Isolation of thermotolerant M. anisopliaeand determination of growth kinetics
The details of how the Evolugator™ achieves continuous culture and the inherent advantages of this technology over other methods of continuous culture are extensively described elsewhere . However, since the technology is so new, it is important to summarize how it works. Briefly, directed selection occurs inside a growth chamber made of 100% silicone tubing (12.7 mm external diameter and 9.5 mm internal diameter, Saint Gobain, France) that is flexible, transparent and gas-permeable. The tubing is filled with growth medium and sterilized prior to mounting into the Evolugator™, where it is subdivided using "gates", which are clamps that prevent the flow of medium and cultured organisms from one subdivision to the next. Between the central gates is the "growth chamber", which has a volume of ~10.8 mL. Oxygenation of the growth chamber is augmented beyond the permeability of the tubing by maintaining a 1.8 mL (± 5%) bubble of filtered air in the growth chamber. Cultures are inoculated into the growth chamber through the tubing using sterilized syringes. The growth medium and the inner surface of the tubing are static with respect to each other, and both are regularly and simultaneously replaced by peristaltic movement of the tubing through the gates. A fresh air bubble is delivered with each dilution cycle by movement of air in predetermined volumes through the unused portion of media upstream of the growth chamber.
In summary, the gates are periodically released allowing unused medium to mix with saturated culture. The tubing is then moved and the gates reclosed – essentially, the majority of the medium and growth chamber are entirely replaced during every dilution cycle. In the "new growth chamber", culture is diluted with unused medium. The "old' growth chamber is now what is called the "sampling chamber" from which samples can be extracted by syringe without fear of contaminating the "new growth chamber".
Dilutions were conducted automatically and controlled through specifically designed software. Dilution can be initiated at a certain cycle duration (chemostat mode), when the culture attains a certain OD (turbidostat mode) or a combination of both. Two turbidimeters (λ = 680 nm, power = 0.7 V) (EFS, Montagny, France) measure the optical density and are zeroed with unused growth medium prior to each experiment.
Since filamentous fungi adhere to solid surfaces, they grow along the inner surface of the "growth chamber". Since the cells from the previous cycle adhere closest to the gate separating the "sampling chamber" and the "new growth chamber", dividing cells will grow along the fresh chamber surface towards the gate separating the "new growth chamber" from unspent medium. Consequently, the cells that reach this gate by growing along the surface are the most recent (and presumably most fit) additions to the population and will be retained in the active culture when the tubing moves again to achieve the next dilution.
For directed evolution of M. anisopliae, the tubing was filled with Sabouraud dextrose (SAB) media and autoclaved prior to use. 2 mL of a growing culture of M. anisopliae 2575 grown in SAB was injected into the first section of the growth chamber and dilution cycles were initiated as described. Temperature was monitored using a PT100 probe (IEC/Din Class A) and regulated via a Proportional Integral & Derivative controller (West P6100). Growth kinetics were determined using a Bioscreen C plate reader system (Growth Curves USA, Piscataway, NJ) in multiple volumes of 250–300 μL. Aliquots of growing cultures were mounted on slides and examined using a PASCAL LSM5 confocal microscope fitted with Nomarski differential interference contrast (DIC) optics.
Identification of recovered adapted fungal strains
Single isolated fungal colonies (corresponding to EVG016, EVG017, and EVG017g) were re-streaked onto fresh Potato dextrose agar plates and used for identification purposes. Fungal identity was confirmed by PCR amplification and sequencing of a portion of the 5.8 S rRNA with its flanking internal transcribed spacer sequences (ITS) and the M. anisopliae specific protease Pr1 as described [35, 36]. Primer pairs used were: (1) ITS5; 5'-gcaagtaaaagtcgtaacaagg, and ITS4; 5'-tcctccgcttattgatatgc-3' and (2) Pr1f, 5'-gccgacttcgtttacgagcac, and Pr1r, 5'-ggaggcctcaataccagtgtc. Genomic DNA was isolated using the Qiagen DNeasy Plant mini-extraction kit according to the manufacturer's protocols (Qiagen Inc., Valencia, CA). PCR reactions were performed using ExTaq DNA polymerase (Takara Corp., obtained from Fisher Sci, Pittsburgh, PA). PCR products were cloned into the pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA) according to the manufacturer's protocols. Plasmid inserts were sequenced at the University of Florida, ICBR, Sequencing Facility.
