As prey and pollinators, insects increase reproduction and allow for outcrossing in the carnivorous plant Dionaea muscipula.

PREMISE: Understanding the factors that limit reproductive success is a key component of plant biology. Carnivorous plants rely on insects as both nutrient sources and pollinators, providing a unique system for studying the effects of both resource and pollen limitation on plant reproduction. METHODS: We conducted a field experiment using wild-growing Dionaea muscipula J. Ellis (Droseraceae) in which we manipulated prey and pollen in a factorial design and measured flower production, number of fruits, and number of seeds. Because understanding reproduction requires knowledge of a plant species' reproductive and pollination biology, we also examined the pollination system, per-visit pollinator effectiveness, and pollen-ovule (P/O) ratio of D. muscipula. RESULTS: Plants that received supplemental prey produced more flowers than control plants. They also had a higher overall fitness estimate (number of flowers × fruit set (total fruits/total flowers) × seeds per fruit), although this benefit was significant only when prey supplementation occurred in the previous growing season. Neither pollen supplementation nor the interaction between pollen and prey supplementation significantly affected overall plant fitness. CONCLUSIONS: This study reinforces the reliance of D. muscipula on adequate prey capture for flower, fruit, and seed production and a mobile pollen vector for reproduction, indicating the importance of considering insects as part of an effective conservation management plan for this species.

Host species differences in the thermal mismatch of host-parasitoid interactions.

Extreme high temperatures associated with climate change can affect species directly, and indirectly through temperature-mediated species interactions. In most host-parasitoid systems, parasitization inevitably kills the host, but differences in heat tolerance between host and parasitoid, and between different hosts, may alter their interactions. Here, we explored the effects of extreme high temperatures on the ecological outcomes - including, in some rare cases, escape from the developmental disruption of parasitism - of the parasitoid wasp, Cotesia congregata, and two co-occurring congeneric larval hosts, Manduca sexta and M. quinquemaculata. Both host species had higher thermal tolerance than C. congregata, resulting in a thermal mismatch characterized by parasitoid (but not host) mortality under extreme high temperatures. Despite parasitoid death at high temperatures, hosts typically remain developmentally disrupted from parasitism. However, high temperatures resulted in a partial developmental recovery from parasitism (reaching the wandering stage at the end of host larval development) in some host individuals, with a significantly higher frequency of this partial developmental recovery in M. quinquemaculata than in M. sexta. Hosts species also differed in their growth and development in the absence of parasitoids, with M. quinquemaculata developing faster and larger at high temperatures relative to M. sexta. Our results demonstrate that co-occurring congeneric species, despite shared environments and phylogenetic histories, can vary in their responses to temperature, parasitism and their interaction, resulting in altered ecological outcomes.

Nonfertilizing sperm in Lepidoptera show little evidence for recurrent positive selection.

Sperm are among the most variable cells in nature. Some of this variation results from nonadaptive errors in spermatogenesis, but many species consistently produce multiple sperm morphs, the adaptive significance of which remains unknown. Here, we investigate the evolution of dimorphic sperm in Lepidoptera, the butterflies and moths. Males of this order produce both fertilizing sperm and a secondary, nonfertilizing type that lacks DNA. Previous organismal studies suggested a role for nonfertilizing sperm in sperm competition, but this hypothesis has never been evaluated from a molecular framework. We combined published data sets with new sequencing in two species, the monandrous Carolina sphinx moth and the highly polyandrous monarch butterfly. Based on population genetic analyses, we see evidence for increased adaptive evolution in fertilizing sperm, but only in the polyandrous species. This signal comes primarily from a decrease in nonsynonymous polymorphism in sperm proteins compared to the rest of the genome, suggesting stronger purifying selection, consistent with selection via sperm competition. Nonfertilizing sperm proteins, in contrast, do not show an effect of mating system and do not appear to evolve differently from the background genome in either species, arguing against the involvement of nonfertilizing sperm in direct sperm competition. Based on our results and previous work, we suggest that nonfertilizing sperm may be used to delay female remating in these insects and decrease the risk of sperm competition rather than directly affect its outcome.

Venus Flytrap Rarely Traps Its Pollinators.

Because carnivorous plants rely on arthropods as pollinators and prey, they risk consuming would-be mutualists. We examined this potential conflict in the Venus flytrap (Dionaea muscipula), whose pollinators were previously unknown. Diverse arthropods from two classes and nine orders visited flowers; 56% of visitors carried D. muscipula pollen, often mixed with pollen of coflowering species. Within this diverse, generalized community, certain bee and beetle species appear to be the most important pollinators, on the basis of their abundance, pollen load size, and pollen fidelity. Dionaea muscipula prey spanned four invertebrate classes and 11 orders; spiders, beetles, and ants were most common. At the family and species levels, few taxa were shared between traps and flowers, yielding a near-zero value of niche overlap for these potentially competing structures. Spatial separation of traps and flowers may contribute to partitioning the invertebrate community between nutritional and reproductive functions in D. muscipula.

