33/-7 promoter elements but lacked similarity to the E. coli sigma(70) promoter elements. The length of the spacer separating the -33 and -7 motifs of the ompA promoter also had a pronounced effect on promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, the GC content of the core promoter sequences had a pronounced effect on Flavobacterium promoter activity. This information was used to conduct a scan of the Flavobacterium johnsoniae and B. fragilis genomes for putative promoters, resulting in 188 hits in B. fragilis and 109 hits in F. johnsoniae.

Characterization of Strong Promoters from an Environmental Flavobacterium hibernum Strain by Using a Green Fluorescent Protein-Based Reporter System

ABSTRACT We developed techniques for the genetic manipulation of Flavobacterium species and used it to characterize several promoters found in these bacteria. Our studies utilized Flavobacterium hibernum strain W22, an environmental strain we isolated from tree hole habitats of mosquito larvae. Plasmids from F. hibernum strain W22 were more efficiently (∼1,250-fold) transferred by electroporation into F. hibernum strain W22 than those isolated from Escherichia coli, thus indicating that an efficient restriction barrier exists between these species. The strong promoter, tac, functional in proteobacteria, did not function in Flavobacterium strains. Therefore, a promoter-trap plasmid, pSCH03, containing a promoterless gfpmut3 gene was constructed. A library of 9,000 clones containing chromosomal fragments of F. hibernum strain W22 in pSCH03 was screened for their ability to drive expression of the promoterless gfpmut3 gene. Twenty strong promoters were used for further study. The transcription start points were determined from seven promoter clones by the 5′ rapid amplification of cDNA ends technique. Promoter consensus sequences from Flavobacterium were identified as TAnnTTTG and TTG, where n is any nucleotide, centered approximately 7 and 33 bp upstream of the transcription start site, respectively. A putative novel ribosome binding site consensus sequence is proposed as TAAAA by aligning the 20-bp regions upstream of the translational start site in 25 genes. Our primary results demonstrate that at least some promoter and ribosome binding site motifs of Flavobacterium strains are unusual within the bacterial domain and suggest an early evolutionary divergence of this bacterial group. The techniques presented here allow for more detailed genetics-based studies and analyses of Flavobacterium species in the environment.