Arena Bioassays Research Articles

Olfactory Response of Sitophilus zeamais Adults to Odours of Semolina Pasta and Semolina Pasta Enriched with Different Amounts of Acheta domesticus Powder.

The behavioural response of adult maize weevil, Sitophilus zeamais, to different types of semolina pasta enriched or not enriched with increasing proportions (5%, 10%, and 15%) of house cricket (Acheta domesticus) powder was investigated in olfactometer arena bioassays by using trap devices. In the five-choice behavioural bioassays, the number of S. zeamais adults attracted to 100% durum wheat semolina pasta was significantly higher than those attracted to the other pasta types enriched with A. domesticus powder. In the two-choice behavioural bioassays, the Response Index for each pasta type was positive and significant. However, although not significant, there was a progressive reduction in the Response Index as the cricket powder content increased. In similar experiments, there were no significant differences between cricket powder alone and the control in the number of attracted S. zeamais, indicating a neutral effect towards insects. These observations suggest that the lower attractiveness of pasta enriched with house cricket powder is mainly due to the masking of host food odours. Solid-phase microextraction coupled to gas chromatography-mass spectroscopy identified a total of 18 compounds in the head-space samples of the different types of pasta, highlighting differences in volatile composition. Some volatile compounds were only present in the pasta produced with cricket powder. In particular, 1-octen-3-ol and phenol were present in the samples containing 5%, 10%, or 15% cricket powder; pentanal, benzaldehyde, and dimethyl disulphide were present in samples containing 10% or 15% cricket powder; and 2,5-dimethyl-pyrazine was present in the sample containing 15% cricket powder. Further investigation with individual compounds and mixtures is needed to define the chemical basis of the differences in the insect olfactory preference observed in this study.

Comparative study on the repellency and chemical profiles of different chilli peppers formulations against Sitophilus oryzae L. (Coleoptera: Curculionidae) in stored wheat

The weevil Sitophilus oryzae L. (Coleoptera: Curculionidae) is the major insect pest of stored grains, including wheat. This study tested the repellency effectiveness of three formulations, including chilli dry powder (CDP), chilli ethanol extract (CEE), and chilli crude extract (CCE), from different chilli peppers of Capsicum annuum L. and Capsicum chinense Jacquin varieties (Solanaceae), against S. oryzae using a modified cup bioassay arena. Repellency responses varied significantly (P ≤ 0.001) within the three different formulations, where the CEE formulation at the concentration of 5 g (w/w) demonstrated the highest effectiveness, effectively deterring the highest number of insects (mean number of repelled insects ± SE) after 24 h of treatment across tested chilli varieties such as Guntur Sannam chilli (GSC) (15.0 ± 0.6), Kashmiri chilli (KC) (16.0 ± 0.0), and Bhut Jolokia chilli (BJC) (18.0 ± 0.6). CDP formulation closely followed, displaying notable repellency activity against the same varieties. Conversely, the CCE formulation exhibited the least effectiveness in repelling the adult weevils. Thus, the repellency activity observed was time-, concentration-, and formulation-dependent. Further, the percentage repellency (PR) assay conducted by following the area preference method demonstrated that the Bhut Jolokia chilli exhibited the highest repellency percentage (61.2%) at a concentration of 0.251 μg/cm2, by categorizing it as a class IV repellent. Gas chromatography-mass spectrometry (GC-MS) analysis detected the presence of capsaicinoids like capsaicin and dihydrocapsaicin, alongside various non-capsaicinoids such as n-hexadecanoic acid and n-pentadecylacetamide, etc., in the ethanolic fraction of chilli pepper varieties, suggesting that the observed differences in the repellency activity might be attributed to varying chemical compositions, warranting further detailed exploration in this domain. These findings provide valuable primary insights into the prospective application of chilli pepper formulations from different varieties as a repellent against S. oryzae.

