s2fieldsofstudy:
Biology
Agricultural And Food Sciences

text:
Development of a Microbial-Based Integrated Pest Management Program for Helicoverpa spp. (Lepidoptera: Noctuidae) and Beneficial Insects on Conventional Cotton Crops in Australia

Entomopathogenic fungi, when used as a microbial control agent against cotton pests, such as Helicoverpa spp., may have the potential to establish and spread in the environment and to have an impact on both pests and beneficial insects. Information on the effect of entomopathogenic fungi on pests and beneficial insects is crucial for a product to be registered as a biopesticide. The effect of the entomopathogenic fungus BC 639 (Aspergillus sp.) against Helicoverpa spp. and beneficial insects (mostly predatory insects) was studied in the laboratory and in cotton field trials. The results show that when Helicoverpa spp. second instar larvae were exposed to increasing concentrations (from 102 to 109) of the entomopathogenic fungus BC 639, the optimum dose required to kill over 50% of the insects was 1.0 × 107 spores/mL. In the field trials, the number of Helicoverpa spp. per metre on plots treated with 1.0 or 0.50 L/ha of BC 639 was the same as on plots treated with the recommended rate of the commercial insecticide, Indoxacarb. However, when plots were treated with 0.25 L/ha of BC 639, this was not as effective at controlling Helicoverpa spp. as 1.0 or 0.5 L/ha BC 639 or Indoxacarb. BC 639 had less effect on predatory insects when applied at lower rates (0.50 and 0.25 L/ha) than at higher rates (1.0 L/ha). Thus, BC 639 was more selective against predators when applied at lower rates than at the higher rate, but was also more selective than Indoxacarb. Thus, the ability of BC 639 to control Helicoverpa spp. effectively with a minimal effect on predatory insects indicates its potential for enhancing integrated pest management programs and to sustain cotton production.

Introduction
Cotton crops in Australia are attacked by a wide range of pests, the major ones being the two lepidopteran pests, Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) [1]. These two pests are responsible for the use of synthetic insecticides in the Australian cotton industry from the 1960s until 1996-1997 [2]. In 1996, transgenic cotton containing genes from Bacillus thuringiensis (Bt) were introduced and then taken up by 90% of Australian cotton farmers. This cotton expresses the toxins Cry1Ac (Ingard ® ) (in transgenic cotton from 1996) and Cry 1Ac + Cry2Ab (Bollgard II ® ) (in transgenic cotton from 2004). The use of transgenic cotton has reduced the impact of the major pest (Helicoverpa spp.) on cotton crops in Australia [3][4][5][6]. These transgenic cotton crops are toxic to Helicoverpa spp. and other lepidopteran pests when ingested [7]. Thus, the introduction of transgenic cotton crops has decreased the use of synthetic insecticides against Helicoverpa spp. by 75%-80% [6]. In contrast, 10% of cotton farmers in Australia still grow non-transgenic cotton and rely exclusively on the use of synthetic insecticides to manage Helicoverpa spp. For these non-transgenic crops, there is a need to control Helicoverpa spp. with synthetic insecticides early in the season, which inadvertently suppresses populations of beneficial insects and potentially causes outbreaks of secondary pests, such as green mirids (Creontiades dilutus), cotton aphids (Aphis gossypii) and green vegetable bugs (Nezara viridula). The negative impact of this practice was of great concern to the cotton industry [6][7][8] until the adoption of transgenic (Bt) cotton crops.
Many beneficial insects, particularly predatory insects and specialist parasitoids, have been recorded in cotton crops worldwide. The role of beneficial insects in regulating pests in cotton agro-ecosystems has become increasingly important [9][10][11][12][13]. Although these beneficial insects do not show tightly coupled dynamics with their prey, because their population dynamics are not solely dependent on target pests, they often provide effective control, particularly in agro-ecosystems with non-chemical insecticide regimes [10,11,[14][15][16].
In Australia, cotton crops are often grown in remote areas that have very sparse natural vegetation [11,14] and that lay fallow for most of the year. With no natural refuges and food sources for the adult natural enemies of pests, beneficial insect populations decline quickly, thereby limiting natural biological control [11,14,17]. Presently, cotton farmers in Australia are inadvertently discriminating against the natural enemies of Helicoverpa spp. and other pests by exclusively using synthetic insecticides on monocultured crops to control Helicoverpa spp. on conventional cotton and to control sucking pests on transgenic (Bt) cotton.
Helicoverpa spp. are difficult to manage on conventional cotton crops: first, because of the lack of diversity and stability in the cotton agro-ecosystem; second, because Helicoverpa spp. are highly mobile and highly migratory and can rapidly infest cotton crops; and third, because their eggs can escape natural enemy attack, as the natural enemies may not be present or established in sufficiently high numbers [2,11,14,18,19]. These factors have encouraged the use of synthetic broad-spectrum insecticides.
As a result, cotton farmers require new pest control methods to manage Helicoverpa spp. and to complement integrated pest management (IPM) on conventional cotton crops. Entomopathogenic fungi are known to be important natural enemies of many pests of agricultural crops [20,21]. Over 750 different species of fungi have been identified to date; these are cosmopolitan organisms that have been isolated from soils and have infected insects around the world [22][23][24]. All stages of insects, from the egg, larva or nymph to adult, can be killed by fungal pathogens. Fungi, unlike other pathogens, such as viruses and bacteria, do not need to be ingested to infect and kill the host. The advantage of using fungi for biological control is that the fungi are self-generating within or on the surface of the plant and can potentially provide ongoing protection as the plant grows, with little effect on non-herbivorous predators [5]. Several species of entomopathogenic fungi have been developed as alternatives to synthetic insecticides because of the low risk to humans, low risk to the environment and low pest resistance [22]. However, in Australia, only two isolates of Metarhizium anisopliae are commercially available, for the control of plague locusts and sugar cane grubs.
The present study evaluated the efficacy of a naturally occurring entomopathogenic fungus, BC 639 (Aspergillus sp.), against Helicoverpa spp. populations and on retaining populations of beneficial insects (e.g., predatory beetles, predatory bugs, predatory lacewings and spiders) on commercial conventional cotton crops. The study objectives were: (1) to assess the dose-response of the BC 639 fungus on Helicoverpa spp. larvae; (2) to assess the efficacy of different application rates of the BC 639 formulated product; (3) to identify the optimum rate of the BC 639 formulated product needed to control Helicoverpa spp. populations; and (4) to assess the impact of the BC 639 formulated product on the natural enemies of the target pests.

Isolation and Formulation of the Entomopathogenic Fungus BC 639
The BC 639 fungus was originally isolated from an infected insect in a commercial cotton farm [13]. The methods of Bahar et al. [25] and Mensah and Austin [13] were followed for isolating the spores for culturing and for preparing the formulation. Using a haemocytometer, the spore concentration was estimated to be 1.0 u 10 7 . The conidial germination was determined from 100 spore counts, and 100% germination was observed [5].

Experiment 1: Dose-Response of Helicoverpa spp. Second Instar Larvae in the Laboratory to BC 639
The experiment was conducted in the laboratory at the Australian Cotton Research Institute in Narrabri, NSW, Australia, from 10 to 28 February 2006. Helicoverpa armigera larvae were cultured in the laboratory and used in the study at the second instar stage.
BC 639 was tested at 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 8 and 10 9 spores/mL. For each treatment dose, 10 H. armigera second instar larvae were placed in a 90-mm Petri dish lined with filter paper and sprayed separately. The larvae from each treatment were then each transferred into separate 45-mm Petri dishes containing a fresh 19-mm conventional cotton leaf disc on filter paper. The Petri dishes were sealed and placed in an incubator set at 26-28 °C. Therefore, each treatment had 10 larvae placed individually in 10 Petri dishes.
The larvae were checked on 1, 2 and 3 days after treatment (DAT), and the number of dead and alive larvae were recorded. The mortality for each treatment was calculated, and a dose-response curve was developed for the concentration of BC 639 (log spores/mL) per mortality (%) of Helicoverpa spp. using GraphPad Prism 6 (GraphPad Software, Inc. San Diego, CA, USA). The number of spores/mL required to cause 50% mortality was extrapolated from the dose-response curve.