Invertebrate community response to coarse woody debris removal for bioenergy production from intensively managed forests.

Increased market viability of harvest residues as forest bioenergy feedstock may escalate removal of coarse woody debris in managed forests. Meanwhile, many forest invertebrates use coarse woody debris for cover, food, and reproduction. Few studies have explicitly addressed effects of operational-scale woody biomass harvesting on invertebrates following clearcutting. Therefore, we measured invertebrate community response to large-scale harvest residue removal and micro-site manipulations of harvest residue availability in recently clearcut, intensively managed loblolly pine (Pinus taeda) forests in North Carolina (NC; n = 4) and Georgia (GA; n = 4), USA. We captured 39,794 surface-active invertebrates representing 171 taxonomic groups using pitfall traps situated among micro-site locations (i.e., purposefully retained piles of hardwood stems and piles of conifer stems and areas without coarse woody debris in NC; windrows and no windrows in GA). Micro-site locations were located within six, large-scale treatments (7.16-14.3 ha) in clearcuts. Large-scale treatments represented intensive harvest residue removal, 15% and 30% harvest residue retention, and no harvest residue removal. In NC, ground beetles (Coleoptera: Carabidae) and crickets (Orthoptera: Gryllidae) were three times more abundant in treatments with no harvest residue removal than those with the most intensive harvest residue removal and were reduced in treatments that retained 15% or 30% of harvest residues, although not significantly. Invertebrate taxa richness was greater at micro-site locations with retained hardwood and pine (Pinus spp.) harvest residues than those with minimal amounts of coarse woody debris. In both states, relative abundances of several invertebrate taxa, including cave crickets (Orthoptera: Rhaphidophoridae), fungus gnats (Diptera: Mycetophilidae and Sciaridae), millipedes (Diplopoda), and wood roaches (Blattodea: Ectobiidae), were greater at micro-site locations with retained harvest residues than those with minimal coarse woody debris. Intensified woody biomass harvesting without retention of ≥15% of harvest residue volume may reduce invertebrate taxa richness and abundances of some key invertebrate taxa in regenerating stands. Further, harvest residue management during and after woody biomass harvesting may be an important consideration for maintaining invertebrate diversity and conserving invertebrates that are influential in the maintenance of ecosystem function and integrity in young forests.

Effects of the insect growth regulator, methoprene, on Onthophagus taurus (Coleoptera: Scarabaeidae).

A bioassay was conducted to determine the impact of methoprene, an insect growth regulator (IGR), on fecundity, larval survival, and size of progeny for Onthophagus taurus Schreber. Adult O. taurus dung beetles were offered methoprene-treated manure in three to five replications each at concentrations of 0.08, 0.45, and 4.5 ppm, respectively. An additional group of adult beetles was immersed in a methoprene-water solution and allowed to reproduce in containers with untreated manure. Data from all treatment groups were compared with untreated control groups. Methoprene did not seem to hinder brood production at 0.45 ppm. Survival of O. taurus was not affected by methoprene-treated manure at 0.08 ppm or when parent beetles were immersed in methoprene-water solution. However, progeny survival was significantly reduced on manure treated with methoprene at 4.5 ppm. Mean pronotal width of O. taurus progeny was significantly smaller in beetles fed methoprene-treated manure (4.5 ppm). The low dose of 0.08 ppm did not affect pronotal widths nor did topical application of methoprene to adults affect pronotal widths in resulting offspring. Although some adverse effects of methoprene were observed at higher concentrations, use of methoprene at concentrations of 0.08 ppm as part of a horn fly control program likely would not greatly affect populations of O. taurus, the most common paracoprid dung beetle in North Carolina.

Helicoverpa zea (Lepidoptera: Noctuidae) Preference for Plant Structures, and Their Location, Within Bt Cotton Under Different Nitrogen and Irrigation Regimes.

Helicoverpa zea Boddie is a common economic pest of cotton (Gossypium hirsutum L.), including transgenic cotton varieties that express Bacillus thuringiensis (Bt). Helicoverpa zea oviposition is similar in Bt and non-Bt cotton, but behavior of H. zea larvae can be different in the presence of Bt, with neonates moving away from terminals faster in single-toxin Bt than non-Bt cotton or avoiding Bt-treated diet in the lab. We quantified H. zea oviposition and larval distribution on structures within cotton plants in small plot experiments of Cry1Ac + Cry1F cotton for 2 yr under different irrigation and nitrogen treatments. More eggs were oviposited on plants receiving nitrogen application during 2016 and on leaves in the top section of irrigated plants during 2017, but other treatment effects on eggs or larvae were minimal. Helicoverpa zea eggs were most common on leaves in the top third of plants at position zero and middle section of cotton plants throughout the season, but some oviposition occurred on fruiting structures as well. First and second instars were more common on squares in the top section of plants during 2016 and bolls in the middle and lower sections during 2017 due to oviposition lower in the canopy during 2017. During both years, third through fifth instars were more common on bolls in the middle and lower section of plants closer to the main stem. These findings have resistance management implications as extended larval feeding on bolls could optimize nutrition, decrease Bt susceptibility, and potentially influence behavioral resistance.