Evaluation of pheromone co-attractants for capturing Lasioderma serricorne (F.) (Coleoptera: Anobiidae)

The cigarette beetle, Lasioderma serricorne (F.), poses a significant threat to food storage, tobacco industries, and herbaria. The development of effective trapping methods with efficient lures is crucial for promptly monitoring this species and exploring potential mass trapping programs. The study aims to examine the potential synergistic effect of combining the synthetic sex pheromone (serricornin) with a blend of α-ionone and β-ionone as an attractant for the cigarette beetle, with the purpose of determining the capture potential of female individuals and exploring an increase in male response. Both laboratory dual-choice arena bioassays and field trap tests were conducted to assess the effectiveness of this attractant combination. The bioassays were performed in Plexiglas cages with traps baited with serricornin, α-ionone, and β-ionone. Field trap tests were conducted in a bakery plant, comparing pheromone traps with a mixture of α-ionone and β-ionone to pheromone traps lured with β-ionone alone. On the one hand, the results of the laboratory bioassays showed that traps with serricornin supplemented with a combination of α-ionone and β-ionone captured a significantly higher number of beetles compared to traps with serricornin alone. The same trend was observed for both males and females. On the other hand, field trap tests, while capturing more individuals with the combined attractant, did not show statistical significance compared to traps with only β-ionone. The study concludes that the combination of serricornin with α-ionone and β-ionone shows promise as an attractant for L. serricorne, as evidenced by significant captures in laboratory bioassays. However, the transition to field conditions reveals a need for further exploration and optimization to fill the observed gap between controlled and real-world settings.

Spectral sensitivity of click beetles (Coleoptera: Elateridae) and their responses to light stimuli in laboratory and field experiments.

With increasingly fewer insecticides registered to control the larvae of pest click beetles (Coleoptera: Elateridae), integrative beetle management, including pheromone- and light-based trapping of adult beetles, must be explored as an alternative strategy. Here, we analyzed the spectral sensitivity and color preference of 9 elaterids across 6 genera in electrophysiological recordings and in behavioral bioassays. In electroretinogram recordings (ERGs), dark-adapted beetles were exposed to narrow wavebands of light in 10-nm increments from 330 to 650 nm. All beetles proved most sensitive to green (515-538 nm) and ultraviolet (UV) light (~360 nm). In 4-choice bioassay arenas with 3 light emitting diodes (LEDs; green [525 nm], blue [470 nm], red [655 nm]) and a dark control as test stimuli, beetles discriminated between test stimuli, being preferentially attracted to green and blue LEDs. In field experiments, Vernon pitfall traps fitted with a green, blue or white LED captured significantly more male and female Agriotes lineatus and A. obscurus than dark control traps. When traps were baited with green or blue LEDs at light intensities that differed by 10-fold, the traps baited with higher light intensity lures captured numerically more beetles but trap catch data in accordance with light intensity did not differ statistically. Light-based trapping may be a viable tool for monitoring elaterid species known not to have pheromones.

Study of sexual behaviour and characterization of female sex pheromone of fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) collected from different locations of Tamil Nadu, India

The fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is considered a highly destructive pest of maize. It has its origin in the tropical and subtropical regions of America and has recently invaded many agricultural regions of India. One of the struggles in managing the pest is pheromone divergence between strains and pheromone blend variation between geographically different populations. The calling pattern, copulation behaviour and mating compatibility of different populations of S. frugiperda were investigated using behavioural methods. EAG and GC-EAD were used to analyze the chemical composition of the female gland extracts and GC–MS was used to identify the active compounds. In closed arena bioassays, the virgin males' behavioural responses to the GC-EAD compounds were further assessed. Three active compounds, Z9-14:OAc, Z11-16:OAc and Z7-12:OAc, were identified as major constituents of sex pheromone extracted from rice strain FAW which were found to be feeding on maize. All three compounds triggered intense male antennation. This study provides a deeper insight into the sexual behaviour of fall armyworm which will help in the development of more efficient lure-based pest management strategies.