Experiment 2: Efficacy of BC 639 against Helicoverpa spp. First and Second Instar Larvae in the Laboratory
The experiment was conducted in the laboratory at the Australian Cotton Research Institute in Narrabri, NSW, Australia. Helicoverpa armigera first and second instar larvae were used for the experiment. The spore concentration used for this study was 1.0 u 10 7 . The conidial germination was 100%.
BC 639 was formulated into an oil-based product by Becker Underwood Pty. Ltd., Australia. The product was evaluated at 1% v/v (1.0 L/ha), and water was used as the control treatment. First instar larvae were used in the first experiment, and second instar larvae were used in the second experiment. For each experiment and each treatment, one larva per plant was placed on a cotton leaf and sprayed until run-off. Each treatment used 40 cotton plants infested with 40 larvae. After spraying, each larvae was transferred into a 35-mL clear plastic container (P10M; Solo, Urbana, IL, USA) containing a soybean-based artificial diet. The number of dead larvae were counted and recorded daily until all of the larvae had pupated. The mortality (%) was calculated for each treatment.

Experiment 3: Efficacy of Different Rates of BC 639 on the Survival of Helicoverpa spp. and Beneficial Insects on Commercial Cotton Crops
The trial was conducted on a dryland, commercial, conventional cotton farm at Getta Getta near Goondiwindi in the Macintyre Valley in Queensland in the 2006-2007 growing season.
The first foliar treatment was applied on 31 January 2007 and the second on 25 February 2007. Foliar treatments were applied using a ground rig sprayer fitted with flat fan nozzles (3 nozzles/row of cotton) to achieve a droplet size of 200 µm and an application volume of 100 L/ha. The treatment was applied in the morning when the temperature was between 20 °C and 28 °C. The decision for when to apply the treatment was based on the IPM Guidelines and the economic threshold of 2.0 larvae per metre as recommended by CottonLogic [1].
Visual counts of Helicoverpa spp. eggs and larvae, predatory beetles, bugs, lacewings and spiders on cotton plants for each treatment were made at approximately weekly intervals. For each of the six treatment replicates, a randomly selected 1-m length of planted row was examined. The number of Helicoverpa spp., predatory beetles, predatory bugs, predatory lacewings and spiders per metre for each treatment was determined.

Experiment 4: Efficacy of Different Rates of BC 639 against Helicoverpa spp. and Beneficial Insects on Conventional Cotton Crops at Norwood in 2007-2008
The trial was conducted on a commercial conventional cotton farm at Norwood, near Moree. The trial was conducted from 16 November 2007 to 11 March 2008.
The following treatments were evaluated for their effect on Helicoverpa spp. eggs, very small and small, medium and large larvae and beneficial insects: (1) 1.0 L/ha BC 639; (2) 0.50 L/ha BC 639; (3) 0.25 L/ha BC 639 (4) 0.85 L/ha Indoxacarb; and (5) unsprayed (untreated) control. The treatment plots were arranged in a randomized complete block design with four replicates per treatment. Each replicated plot measured 40-m wide (equivalent to the number of rows) and 90-m long. A 40-m wide buffer separated each of the Indoxacarb-treated plots, fungus plots and unsprayed plots.
The number of insects were counted 24 h before treatment and then 3, 7 and 14 DAT for each spray application. Foliar treatments were applied on 30 January 2008 (first spray) and 25 February 2008 (second spray) using a ground rig sprayer fitted with flat fan nozzles (3 nozzles/row of cotton plants) to achieve a droplet size of 200 µm and an application volume of 100 L/ha. The treatments were applied in the morning when the temperature was between 20 °C and 28 °C. On each occasion, the treatments were applied using 100 L of water per hectare. The control plot was unsprayed.
Visual counts of Helicoverpa spp. eggs and larvae, predatory beetles, bugs, lacewings and spiders for each treatment were made at approximately weekly intervals. For each of the four treatment replicates, a randomly selected 1-m length of planted row was examined. The number of Helicoverpa spp., predatory beetles, predatory bugs, predatory lacewings and spiders per metre for each treatment was determined.

Data Analysis
All experimental data were analysed using repeated measures analysis of variance (v. 2.03; Graphpad Instat and Prism Software Inc., San Diego, CA, USA). Treatments and sample dates were the independent variables. Tukey-Kramer multiple comparison tests were used to compare the treatment means.

Experiment 1: Dose-Response of Helicoverpa spp. Second Instar Larvae to BC 639 in the Laboratory at the Australian Cotton Research Institute
Exposure to progressively increasing concentrations of BC 639 from 10 2 to 10 9 spores/mL resulted in the deaths of Helicoverpa spp. second instar larvae ( Figure 1). The optimum dose required to kill over 50% of the insects intercepts the curve at 10 7 and was estimated to be 1.0 u 10 7 spores/mL ( Figure 1).

Experiment 2: Efficacy of BC 639 against Helicoverpa spp. First and Second Instar Larvae in the Laboratory
BC 639 caused 80% and 82% mortality of H. armigera first and second instar larvae, respectively, and was significantly different from the control (water)-treated larvae with 5% and 7.5% mortality, respectively (Table 1).

Helicoverpa spp. Eggs
After the first spraying, oviposition by Helicoverpa spp. was the same among treated and control plots at 3 DAT, but was significantly lower (p < 0.003) on the BC 639-and Indoxacarb-treated plots than the unsprayed plots at seven and 14 DAT (Figure 2A). No significant difference (p > 0.05) was detected after the first spraying among cotton plants treated with different rates of BC 639 and Indoxacarb (Figure 2A).
After the second spraying, the number of Helicoverpa spp. eggs per metre on plots treated with BC 639 and 0.85 L/ha Indoxacarb was the same at 3, 7 and 14 DAT, but they were significantly lower (p < 0.02) than the unsprayed plots at three DAT ( Figure 2A). Overall, there was no strong evidence that application of the fungus deterred egg laying by Helicoverpa spp. on treated cotton plants ( Figure 2A).

Helicoverpa spp. Very Small + Small (First to Third) Instar Larvae
Significant differences (p < 0.05) in the number of first and second instar larvae per metre were found among treatments at seven DAT ( Figure 2B). Plots treated with 1.0 L/ha, 0.50 L/ha and 0.85 L/ha Indoxacarb recorded the lowest number of very small and small (VS + S) (first to third) instar larvae per metre after the first spraying ( Figure 2B). No significant differences were detected between plots treated with 0.25 L/ha BC 639 and the control (unsprayed) plots after the first spraying ( Figure 2B). The numbers of VS + S instar larvae per metre recorded on plots treated with 1.0 L/ha and 0.50 L/ha BC 639 was the same as plots treated with Indoxacarb after the first spraying. At 14 days after the first spraying, the number of VS + S larvae per metre was the same among treated and control plots ( Figure 2B). In contrast, no significant differences were detected between the BC 639-treated plots and the control (unsprayed) plots ( Figure 2B).

Helicoverpa spp. Medium + Large (Fourth to Sixth) Instar Larvae
Following the first spraying, the number of medium + large (M + L) larvae was significantly lower (p < 0.01) on 1.0 L/ha and 0.50 L/ha BC 639-and 0.85 L/ha Indoxacarb-treated plots than plots treated with 0.25 L/ha BC 639 or the control (unsprayed) plots ( Figure 2C). The number of M + L larvae per metre recorded on plots treated with 0.25 L/ha BC 639 was the same (p > 0.05) as the control (unsprayed) plots ( Figure 2C), indicating that the 0.25 L/ha rate of BC 639 was not effective against Helicoverpa spp.
Following the second spraying, the same number of M + L larvae per metre (p > 0.05) was found among treatments at three and 14 DAT ( Figure 2C). At seven DAT, however, the number of M + L recorded on the unsprayed plots was significantly higher (p < 0.03) than the BC 639-and Indoxacarb-treated plots. In contrast, no significant difference (p > 0.05) in the number of M + L larvae was found on 1.0 L/ha 0.50 and 0.25 L/ha BC 639-treated plots, Indoxacarb-treated plots and the unsprayed plots at 14 DAT ( Figure 2C).

Beneficial Insects
Beneficial insects found at the study site were mainly predatory insects ( Table 2). Predators of Helicoverpa spp. identified from the treated plots were predatory beetles (only three species were predominant in the study site), bugs, lacewings and spiders ( Table 2).