Helicoverpa zea (Lepidoptera: Noctuidae) Oviposition and Larval Vertical Distribution in Bt Cotton Under Different Levels of Nitrogen and Irrigation.

In some Bt cotton (Gossypium hirsutum L.) varieties, bollworm (Helicoverpa zea Boddie) larval behavior differs from non-Bt varieties. Laboratory assays indicate bollworm larvae can detect Bt proteins, which may cause behavioral differences. Plant stress from factors including fertility and water availability causes changes in plant physiology and Bt expression. Our objective was to determine whether nitrogen and irrigation influenced bollworm behavior in Bt cotton by recording the vertical distribution of eggs and larvae over time. We conducted small plot experiments with Cry1Ac + Cry1F cotton in 2016 and 2017 with three nitrogen rates, along with irrigated and nonirrigated treatments during 2017. Bollworm locations were determined by in-field examination of 10-20 cotton plants per plot over 6-8 wk. The location of each egg and larva was recorded by node, with instar estimation of each larva. Oviposition was higher in in plots receiving nitrogen; first and second instars were also more common in plots receiving nitrogen or irrigation, whereas older instars had similar numbers among treatments. Oviposition was more evenly distributed throughout the canopy earlier in the sampling period than during later weeks, with more eggs in the top third of the canopy in only three of 14-wk. Early instars were also evenly distributed throughout the canopy. Later, instars moved to the middle portions of the canopy, away from bottom nodes, and did not move toward the terminal. Understanding bollworm behavior can inform both crop scouting and resistance management decisions.

Development and Dispersal of Helicoverpa zea (Lepidoptera: Noctuidae) on Non-Bt and Bt Pyramided Cotton.

Bollworm (Helicoverpa zea Boddie) (Lepidoptera: Noctuidae) can cause economic losses in both non-Bt and Bt cotton. Larvae modify their behavior in the presence of Bt by moving away from terminals faster in Bt cotton compared to non-Bt cotton and avoiding Bt-treated diets. Our objectives were to understand differences in bollworm egg and larvae populations within, and dispersal away from, non-Bt and Bt pyramided-toxin cotton. We conducted small plot experiments in 2016 and 2017 to monitor on-plant egg and larval numbers, and off-plant dispersal of larvae, from non-Bt and different Bt toxin pyramided cotton. Bollworm adults preferred to oviposit in most Bt toxin pyramids compared to non-Bt; this was likely unrelated to detection of Bt by adults, but rather density-dependent aversion from high larval populations. First instar numbers were similar in all non-Bt/Bt toxin pyramids and dispersed at a similar rate. Second through fifth instar numbers were higher in non-Bt than Bt toxin pyramids but dispersed equally from all non-Bt/Bt toxin pyramids, regardless of Bt pyramid type. Development times of larvae were often slower in Bt toxin pyramids compared to non-Bt. Fifth instars were found in, and dispersing from, Bt toxin pyramids containing Vip3A, raising concerns of resistance development. Furthermore, differences in oviposition rate among non-Bt/Bt toxin pyramids and slowed development rate of larvae on Bt varieties could create inconsistencies in generation times emerging from Bt and non-Bt hosts, which could contribute to resistance development.

Effects of vegetated field borders on arthropods in cotton fields in eastern North Carolina.

The influence, if any, of 5m wide, feral, herbaceous field borders on pest and beneficial arthropods in commercial cotton, Gossypium hirsutum (L.) (Malvales: Malvaceae), fields was measured through a variety of sampling techniques over three years. In each year, 5 fields with managed, feral vegetation borders and five fields without such borders were examined. Sampling was stratified from the field border or edge in each field in an attempt to elucidate any edge effects that might have occurred. Early season thrips populations appeared to be unaffected by the presence of a border. Pitfall sampling disclosed no differences in ground-dwelling predaceous arthropods but did detect increased populations of crickets around fields with borders. Cotton aphid (Aphis gossypii Glover) (Hemiptera: Aphididae) populations were too low during the study to adequately assess border effects. Heliothines, Heliothis virescens (F.) and Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), egg numbers and damage rates were largely unaffected by the presence or absence of a border, although in one instance egg numbers were significantly lower in fields with borders. Overall, foliage-dwelling predaceous arthropods were somewhat more abundant in fields with borders than in fields without borders. Tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois) (Heteroptera: Miridae) were significantly more abundant in fields with borders, but stink bugs, Acrosternum hilare (Say), and Euschistus servus (Say) (Hemiptera: Pentatomidae) numbers appeared to be largely unaffected by border treatment. Few taxa clearly exhibited distributional edge effects relative to the presence or absence of border vegetation. Field borders like those examined in this study likely will have little impact on insect pest management in cotton under current insect management regimens.