Evaluation of conidial production and production of conidia for bioassays
Conidia of M. anisopliae Strain ARSEF2575 and the two temperature tolerant clones, EVG016 and EVG017, were produced in a biphasic system by ARS at Sidney MT following the methods of Bradley et al 1992. In brief, conidia from agar media were used to inoculate flasks of liquid medium (40 g L-1 glucose, 20 g L-1 yeast extract, 15 ml L-1 corn steep liquor). The liquid cultures were incubated for 3–4 d at 25–26°C and 150 rpm and then used to inoculate flaked barley (Minnesota Grain, Eagan MN) that had been premoistened with reverse osmosis water (3:5 v:w), and autoclaved (103 KPa for 30 min/kg) in vented, plastic mushroom spawn bags (SacO2, Microsac, Belgium). The liquid cultures were mixed with the substrate within the bags under aseptic conditions, at a ratio of 3:10 (v/w) and the open ends of the bags were heat-sealed. The solid substrate fermentation phase proceeded at 26–27°C in constant darkness. Cultures were observed daily and crumbled by hand within spawn bags as needed to prevent binding of the substrate and provide aeration throughout the culture substrate. After 8–10 days the whole cultures were then transferred to kraft paper bags in which they were dried for 10 days at 23–25°C, to a final moisture of < 0.4 Water Activity Units (< 6% gravimetric moisture). Conidia were harvested by mechanical sieving through 20-and 100-mesh sieves under standardized conditions in an ultrasonic sieve shaker (AS200, Retsch Corp., Newton PA) with the conidial fractions < 0.150 mm (100 U.S. Mesh) retained for yield determination and use in bioassays. After conidia were harvested, the mass of harvested conidia and conidial counts were used to calculate yield per kg of dry barley. Three replicate batches of 500 g dry barley were used for each strain as described above. For conidial counts two replicate samples of 0.1 g of harvested conidia were suspended in 0.1% Silwet L77™ (Loveland Chemicals), serially diluted with water as appropriate and counted using an Improved Neubauer hemocytometer under 400× phase contrast microscopy. All conidial preparations were stored at 3°C until use.
Prior to use in bioassays, conidial viabilities were determined by plating dilute aqueous suspensions of each technical powder onto potato dextrose agar, incubating at 27–28°C for 16–19 hr, and then examining the conidia with 400× phase contrast microscopy. A preliminary step to determine fungal viability was performed, in which a small quantity of dry conidia was exposed to 100% relative humidity for 1–2 hr before suspension and plating. A minimum of 400 conidia were examined for germination; a conidium was considered viable (germinated) if it had produced a visible germination peg during the specified incubation time. Viabilities of the two M. anisopliae technical powders were 90 and 92% for 2575 and EVG016, respectively. Because the conidial production of the original EVG017 was insufficient to provide enough conidia for bioassay, the clone was passed through adult Melanoplus sanguinipes, reisolated from a single colony and grown for two cycles on agar media. The passaged fungus, EVG017g, regained ability to sporulate on Sabouraud dextrose agar supplemented with 0.1% yeast extract (SDAY) and Potato dextrose agar (PDA), as well as in solid substrate fermentation and was used to evaluate conidial production and to produce conidia for bioassays as described above. Conidial viability of the conidia powder used for bioassays was 88%.
For bioassays, the dry conidial powders were first formulated in culinary canola oil with the final titers determined by hemocytometer counts of serial dilutions made with kerosene. To determine the relative infectivity (median infectious dose) and virulence (median and average lethal times of one selected dose), a series of conidial suspensions in canola were prepared with the actual spore concentrations determined by hemocytometer count and adjusted for conidial viability. One week old, adult M. sanguinipes from a nondiapausing laboratory colony were used in all bioassays. A topical, 5-dose bioassay with adult Melanoplus sanguinipes was conducted with doses bracketing the estimated LD50 for each strain, based on a preliminary bioassay. In the bioassay a one μl droplet of spores in oil was applied to the front left coxa of each insect, with 20–25 insects per dose. Dosed insects and controls incubated at 28°C in cylindrical cellulose acetate cages (50 cm × 10 cm) with mesh covered openings. Bioassays were replicated twice in their entirety, with a total of 120–150 insect per bioassay. Day 7 mortalities were used to calculate LD50 and associated statistics by probit analysis with Polo-Plus™ (LeOra Software). Median and average survival times were calculated using Kaplan Meier survivorship statistics  with KMSsurv.exe . The replicate tests for each fungus were first compared, and, being not significantly different were combined for further analysis.
To determine the heat tolerance of the parent and both mutant strains in vivo, adult M. sanguinipes were dosed topically at the ~LD95 for each clone or parent and incubated at 36 ± 0.5°C or 28 ± 0.5°C. Daily mortality was recorded for 14 days. Temperatures were monitored continuously by means of a Hobo® temperature logger placed in an empty grasshopper container, which was positioned amidst the grasshopper containers. All assays were replicated twice in their entirety. Median and average lethal times were calculated as described above. The ST50 for each strain was calculated as described above for the 28°C treatments; this parameter could not be calculated for the higher temperature because of the low mortalities. Insect body temperatures were monitored with a thermocouple inserted into a thermal surrogate placed within an empty cage along with the cages with grasshoppers, a technique that has been shown to accurately reflect grasshopper body temperatures during both normal and thermoregulatory behavior [39, 40].
Conidial germination and radial growth studies of the three fungi were conducted in parallel with the insect bioassays to better understand the assay mortalities. The reisolated EVG017g was evaluated along with the original ARSEF2575 and EVG016 fungi. Conidial germination tests were conducted as the viability determinations described earlier but parallel plates were incubated at 36.5°C as well as 28°C and conidia examined at 18, 24, 48, 72 and 96 hr. After the last observation the Petri plates of fungi incubated at 36.5°C were transferred to 28°C and observed daily for another 2–3 days.
Radial growth tests were carried out in several ways. Replicate 60 mm Petri plates of SDAY agar were point-inoculated with the fungi, and initially incubated at 36.5°C. for 4 days, then half of the samples were transferred to 28°C for further observation. Other plates were incubated at 28°C until the colonies were 3–4 mm in diameter at which time one half were transferred to 36.5°C and subsequent radial growth monitored for up to 18 days. In all cases, colony radii were measured daily across two perpendicular axes with a digital Vernier caliper. There were three replicate plates for each treatment.