Predatory Beetles
Following the first spraying on 31 January 2007, the number of predatory beetles per metre on the plots treated with 0.5 and 0.25 L/ha BC 639 (lower rates) was not significantly different (p > 0.05) from the unsprayed (control) plots, although plots treated with 1.0 L/ha BC 639 (higher rate) and Indoxacarb were significantly lower (p < 0.01) than the unsprayed plots at 3, 7 and 14 DAT ( Figure 3A). The number of predatory beetles per metre on plots treated with 1.0 L/ha BC 639 was less than that on unsprayed plots (control), there were significantly more (p < 0.01) than on the Indoxacarb-treated plots ( Figure 3A).
After the second spray, the number of predatory beetles per metre on the BC 639-treated plots was identical (p > 0.05) to the unsprayed plots at three and seven DAT, but was significantly lower (p < 0.01) than the unsprayed plots at 14 DAT ( Figure 3A). In contrast, the number of predatory beetles per metre on the Indoxacarb-treated plots was significantly lower (p < 0.001) than the unsprayed plots at 3, 7 and 14 DAT ( Figure 3A).

Predatory Bugs
Following the first spraying, the number of predatory bugs per metre on 1.0 and 0.5 L/ha BC 639-treated plots and Indoxacarb-treated plots was the same, but it was significantly lower (p < 0.01) than the 0.25 L/ha BC 639 and unsprayed (control) plots at three DAT ( Figure 3B). However, at seven and 14 DAT, the number of predatory bugs per metre on the BC 639-treated plots (all rates) was the same as the unsprayed plots, but significantly higher (p < 0.001) than the Indoxacarb-treated plots ( Figure 3B). The Indoxacarb-treated plots consistently showed fewer predatory bugs per metre after the first spraying ( Figure 3B).
Following the second spraying, the number of predatory bugs per metre was not significantly different (p > 0.05) between the BC 639-treated plots, Indoxacarb-treated plots and the unsprayed plots ( Figure 3B). There could have been an influx of bugs from the unsprayed and BC 639-treated plots into the Indoxacarb-treated plots or the effect could have been due to spray drift.

Predatory Lacewings
The predominant predatory lacewings identified from the study plots were Chrysopa spp. and Micromus tasmaniae ( Table 2). Following the first spraying, the number of predatory lacewings per metre on plots treated with 1.0 L/ha BC 639 and Indoxacarb was the same, but it was lower than 0.50 and 0.25 L/ha BC 639 and the unsprayed plots at 3 DAT (p < 0.0001), 7 DAT (p < 0.001), 14 DAT (p < 0.01) and 25 DAT (p < 0.001 ( Figure 3C)). In contrast, plots treated with 0.50 and 0.25 L/ha BC 639 had the same number of predatory lacewings as the unsprayed plots ( Figure 3C).  Following the second spraying, the number of lacewings per metre in plots treated with all rates of BC 639 and the unsprayed plots was the same at three DAT, but was significantly different (p < 0.01) from the Indoxacarb-treated plots ( Figure 3C). At seven DAT, the number of predatory lacewings found on 0.5 and 0.25 L/ha and the unsprayed plots was the same (p > 0.05), and this was significantly different from the 1.0 L/ha (p < 0.01) and Indoxacarb-treated plots (p < 0.001) ( Figure 3C). No significant difference (p > 0.05) was recorded among the BC 639 rates, Indoxacarb and the unsprayed plots at 14 DAT ( Figure 3C).

Spiders
The spiders identified from the study plots were Lycosa spp., Oxyopes spp., Salticidae spp. and Araneus spp. ( Table 2).The number of spiders per metre on plots treated with the BC 639 rates and the unsprayed plots was the same following the first (31 January 2007) and second (25 February 2007) treatments ( Figure 3D). However, the number of spiders per metre on the Indoxacarb-treated plots following the first and second treatments was significantly lower (p < 0.01) than the BC 639 and unsprayed plots at seven DAT after the first and second spraying ( Figure 3D).

Helicoverpa spp. Eggs
The first spraying was applied when the density of Helicoverpa spp. eggs ranged from 18 to 28 eggs per metre ( Figure 4A). After the first spraying, the number of eggs recorded on plots treated with BC 639 rates, Indoxacarb and the unsprayed plots was the same at 3, 14 and 25 DAT (p > 0.05) ( Figure 4A). However, the number of Helicoverpa spp. eggs per metre recorded on plots treated with 1.0 and 0.5 L/ha BC 639 and Indoxacarb was significantly lower (p < 0.01) at seven DAT than plots treated with 0.25 L/ha and unsprayed plots ( Figure 4A). However, after the second spraying, the number of eggs per metre was the same among treated and unsprayed plots ( Figure 4A).

Helicoverpa Very Small and Small Larvae
After the first spraying, the plots treated with BC 639 rates and Indoxacarb had a significantly lower (p < 0.01) number of VS + S larvae at 3 (p < 0.0001), 7 (p < 0.003) and 14 DAT than the unsprayed plots ( Figure 4B). Similar results were found for plots treated with BC 639 rates, Indoxacarb and the unsprayed plots at 3, 7 and 14 DAT ( Figure 4B). After the second spray, the number of Helicoverpa VS + S larvae recorded on plots treated with BC 639 rates and Indoxacarb was the same and below the recommended threshold (two larvae per metre) used to manage these pests on conventional cotton crops [1]. and 14 DAT (p < 0.003) than the unsprayed plots ( Figure 4C). Similarly, the number of M + L larvae per metre on plots treated with BC 639 and Indoxacarb was the same after the second spraying ( Figure 4C). The unsprayed plots had the highest number of M + L larvae per metre at 3-14 DAT (3.50-3.75 per metre) ( Figure 4C).

Predatory Beetles
Predatory insects identified from the study plots are listed in Table 2. The predominant predatory beetles identified from the study plots were C. transversalis, D. notescens and D. bellulus (Table 2), and the number of the predatory beetles per metre is given in Figure 5A. No significant differences (p > 0.05) were found among BC 639 rates, Indoxacarb and the unsprayed plots at three and seven DAT after the first spraying ( Figure 5A). However, the number of predatory beetles per metre on the unsprayed plot (3.25 per metre) was significantly higher (p < 0.001) than that on the plots treated with BC 639 rates and Indoxacarb at 14 DAT ( Figure 5A). Similarly, no significant differences were recorded in the number of predatory beetles per metre among treatments at three and seven DAT, but there were significantly different (p < 0.01) numbers between the unsprayed plots, the plots treated with BC 639 and Indoxacarb at 14 DAT after the second spraying ( Figure 5A). The effect could have been a result of spray drift. The number of predatory beetles per metre on plots treated with Indoxacarb and BC 639 rates at 14 DAT was not significantly different (p > 0.05) ( Figure 5A).

Predatory Bugs
The predominant predatory bugs identified from the study plots are given ( Table 2). No significant differences (p > 0.05) were found among the unsprayed plots, BC 639-and Indoxacarb-treated plots after the treatments ( Figure 5B).

Predatory Lacewings
The predominant predatory lacewings identified from the study plots were Chrysopa spp. and Micromus tasmaniae (Table 2). No significant differences were found among BC 639-treated plots and the unsprayed plots after the first and second treatments ( Figure 5C). The Indoxacarb-treated plots consistently showed the lowest number of predatory lacewings per metre (about 50% lower than the BC 639-treated plots) throughout the study. However, it was only at three DAT since the first spray that the difference between the Indoxacarb-treated plots was significantly lower (p < 0.001) than the BC 639-treated plots and the unsprayed plots ( Figure 5C). This indicates that BC 639 had minimal impact on predatory lacewings compared with Indoxacarb ( Figure 5C).

Spiders
The predominant spiders identified from the study plots are given ( Figure 5D). No significant differences were found among the unsprayed plots, BC 639-and Indoxacarb-treated plots after the first and second treatments ( Figure 5D).