Effect of tobacco budworm (Lepidoptera: Noctuidae) infestation level on budworm-resistant and -susceptible varieties of flue-cured tobacco in North Carolina.

Field experiments were conducted from 1972 to 1978 and from 1998 to 1999 to evaluate tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae), larval feeding on flue-cured tobacco, Nicotiana tabacum (L.), yield in eastern North Carolina. In the earlier studies, using variety Coker 319, treatment plots were evaluated when either 0 or 100% of plants in a plot were infested with H. virescens larvae. Treatment differences based on actual yield loss (kilograms per hectare) were compared with estimations of yield loss based on leaf consumption and leaf loss. Results indicate actual yield loss when 100% of plants were infested was less than the corresponding estimates of yield loss. In the later experiments, two tobacco budworm-resistant lines, 'CU 263' and 'CU 370', were compared with a commercial susceptible variety, K 326, when 0, 10, 20, or 40% of plants were infested (1998) and 0, 10, 40, 75, or 100% of plants were infested (1999). Although significant increases in leaf equivalents consumed were associated with infestations exceeding the recommended threshold, differences were not detected for yield (kilograms per hectare), quality (dollars per kilogram), and value (dollars per hectare) within each tobacco line. Additionally, there was not a significant correlation between value and infestations level for any of the tobacco lines. These results provide economic support for tolerance of a higher treatment threshold. Although K 326 sustained more leaf equivalent loss than CU 263 and CU 370, the value of K 326 harvested was higher than that of CU 263 and CU 370. To justify use of resistant varieties, the combination of pest pressure and the benefit of host plant resistance must be greater than the capacity of a susceptible variety to produce competitive yields, despite sustaining significantly higher loss.

Trap-mulching Argentine ants.

Argentine ant, Linepithema humile (Mayr), management is constrained, in large part, by polydomy where nestmates are distributed extensively across urban landscapes, particularly within mulch. Management with trap-mulching is a novel approach derived from trap-cropping where ants are repelled from a broad domain of nest sites to smaller defined areas, which are subsequently treated with insecticide. This concept was field-tested with mulch surrounding ornamental trees replaced with a narrow band of pine (Pinus spp.) needle mulch (trap) within a much larger patch of repellent aromatic cedar (Juniperus spp.) mulch. After ants reestablished around the trees, the pine needle mulch band was treated with 0.06% fipronil (Termidor). Poor results were obtained when the trap extended from the tree trunk to the edge of the mulched area. When the trap was applied as a circular band around the tree trunk reductions in the number of foraging ants were recorded through 14 d compared with an untreated mulch control, but not for longer periods. Reductions in the number of ant nests within mulch were no different between the trap mulch and any of the other treatments. We conclude that trap-mulching offers limited benefits, and that successful management of Argentine ants will require implementation of complementary or perhaps alternative strategies.

Systemic Imidacloprid Affects Intraguild Parasitoids Differently.

Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) and Campoletis sonorensis (Cameron) (Hymenoptera, Ichneumonidae) are solitary endoparasitoids of the tobacco budworm, Heliothis virescens (Fabricius) (Lepidoptera, Noctuidae). They provide biological control of H. virescens populations in Southeastern US agricultural production systems. Field and greenhouse experiments conducted from 2011-2014 compared parasitism rates of parasitoids that developed inside H. virescens larvae fed on tobacco plants treated with and without imidacloprid. The parasitoids in our study did not have a similar response. Toxoneuron nigriceps had reduced parasitism rates, but parasitism rates of C. sonorensis were unaffected. Preliminary data indicate that adult female lifespans of T. nigriceps are also reduced. ELISA was used to measure concentrations of neonicotinoids, imidacloprid and imidacloprid metabolites in H. virescens larvae that fed on imidacloprid-treated plants and in the parasitoids that fed on these larvae. Concentrations were detectable in the whole bodies of parasitized H. virescens larvae, T. nigriceps larvae and T. nigriceps adults, but not in C. sonorensis larvae and adults. These findings suggest that there are effects of imidacloprid on multiple trophic levels, and that insecticide use may differentially affect natural enemies with similar feeding niches.