Discussion
The study has shown that the application of a formulated product of BC 639 entomopathogenic fungus effectively controlled populations of Helicoverpa spp. on commercial cotton crops with minimal or no effect against predators of Helicoverpa spp. One exception occurred when the product was applied at 1.0 L/ha (the highest rate in this study), and some negative effect was evident on predatory insects at the Getta Getta study site. In our study, the fungus killed Helicoverpa spp. larvae within 3-7 DAT. Application of BC 639 at 1.0 and 0.50 L/ha reduced the number of Helicoverpa spp. per metre, and these rates were consistently more effective than the lower 0.25 L/ha rate. Thus, the optimum effective rate of application of BC 639 against Helicoverpa spp. on cotton crops to reduce pest damage was 0.50 L/ha. Similar results were obtained when BC 639 was applied to Creontiades dilutus on commercial conventional cotton crops [15].
According to [5,25,26], entomopathogenic fungi, such as BC 639, can negatively affect the survival of soft-bodied beneficial insects. In the present study, application of BC 639 at 0.5 and 0.25 L/ha did not significantly affect predatory beetle population, but when BC 639 was applied at 1.0 L/ha, it affected the number of predatory beetles per metre, with a similar effect to that of Indoxacarb (see Figure 3A). Although the application of 1.0 L/ha BC 639 reduced the number of predatory beetles per metre, the negative effect of the 1.0 L/ha was significantly less than the negative effect of Indoxacarb. The present results also showed that following the first spraying, 1.0 and 0.50 L/ha BC 639 had a negative effect on the predatory bugs at three DAT, but the population recovered at seven and 14 DAT. In contrast, BC 639 applied at 0.25 L/ha had no effect on the predatory bugs. In the case of predatory lacewings, the 1.0 L/ha BC 639 had a similar negative effect on predatory lacewings as the Indoxacarb treatment. Furthermore, Indoxacarb was consistently disruptive to predatory bugs and spiders, whereas BC 639 had no effect on the spiders. These results suggest that the application of lower rates of BC 639 had a more selective effect on predatory beetles, bugs, lacewings and spiders than the Indoxacarb. Thus, the number of predatory insects recorded for the lower rates of BC 639-treated plots and the unsprayed plots was the same and significantly higher than the 1.0 L/ha BC 639 rate, which prevents us from speculating that BC 639 is essentially benign at all rates on soft-bodied insects. In the case of Indoxacarb, our study found that the selectivity of Indoxacarb only occurred in the treated plots that were adjacent to the unsprayed plots, indicating a possible influx of predators from the unsprayed plots to the Indoxacarb-treated plots.
Entomopathogenic fungi in general are known to be important natural enemies of many pests of agricultural crops [20] and have an advantage over synthetic insecticides in the conservation of beneficial insects [20]. The BC 639 fungus used in this study is self-generating on the surface of the plant and can potentially provide ongoing protection as the plant grows, with little effect on non-herbivorous predators [15,5]. Cotton ecosystems contain many strains of fungi, such as in the soil and on plant debris, but long-term research is required to identify potential fungi and to increase its pathogenicity and adaptability to a particular environment, so as to enhance its ability to infect and control insects on agricultural crops.
The selectivity of BC 639 against predatory insects indicates that this fungus has the potential to enhance conservational biological control, to support IPM and to reduce synthetic insecticide use on conventional cotton crops. IPM involves using all means of managing pest populations with the aim of reducing insecticide use while maintaining profitability, yield and fibre quality [15].
Similar to many other cotton production systems worldwide, the Australian cotton cropping system is strictly monoculture. This practice and the use of synthetic insecticides inadvertently discriminates against beneficial arthropods. Therefore, there is a need to use biological pesticides, such as lower rates of BC 639, to conserve beneficial insects and support IPM in cotton agro-ecosystems. By integrating BC 639 fungal biopesticide with beneficial insects, rather than relying on beneficial insects alone, we can effectively enhance the control of Helicoverpa spp. in monoculture cropping systems, such as cotton. This is because Helicoverpa spp. are highly migratory and can quickly infest cotton crops and lay their eggs. Such behaviour by Helicoverpa spp. means that by the time beneficial insects arrive and establish in the cotton crops, the pest might have developed into medium and large larvae (fourth to sixth instar), which are too large for the beneficial insects (mostly predators) to feed on and, thus, to control. Hence, beneficial insects (particularly predatory insects) need to be well established in the cotton crops prior to the arrival of the pest (Helicoverpa spp.) to enable a sufficiently rapid response for population control. Thus, an IPM strategy developed against Helicoverpa spp. should involve tools and strategies that enhance the control of these pests; these could include fungal biopesticides, such as BC 639, which is selective against beneficial insects.
An IPM program for large-, medium-and small-scale monoculture crops, such as cotton, should develop in a stepwise progression that initially involves strategies to establish beneficial insects in the cotton field [13], followed by integration with IPM-compatible tools, such as the BC 639 entomopathogenic fungus that controls the target pest, but is selective for beneficial insects. Intervention with broad-spectrum synthetic pesticides should be used as a last resort when pests exceed thresholds and when no other effective selective management options are available.
The success of IPM programs in many cropping systems, especially conventional cotton systems, will depend on the availability of IPM-compatible tools and strategies to farmers. To adopt a true IPM program, farmers will need alternative pest control tools that have a minimal impact on the beneficial insects, whatever crop is grown. For cotton farmers in Australia and other parts of the world, the increase in Helicoverpa spp. populations on conventional cotton crops, yield loss, high cost of cotton production, insecticide resistance, disruption of beneficial species and environmental impacts are considered to be the major factors influencing the implementation of a sustainable IPM program for cotton crops. Thus, it is crucial that research continues to identify and develop new IPM-compatible tools and alternative strategies for managing and controlling Helicoverpa spp. and other pests [6,12,13]. Hence, the use of biological pesticides, including lower rates of BC 639 entomopathogenic fungus, is very important to support IPM in cotton agro-ecosystems.
The microbial biopesticide BC 639 is selective at lower application rates of 0.5 and 0.25 L/ha against predatory insects and has been used to effectively control Helicoverpa spp. on commercial cotton crops.

Conclusions
Application of a formulated entomopathogenic fungus (Aspergillus sp. BC 639) effectively controlled populations of Helicoverpa spp. on commercial cotton crops with minimal or no effect against predators of Helicoverpa spp. when the product was applied at 0.5 and 0.25 L/ha (the lower rates in this study). The fungus killed Helicoverpa spp. larvae within 3-7 DAT. Application of BC 639 at 1.0 and 0.50 L/ha was more effective than applications at the lower rate of 0.25 L/ha. The 1.0 and 0.50 L/ha rates of BC 639 had a similar efficacy against Helicoverpa spp. larvae as the 0.85 L/ha treatment with the commercial conventional synthetic insecticide Indoxacarb. This result suggests that the optimum effective rate for the application of BC 639 to control Helicoverpa spp. infestations on cotton crops was 0.50 L/ha. The microbial biopesticide BC 639 was selective against predatory insects at lower rates, and it can effectively control Helicoverpa spp. on commercial cotton crops and, thus, has a great potential to complement IPM programs in cotton crops.


extfieldsofstudy: Biology Medicine

text:
Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms

Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, GltPh, in several different conformational states have revealed that the protein structure is asymmetric in the outward- and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called “repeat-swap modeling” to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeat-swap homology modeling protocol, and calibrate the accuracy of the method using GltPh, for which both inward- and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of GltPh. The repeat-swapped model of VcCNT predicts that nucleoside transport also occurs via an elevator-like mechanism.

Secondary active transporters are critical for neurotransmitter clearance and recycling during synaptic transmission and uptake of nutrients. These proteins mediate the movement of solutes against their concentration gradients, by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously. Structures of a bacterial homolog of neuronal glutamate transporters, Glt Ph , in several different conformational states have revealed that the protein structure is asymmetric in the outward-and inward-open states, and that the conformational change connecting them involves a elevator-like movement of a substrate binding domain across the membrane. The structural asymmetry is created by inverted-topology repeats, i.e., structural repeats with similar overall folds whose transmembrane topologies are related to each other by two-fold pseudo-symmetry around an axis parallel to the membrane plane. Inverted repeats have been found in around three-quarters of secondary transporter folds. Moreover, the (a)symmetry of these systems has been successfully used as a bioinformatic tool, called "repeat-swap modeling" to predict structural models of a transporter in one conformation using the known structure of the transporter in the complementary conformation as a template. Here, we describe an updated repeatswap homology modeling protocol, and calibrate the accuracy of the method using Glt Ph , for which both inward-and outward-facing conformations are known. We then apply this repeat-swap homology modeling procedure to a concentrative nucleoside transporter, VcCNT, which has a three-dimensional arrangement related to that of Glt Ph . The repeat-swapped model of VcCNT predicts that nucleoside transport also occurs via an elevator-like mechanism.

Introduction
Neurotransmitters are required for signal transduction between neurons; however, to prevent continuous signaling and neuronal toxicity, they are rapidly removed from the synaptic cleft by membrane transporters. Transporter proteins are also required for TM movement of essential nutrients such as citrate, into the brain. The involvement of transporters in diseases such as depression and autism, render them important therapeutic targets for neurological and psychiatric disorders. Secondary transporter proteins mediate the movement of neurotransmitters and nutrients against their concentration gradients by using the energy released in the movement of ions down pre-existing concentration gradients. To achieve this, transporters conform to the so-called alternating-access hypothesis, whereby the protein adopts at least two conformations in which the substrate binding sites are exposed to one or other side of the membrane, but not both simultaneously.
A significant bottleneck in the design of therapeutic treatments, however, is the limited amount of structural information available for these transporters. The overexpression and crystallization of membrane proteins is notoriously challenging (Bolla et al., 2012). Transporter proteins in particular, tend to crystallize in only one of the two major states, making it difficult to resolve their structures in the other conformations, and limiting our understanding of transporter mechanism. For example, the structure of a sodium-coupled dicarboxylate transporter from Vibrio cholerae, VcINDY, which is related to the neuronal citrate transporter NaCT, has only been determined in one conformation to date (Mancusso et al., 2012). In this regard, Glt Ph , a homolog of the neuronal EAATs of the SLC1 family, from Pyrococcus horikoshii, has become an important model system. The relatively large number of X-ray structures of Glt Ph in different states (Yernool et al., 2004;Reyes et al., 2009;Verdon and Boudker, 2012;Verdon et al., 2014) makes this transporter an ideal system for the study of transport mechanism in neuronal transporters, from a structural point of view.
Analysis of the structure of Glt Ph revealed an invertedrepeat architecture (Yernool et al., 2004;Crisman et al., 2009;Boudker and Verdon, 2010), a feature observed in around threequarters of secondary active transporters of known fold (see Forrest et al., 2011;Forrest, 2015 for reviews). Specifically, large segments of the polypeptide chain are related by structure, but are oriented with the opposite TM topology. This confers the structure with an inherent two-fold pseudo-symmetry, which turns out to be related to the ability of the transporter to adopt two major conformations during the transport cycle, namely outward-and inward-facing states. Specifically, the two repeats adopt a distinctive asymmetry in their conformations, which results in the overall formation of, e.g., an outwardfacing state (Fleishman et al., 2006;Chen et al., 2007;Forrest et al., 2008;Crisman et al., 2009;Radestock and Forrest, 2011;Liao et al., 2012). By exchanging their conformations, the two repeats can create a new asymmetric state that is open to the opposite side of the membrane, e.g., inwardfacing (Fleishman et al., 2006;Chen et al., 2007;Forrest et al., 2008;Crisman et al., 2009;Radestock and Forrest, 2011;Liao et al., 2012;Schushan et al., 2012). We refer to this process of alternating access by inverted-topology transporters as "asymmetry exchange." The asymmetry-exchange concept has been successfully used as a bioinformatics tool to obtain models of a given conformation using the X-ray structure of the transporter in the complementary conformation as a template (Forrest et al., 2008;Crisman et al., 2009;Radestock and Forrest, 2011;Liao et al., 2012;Schushan et al., 2012;Yaffe et al., 2013;Fowler et al., 2015). In this so-called repeat-swap homology modeling technique, the first structural repeat is modeled using the second structural repeat as a template, while the second repeat is concurrently modeled using the first repeat as a template. Given the difficulties of transporter crystallization mentioned above, the ability to predict one state of a transporter once structural data is available for the complementary state provides a very valuable tool.
This repeat-swap technique was first applied to the bacterial amino-acid transporter LeuT from Aquifex aeolicus, which is a homolog of neuronal NSSs from the SLC6 family, such as SERT. Comparison of the outward-facing X-ray structure with the inward-facing model of LeuT accurately predicted the cytoplasmic pathway in NSS transporters (Forrest et al., 2008), and led to the proposed "rocking-bundle" mechanism ). This mechanism involves the movement of a so-called "transport" domain (which in the case of LeuT, consists of a four-helix bundle, leading to the name "rocking bundle"), relative to a "scaffold" domain, which remains essentially static with respect to the membrane. Although the rocking-bundle concept is somewhat simplistic, the major states of the transport cycle in LeuT-like transporters were subsequently found to be consistent with the overall changes predicted by the repeat-swap mechanism, at least with respective to the relative positions of domains (Weyand et al., 2008;Shimamura et al., 2010;Krishnamurthy and Gouaux, 2012). Similar rigid-body movements, also referred to as a "rocker-switch" mechanism, have been proposed for other transporter families such as the major facilitator superfamily (Abramson et al., 2003;Radestock and Forrest, 2011). In all these cases, structures of the end states confirm that the asymmetry exchange hypothesis predicts the changes in relative positions of domains (see e.g., Dang et al., 2010;Radestock and Forrest, 2011), and predicts the helices that line the access pathways.
One of the most dramatic conformational changes identified in a secondary transporter has been for Glt Ph . The mechanism for Glt Ph is unlike the aforementioned mechanisms, in which the substrate is expected to maintain its position, while domains in the protein called "gates" open and close on either side of the substrate binding site. Comparison of outward-and inwardfacing structures of Glt Ph instead reveals a mechanism in which a domain containing the substrate binding site (known as the "transport" domain) moves up and down within the protein, reminiscent of an elevator or a piston, in order to transport the substrate and ion binding sites across the hydrophobic barrier provided by the protein and membrane (Reyes et al., 2009). Specifically, the transport domain moves perpendicular to the membrane plane by ∼16 Å, and rotates by ∼40 • . Remarkably, even this extensive conformational change was predicted by a repeat-swap modeling procedure, in which a cytoplasm-facing state of the protein was modeled using the inverted repeats from a Glt Ph crystal structure in an outward-facing conformation as a template (Crisman et al., 2009;Forrest et al., 2011).
The procedure used by Crisman et al. (2009) was developed ad hoc since the goal of that study was to provide proof of principle that repeat-swapping could lead to an alternate-conformation model of a transporter with such a complex fold. In the present work, we describe a refined and standardized repeat-swap homology modeling protocol. We then focus on the application of this protocol to proteins that potentially undergo an elevatorlike movement during their transport cycle, although the updated protocol is applicable to any protein with an inverted-repeat topology, independent of the type of conformational mechanism. The availability of X-ray structures of Glt Ph in both outward-and inward-open conformations makes this protein a suitable model system on which to validate, refine, and calibrate the protocol. We therefore use the protocol to predict the outward-facing conformation of Glt Ph based on an inward-facing conformation, i.e., the converse of the model built during the previous work (Crisman et al., 2009). The model constructed using the refined protocol differs by ∼4 Å (averaged over all Cα positions) from the X-ray structure in the same conformation, which is a remarkably small error given that the conformational changes are on the order of 15-20 Å. We demonstrate that a large proportion of that "error" is in fact a difference in the extent of the excursion, which depends on the initial state that is used as a template.
We then apply the standardized protocol to VcCNT, a CNT from V. cholerae, whose structure is available only in an inward-facing conformation (Johnson et al., 2012). VcCNT has also been proposed to transport its substrate and ions via an elevator-like movement. Although not related by sequence, or even TM topology (Johnson et al., 2012), several features of VcCNT are similar to those of Glt Ph , namely that it appears to comprise scaffold/oligomerization and transport domains, as well as helical hairpin elements that dip into, but do not cross, the membrane (Figure 1). In addition, comparison of the model obtained for VcCNT in an outward-open state to the inward-open X-ray structure, suggests that a similar elevatorlike mechanism also occurs in the CNT transporter family. Our results therefore show that the new protocol may be useful for predicting conformational mechanisms in other neuronal transporters whose structures that are undoubtedly to follow. Such information will be important in the design of effective therapies targeted to neuronal transporters.

Materials and Methods
The eight steps (a-h) of the standardized repeat-swap homology modeling protocol, illustrated in Figure 2, are as follows: (a) Identification of structural repeat units. The starting point of this methodology is the identification of the residues that constitute each of the inverted repeats in the known structure ( Figure 3A). The underlying assumption is that these repeat units adopt different internal and relative conformations in order to create the asymmetry of an outward-or inwardopen state; exchanging between these two conformations is therefore what enables the major conformational change required for TM transport. To date, no reproducible strategy has been developed to identify structural repeats reliably, mainly because the asymmetry and low sequence identity between the domains can stymie even the most sensitive structure alignment programs. Thus, the repeats may be identified in a number of, essentially manual, ways. Two tools are available to aid this process: SymD (Tai et al., 2014) and CE-symm (Myers-Turnbull et al., 2014), which take as input a single protein structure file in Protein Data Bank (PDB) format, and then report internal pseudo-symmetry within the structure ( Figure 3B). Progressively dividing the structure into smaller domains can be a useful strategy for identifying smaller repeated elements such as those in Glt Ph ( Figure 3A).
FIGURE 2 | Repeat-swap homology modeling protocol. To build a molecular model of membrane transporters with internal structural pseudo-symmetry, the crystal structure of a protomer of the protein (e.g., Glt Ph ) is required as an input. In step (a) the protomer must be carefully analyzed in order to identify the internal repeats. This information is often provided by the authors that report the structure, however, additional insights may be obtained using SymD and CE-symm. In preparation for the modeling, (b,c) an initial sequence alignment is built from a structural alignment of the repeat units (e.g., RU1 and RU2) using a structure alignment program such as TM-Align. In addition, (d) a template structure is built by changing the order of the repeat units in the PDB file and including the regions of the protein that do not belong to the repeat units. At this point, (e) an initial swapped model of the protein can be built, using Modeler. (f) The input alignment is then carefully refined to optimize the agreement with the secondary structure definition and the evolutionary conservation pattern, obtained with DSSP and ConSurf, respectively. Once a model is built using the optimal alignment, the scaffold and transport domains are identified, where possible. (g) Cα-Cα distance restraints can then be defined for each of these domains according to the crystal structure values, and the model is rebuilt using those restraints. Finally, (h) substrates are added to the binding site, if known, and the final repeat-swapped model can be compared with the crystal structure.
(b) Structural and sequence alignment of the repeat units. After structural superimposition of the repeat fragments using a structure alignment algorithm (e.g., TM-Align Zhang and Skolnick, 2005), a sequence alignment between the repeats can be extracted, based on the pairs of residues that are close in space in the structure alignment. Such a pairwise sequence alignment is typically reported by the structure alignment program. (c) Preparing the initial full-length sequence alignment. The initial sequence alignment of the full-length protein to the template is constructed by duplicating the sequence alignment obtained in step b. Specifically, the first repeat of the protein is modeled based on -and is therefore aligned to -the second repeat, while the second repeat is modeled based on the first repeat (see Figure 3C). (d) Preparing the template coordinate file. The "swapped" template used in this procedure is obtained by simply changing the order of the repeats in the PDB-formatted coordinate file: in this way, the first part of the coordinate file contains the residues from the second repeat, and is followed by those that constitute the first repeat. To model fragments of the protein that are not part of the structural repeats and therefore have no counterpart (e.g., helices 4a and 4b in Glt Ph , Figures 3A,B, or other peripheral TM helices), the template can be the same fragment in the X-ray structure ( Figure 3C). Note that a characteristic of the repeat-swap modeling process is that the model obtained is oriented "upside-down" with respect to the template (for example, in Glt Ph , the N-terminal end of TM1 is modeled to have the same coordinate space as the N-terminal end of TM4c in the template); as a consequence an additional step is required to reorient the additional or peripheral template fragments to the position in space that they need to be in the model. In practice, structurally aligning the X-ray Repeat-swap modeling of the Glt Ph and VcCNT transporters in alternate conformations. (A) A schematic representation of the topology of each protein is shown colored according to the structural repeats. In Glt Ph , the helices on the blue and cyan triangular backgrounds comprise repeat unit 1 (RU1), while repeat unit 2 (RU2) is composed of the helices on the red and orange triangular backgrounds. In VcCNT, the blue helices form RU1 and the red helices comprise RU2. (B) In both cases, RU1 is related to RU2 by two-fold pseudo-symmetry, with the symmetry axis (black line) parallel to the membrane. (C) A structural alignment, built with TM-Align, of the repeats of each protein is shown in cartoon representation with the helices colored according to the topology. The initial sequence alignment used to build a swapped-repeat model was generated based on this structural alignment. A schematic of the sequence alignment is shown above, with peripheral helices in the model and template shown in gray. (D) Refined sequence alignment between model and template sequences. The percentage of identical sequences is 21.2 and 46.4% for Glt Ph and VcCNT, respectively, for the full length alignment, including peripheral elements; within the repeats, 14.0 and 7.3% of the residues are identical. Note that the template sequence is the input X-ray protein structure with the order of the repeats reversed. The sequence range of each repeat is displayed with transparent rectangles under the target sequences colored according to the triangular backgrounds in (A). The secondary structure (helix) assignment obtained with DSSP for the X-ray structure is indicated by dark blue rectangles.
structure onto an initial swapped model using the same repeats (e.g., repeat 1 in the X-ray onto repeat 1 in the model) is a straightforward way to reorient the peripheral segments; these must then be added to the template coordinate file in the new orientation, for inclusion in a subsequent round of modeling. The approximate orientation of these peripheral segments will be corrected by adding restraints in a later step of the protocol (step g). Once all the pieces are assembled in order, the residues in the new "swapped" template coordinate file need to be renumbered. (e) Constructing an initial structural model. The initial fulllength alignment, with the sequence of the additional or peripheral fragments correctly included, is used as a guide to build a preliminary homology model based on the "swapped" template. (f) Refinement of the sequence alignment. The initial sequence alignment typically requires refinement, due to the sequence and structural divergence of the repeats, which can make them difficult to align. A common step is to remove gaps within secondary structural elements, where the secondary structure is assigned according to DSSP (Kabsch and Sander, 1983) for the known structure ( Figure 3D). In addition, the conservation patterns obtained from the ConSurf web server (Ashkenazy et al., 2010) can be mapped onto the initial model ( Figures 4B,D), and used to position the most conserved residues so that they are packed inside the protein, while sequence-variant positions face the exterior, mimicking the arrangement in the input X-ray structure. The model built with the refined alignment is useful for identifying the scaffold and transport domains in the case of a two-domain elevator-like mechanism; superimposing the preliminary model onto the known structure using only helices that form the oligomerization interface (Figure 1) can provide a first indication of the mobile elements of the protein. (g) Refinement of the model by introducing distance restraints.
Additional distance restraints between Cα atoms taken from the known structure can be used in the repeatswap modeling process. These restraints are intended to conserve the native helical arrangement and intramolecular packing within domains that move relative to one another, reducing the deviations that arise due to the sequence divergence of the repeats. They also help to fix the orientation of any peripheral segments (see blue helices in Figure 4C). However, identification of suitable restraints requires definition of the scaffold and transport domains, as described above, and should be applied cautiously if these boundaries are not clear. (h) Introduction of the ions and/or substrate. In the final step, any known ions and/or substrates are introduced into the binding site (Figure 4). First, the template substrate position is estimated by superposing the binding site of the known structure on that of the initial model. Then, the model building is repeated using this template substrate, while applying additional distance restraints between substrate atoms and binding site atoms.
The details of the software and parameter choices used are as follows: Model building -Models were built using Modeller v9.13 (Šali and Blundell, 1993). After each of the refinements of the alignment in step f of the protocol, 200 iterations of model building were performed. For steps g and h, 2000 iterations of model building were performed, in order to increase the chances that the sampling creates a model that satisfies all of the input restraints.
Model assessment -Each set of models was evaluated using MolPDF and ProQM scores (Ray et al., 2010), as well as Procheck analysis (Laskowski et al., 1993). The MolPDF score describes how well the model satisfies the input restraints, including those created from the template, the alignment, and any additional applied restraints, and is therefore an arbitrary value dependent on those features; the model with the smallest MolPDF score best satisfies all the restraints. The ProQM score measures the degree to which a set of coordinates is consistent with a number of required features, including TM segments predicted using TOPCONS (Bernsel et al., 2009), the distance to the membrane center for residues in α-helical segments predicted with ZPRED (Granseth et al., 2006), secondary structure elements predicted by PSIPRED (Jones, 1999), burial of conserved residues, and exposure of variant residues. The ProQM score is assigned as a 21-residue window-average to each residue, and is then summed to give a total score per model, with values ranging from 0 to 1, where values of 0.7 are typical of membrane protein structures solved by X-ray crystallography. Procheck assesses the degree to which the features of the model are consistent with those of known protein structures in terms of bond distances, angles, dihedrals and overlapping atoms. In this work, Procheck is used FIGURE 4 | Refinement of repeat-swapped outward-facing models of Glt Ph and VcCNT compared with input inward-facing structures. Models of Glt Ph (A,B) and VcCNT (C,D) built using initial or refined alignments, both before and after applying restraints within the scaffold and transport domains. Models are shown as cartoon helices, colored with separate colors for each helix (see Figure 5) in (A,C), and using ConSurf coloring in (B,D). Substrates are shown as spheres, with their pathways illustrated using arrows. ConSurf conservation patterns are mapped to the helices using colors from cyan (most variant residues) to dark pink (most conserved residues); yellow indicates positions with insufficient sequence information to allow a conservation assignment. As seen for the X-ray structures (right), conserved residues cluster on the inside of the protein, and at oligomerization interfaces, while variant residues are found primarily on the surface. This observation, and the patterns seen in the template structure were used to guide the refinement of the alignments. The position of the proteins in the membrane was determined with the OPM server.
to identify the fraction of backbone groups that lie outside the favored regions of the Ramachandran plot.
The final model was selected as that in step h with the lowest MolPDF score, and/or the highest Procheck and global ProQM scores.
Distance restraints -All distance restraints applied in steps g and h were represented as a Gaussian with a standard deviation of 0.1 Å. Distance restraints between Cα atoms were applied to  (Dahl et al., 2012). To quantify the conformational change of the two transporters, the rotation axis, rotation angle, and the displacement of the helices were evaluated. The black line shows the rotation axis, which lies approximately perpendicular to the symmetry axis in each transporter (see Figure 3B). The displacement values suggest an elevator-like movement of the transport domain containing the substrates (spheres), and this displacement is associated with a rotation of 43.5 and 11.8 • , for Glt Ph and VcCNT, respectively. For reference, the conformational change in the transport domain between two X-ray structures of Glt Ph (PDB IDs 3KBC and 1XFH) involves a rotation of 39.6 • and a displacement by x = 3.9, y = 2.8, z = 16.4 Å. (C,D) Displacement per residue determined as the distance between Cα atoms in the crystal structure and the model after fitting using the scaffold domains (black), for Glt Ph (C) and VcCNT (D). In the case of Glt Ph , the conformational change between known inward-facing and outward-facing substrate-bound structures is shown (red).
preserve the internal structure of the scaffold/oligomerization and transport domains, independently, and were assigned according to the input crystal structure for all pairs of Cα atoms < 60 Å apart within each domain.
The distance restraints applied in step h between substrates and binding site atoms were defined as follows: For Glt Ph , restraints were applied between the two Na + ions or any non-hydrogen ("heavy") atom of the aspartate substrate, and any heavy atom of the protein within 5 Å in the known/template structure, PDB (Berman et al., 2000) identifier, ID: 3KBC (Reyes et al., 2009).
For VcCNT, restraints were applied between the Na + ion and the O atoms in the backbone of N149, V152 and I184, the hydroxyl group of S183, and a crystallographic water molecule. Restraints were also applied between any heavy atoms of the uridine and any heavy-atom of the protein within 3.5 Å in the template structure, PDB ID: 3TIJ (Johnson et al., 2012).
Other programs -Structural alignments were obtained using TM-align (Zhang and Skolnick, 2005) and Jalview was used for editing and visualization of alignments (Waterhouse et al., 2009). Secondary structural elements were assigned with DSSP (Edgar, 2010) and conservation patterns were obtained using the ConSurf server with default parameters (Ashkenazy et al., 2010). Molecular figures were generated using PyMol v1.6 (Schrödinger, Inc.). The position of the proteins in the membrane was determined for the X-ray structures with the OPM server (Lomize et al., 2006), and the orientation of the models was defined after superposition of the scaffold domain onto that of the corresponding X-ray structure. Crisman et al. (2009) previously applied an ad hoc repeatswap protocol to construct an inward-facing substrate-bound occluded model of Glt Ph (Protein Model Database (Castrignanò et al., 2006), PMDB identifier: PM0075966) based on an available structure of Glt Ph in an outward-facing substratebound occluded conformation (Boudker et al., 2007, PDB ID: 2NWL). The predicted inward-facing state captured the major conformational change revealed in a structure published around the same time (Reyes et al., 2009), thereby demonstrating that the conformational change is a result of asymmetry exchange. Here, we propose a more standardized and generalized protocol for repeat-swap homology modeling (see Materials and Methods), and calibrate the method by building an outward-facing occluded model of Glt Ph based on the available inward-facing structure (PDB ID: 3KBC), thereby providing a measure of the expected accuracy of the method for that case by comparison with a known structure in the same state (PDB ID: 1XFH). We then apply the FIGURE 6 | The repeat-swapped model of VcCNT contains an extracellular substrate pathway that is not present in the template structure. The trimeric VcCNT X-ray structure (A,C) and model (B,D) are viewed from the extracellular side, colored according to Figure 1 and rendered either as cartoon helices (A,B) or using the van der Waals surface (C,D). The substrate (spheres, arrows) is visible from the extracellular side in the model (D), but not in the inward-facing crystal structure (C). updated protocol to VcCNT, revealing that the mechanism of this secondary transporter is also likely to involve an elevator-like conformational change.

Results and Discussion
An Outward-Facing Model of Glt Ph Built Using the Inward-Facing Structure as a Template As mentioned above, structures of Glt Ph are available in several different conformations, including outward-and inward-open, outward-occluded, intermediate, and apo forms (Yernool et al., 2004;Boudker et al., 2007;Reyes et al., 2009;Boudker and Verdon, 2010). Here, we wished to compare predictions of the major conformational change between outward-occluded and inward-occluded states, with structures of the end points at a similar resolution. Thus, we elected to use the inward-occluded substrate-bound structure of Glt Ph at 3.51 Å resolution (PDB ID: 3KBC, Reyes et al., 2009) as a template to model the outward-occluded substrate-bound state, which could then be compared with a known structure also at 3.50 Å resolution (PDB ID: 1XFH, Yernool et al., 2004). This we did by following the protocol described in the Section "Materials and Methods." As mentioned above, a few steps in the repeat-swap homology modeling protocol do not yet have reliable automated procedures. Specifically, we defined the inverted-topology repeats in Glt Ph according to Crisman et al. (2009; Figure 3A). We note that this differs slightly from the definition of Reyes et al. (2009), who defined TM3 and TM6 as part of RU1B and RU2B, respectively. We elected to define them this way because the structure superpositions led to more reasonable initial sequence alignments, requiring fewer cycles of refinement. The final refined sequence alignment is shown in Figure 3D.
For assigning the domains that move relative to one another (transport and scaffold domains) we used the definition of Reyes et al. (2009; Figure 1, see Materials and Methods). In an important update to the protocol, restraints were imposed between all pairs of Cα atoms within each of these domains separately during model building. The final model contains aspartate and two sodium ions ( Figure 4A).

Accuracy of the Repeat-Swapped Model of Glt Ph
The model of the outward-occluded state of Glt Ph (Figure 5A) was compared with the inward-occluded (template) X-ray crystal structure by structurally aligning the scaffold/oligomerization domains. This comparison revealed a conformational change consisting of a ∼44 • rotation of the transport domain about an axis running approximately perpendicular to the symmetry axis, and a ∼10 Å translocation (normal to the membrane), relative to the scaffold/oligomerization domain. As a result, the substrate binding site is exposed to the opposite side of the membrane. This domain movement translates to positional changes of between 5 and 23 Å for the individual Cα atoms in the transport domain ( Figure 5C, black line). The helical hairpins HP1 and HP2, which are at the interface with the scaffold domain, undergo the largest movements, whereas the helices facing the lipids (TM3 and TM6) undergo the smallest changes.
These predicted changes recapitulate those observed in a comparison of the template inward-occluded structure (PDB ID: 3KBC), with an outward-occluded substrate-bound structure of similar resolution (PDB ID: 1XFH), which reveals a rotation of ∼40 • and a translation perpendicular to the membrane of ∼16 Å, corresponding to positional changes of between 10 and 25 Å per Cα atom ( Figure 5C, red line). Thus, the degree of rotation and direction of translocation are very similar. Moreover, this comparison allows a quantitative analysis of the accuracy of the repeat-swap method. Two factors determine the accuracy of the model: first, the degree to which the two repeats are structurally similar, which in turn is related to their sequence similarity; and second, the degree of symmetry that relates the two states.
With respect to the first factor, the similarity between the structures of the repeats is related to the expected accuracy of a typical homology model, which decreases as a function of decreasing sequence identity (Forrest et al., 2006). In Glt Ph , the sequence identities between repeats are only ∼9-14% (Figure 3), leading to an expected accuracy of ∼1.5-4 Å in the Cα positions in the TM segments (Forrest et al., 2006). However, with the new protocol we have also captured information about the known structural features of each domain, by incorporating restraints within the oligomerization/scaffold and transport domains separately, based on intra-domain distances in the known structure (see Materials and Methods). During the modeling, these intra-domain distance restraints will be satisfied as long as they do not lead to significant violations of the other restraints obtained from the use of the other repeat as a template (for which there are a larger number of distance restraints). Applying these new intra-domain restraints in the case of Glt Ph leads to a significant improvement in the accuracy of the individual domains. Specifically, after applying these restraints the root mean squared deviation (RMSD) between the model and the crystal structure of the outward-facing conformation decreases from 4.8 Å and 6.7 Å, to 2.5 Å and 2.9 Å over all Cα atoms in the scaffold/oligomerization and transport domains, respectively. This improvement occurs without altering the overall elevatorlike movement; before incorporating the intradomain restraints, the rotation and translocation were ∼45 • and ∼11 Å, respectively (cf. ∼44 • and ∼10 Å for the final model described above).
The above results show that the error in the new model is only slightly higher than the difference between the two known structures in different conformations (PDB IDs: 1XFH and 3KBC), which is 1.2 and 3.4 Å for the scaffold/oligomerization and transport domains, respectively. Using a length-independent measure for the same comparison, the template modeling score (TM-score) increases from 0.62 and 0.64 to 0.83 and 0.86, for the models built without and with intra-domain restraints, respectively. (A TM-score of 1.0 indicates that the Cα atoms have identical positions.) For comparison, the TM-scores when comparing the inward-and outwardfacing crystal structures domains are 0.91 and 0.73, for the scaffold/oligomerization and transport domains, respectively. The remarkable accuracy of this modeling procedure is achieved in spite of the extremely low (<15%) sequence identity, and even while the overall conformational change is captured ( Figure 5).
As mentioned, the second factor determining repeat-swap model accuracy is the particular conformational state of the template. Thus, if the input structure is semi-occluded, then the output model will also be semi-occluded. In the case of the swapped model of Glt Ph , the rotation of the transport domain is very similar to that observed in comparing the two structures, but the translocation of that domain implied by the model is slightly (∼7 Å) smaller ( Figure 5C, compare red and black lines) demonstrating that the modeled conformational change is less extreme than expected based on the crystal structures. In other words, the (template) inward-facing structure is less "inward" than the known outward-facing structure is "outward, " resulting in a model that is also less "outward." Thus, the major value of the repeat-swap modeling is to identify the elements involved in the conformational change, and the directionality of their movement, which are clearly both well captured by this approach.

A Model of the Outward-Facing Conformation of VcCNT
In Johnson et al. (2012), a structure of a CNT from V. cholerae (VcCNT) was reported at 2.4 Å resolution, with a substrate binding site that is exposed toward the presumed cytoplasm.
VcCNT is related to transporters of the human solute carrier SLC28 family. To date, all other structures reported for this transporter fold reflect the same state of the transporter (Johnson et al., 2014) and therefore the mechanism of conformational change is unknown. It has been proposed, based on visual analysis of the structure and binding site accessibility data, that CNTs undergo a similar elevator-like movement (Johnson et al., 2012), with two helical hairpins moving above and below TM helix 6, which serves as a hydrophobic barrier. In particular, the overall "design principle" of the CNTs appears to be similar to that of Glt Ph , with a trimeric quaternary structure, helical hairpins, and clear transport-and scaffoldlike boundaries (Johnson et al., 2012, Figure 1). However, at the level of individual helices, a structural comparison revealed few commonalities (Johnson et al., 2012). Nevertheless, the VcCNT fold clearly contains inverted-topology repeats, which together assemble into the transport domain (Figures 3A,B). Internal symmetry within the scaffold/oligomerization domain of VcCNT is less clear, except for the interfacial helices IH2 and IH3, and therefore, if additional internal symmetry ever existed, it appears to have been lost during evolution, perhaps because trimerization provides sufficient scaffolding without compromising the transport mechanism. As in Glt Ph , the scaffold domain of VcCNT also contains within it the helices involved in forming the physiologically relevant trimer interface ( Figure 1B).
To pursue the hypothesis that VcCNT undergoes an elevator movement, we built a repeat-swapped structural model using the inward-facing structure as a template, and using the definition of repeats shown in Figures 3A,C, by applying our updated protocol (Figure 2, see Materials and Methods). The final alignment after initial model-building and refinement (Figures 4C,D) is shown in Figure 3D.
We defined repeat units 1 and 2 as comprising the transport domain, with the exception of interfacial helices IH2 and IH3, which were assigned to the scaffold domain, together with the peripheral trimerization helices (Figures 1A  and 3A-C). Along with restraints to position a uridine substrate and a sodium ion in binding sites formed by the same residues as in the template, we also imposed restraints within each domain to build a refined model that matches as many aspects of the known structure as possible, while capturing the same overall conformational change. The final model of VcCNT (Figures 5B,D) is freely available from the PMDB (https://bioinformatics. cineca.it/PMDB/) with the identifier PM0080188, and the model generated without restraints is available upon request.
The repeat-swapped model of VcCNT adopts a conformation in which the substrates are visible via an aqueous pathway connected to the extracellular solution (Figure 6). The conformational change predicted by the VcCNT model also involves an elevator-like movement of the hairpin domains, similar to that seen for Glt Ph as suggested by Johnson et al. (2012). These changes arise due to a ∼12 • rotation of the transport domain around a similar axis to that found in Glt Ph , as well as lateral and displacements along the membrane normal of ∼3-4 Å ( Figure 5B). Note that very similar changes are found when the intradomain restraints were not included; specifically, the transport domain rotates by ∼15 • , and is also translocated by 3-4 Å. Interestingly, the extent of this conformational change is more modest than that of Glt Ph , with Cα displacements of between 4 and 10 Å, after superposition on the scaffold helices ( Figure 5D).
It is entirely possible that VcCNT undergoes more extreme movements than can be predicted based on the available structural data. Nevertheless, as noted above, the directionality and composition of the moving domains predicted by the repeat-swap model are likely to be correct, based on our observations using Glt Ph . Therefore, this model of the outwardfacing state of VcCNT should provide a useful starting point for experimental studies of the conformational change required for transport in CNTs, by illustrating which elements become buried or exposed, and which elements move relative to one another.

Conclusion
Repeat-swap modeling has proven to be helpful for analyzing the conformational changes in various homologs of neuronal transporters, including Na + /Cl − -dependent transporters of the NSS family (Forrest et al., 2008), EAATs (Crisman et al., 2009), and vesicular monoamine transporters (Yaffe et al., 2013). Although the models generated may not be highresolution enough for drug design, the identification of alternate conformations and conformational mechanisms is essential for the development of treatments against such transporters, as it provides insight into changes in the transporter structure that may affect inhibitor binding. For example, inhibitors of serotonin uptake such as ibogaine and cocaine bind to different conformations of SERT (Tavoulari et al., 2009), and similar conclusions have been evoked for reserpine and tetrabenazine binding to the vesicular monoamine transporter VMAT2 (Schuldiner et al., 1995). Therefore, molecular models based on repeat-swap modeling can be extremely useful to help design further experiments such as cross-linked constructs for crystallography, whose structures can then be used for drug design.
The current study provides an updated and more systematic protocol for repeat-swap modeling of secondary transporters with inverted-repeats. Our analysis of the Glt Ph model reveals that the model accuracy can be dramatically improved by inclusion of restraints extracted from the input structure. Those restraints can be readily defined if there is a distinct boundary between the mobile and static segments of the transporter. In the cases of Glt Ph and VcCNT this boundary became clear after producing an initial repeat-swapped model of each protein, and comparing that model with the input crystal structure by superposition of the helices forming the oligomerization interface. With the benefit of this insight, this boundary becomes relatively clear merely from analysis of the architecture viewed from above the membrane ( Figure 1B).
The inward-facing crystal structure of VcCNT and the swapped model in an outward-facing conformation that we present here provide a molecular-level description of the conformational changes in CNTs. We expect that this insight will prove to be important for understanding the mechanism of TM transport of nucleosides and of nucleoside-derived drugs, such as anticancer drugs like gemcitabine. More broadly, our model strongly suggests that the remarkable elevator-like movements identified in the glutamate transporter family are not unique, and that transporters in the SLC28 CNT family also undergo elevatorlike movements during their alternating access mechanism. This raises the possibility that other transporter families may use a similar elevator-like mechanism. For example, the sodiumcoupled dicarboxylate transporter VcINDY (Mancusso et al., 2012), which is a homolog of the neuronal sodium-coupled citrate transporter NaCT, contains inverted-topology repeats with reentrant helical hairpins similar to those observed in Glt Ph and VcCNT. It will be of interest to examine which other neuronal transporters use elevator mechanisms for TM transport of their substrates.