Contact chemoreceptive mate recognition in Cerambyx welensii Küster (Coleoptera: Cerambycidae)

Abstract Cerambyx welensii Küster (Coleoptera: Cerambycidae) is a wood‐borer responsible for the decline of Mediterranean oaks in open woodlands. To establish that contact pheromones are involved in mate recognition of C. welensii, we extracted the cuticular hydrocarbons by solid phase microextraction at pre‐reproductive, beginning and ending of the reproductive period, and by solvent extraction of prothorax and elytra. The extracts were analysed by GC–MS under electron impact and chemical ionization conditions. Cuticular hydrocarbon profiles varied according to the reproductive period, although differences between sexes were not significant. Two compounds, 11/13‐methylheptacosane and 11‐methylnonacosane were more abundant in females at the beginning and ending of the reproductive periods. Compound 11/13‐methylheptacosane was also more representative in female prothorax than in males, and 2‐methyloctacosane was richer in male elytra than in females. We also studied the role of cuticular hydrocarbons in mate recognition in arena bioassays. Treatment of solvent‐washed dead females and glass dummies with one female equivalent (FE) of cuticular extract elicited mating responses in males, especially at the beginning of the reproductive period, with copulation attempts reaching 61.9% on solvent‐washed dead females and 23.8% on dummies. The successive treatment with synthetic compounds approaching a male cuticular profile inhibited male response. Our results confirm that contact pheromones mediate mate recognition in C. welensii. Knowledge of the precise role played by the major compounds 11/13‐methylheptacosane and 11‐methylnonacosane and other minor compounds representative in female prothorax may contribute to the development of novel management strategies against C. welensii.

Involvement of Holotrichia parallela odorant-binding protein 3 in the localization of oviposition sites

Organic fertilizers-derived volatiles attract Holotrichia parallela during oviposition. However, the mechanisms underlying the perception of oviposition cues in H. parallela remain unclear. Here, H. parallela odorant-binding protein 3 (HparOBP3) was identified as a key OBP. Bioinformatics analysis showed that HparOBP3 clustered together with Holotrichia oblita OBP8. HparOBP3 was mainly expressed in the antennae of both sexes. Recombinant HparOBP3 exhibited distinct binding affinities towards 22 compounds released by organic fertilizers. After 48 h of RNA interference (RNAi), the expression of HparOBP3 in male and female antennae was decreased by 90.77 % and 82.30 %, respectively. In addition, silencing of HparOBP3 significantly reduced the electrophysiological responses and tropism of males to cis-3-hexen-1-ol, 1-hexanol, and (Z)-β-ocimene as well as females to cis-3-hexen-1-ol, 1-hexanol, benzaldehyde, and (Z)-β-ocimene. Molecular docking indicated that hydrophobic residues Leu-83, Leu-87, Phe-108, and Ile-120 of HparOBP3 were important amino acids for interacting with ligands. Mutation of the key residue, Leu-83, significantly diminished the binding ability of HparOBP3. Furthermore, acrylic plastic arena bioassays showed that the attraction and oviposition indexes of organic fertilizers to H. parallela were reduced by 55.78 % and 60.11 %, respectively, after silencing HparOBP3. These results suggest that HparOBP3 is essential in mediating the oviposition behavior of H. parallela.

Footprints of the lady beetles Cryptolaemus montrouzieri and Tenuisvalvae notata affect their foraging behavior and predation rate

AbstractThe lady beetles Tenuisvalvae notata (Mulsant) and Cryptolaemus montrouzieri Mulsant (both Coleoptera: Coccinellidae), are used in biological control of mealybugs in many countries, including Brazil. As individuals of these species walk on plant surfaces, they leave chemical footprints that can affect their interactions with conspecifics, as well as other coccinellid species in the same environment. Therefore, this study aimed to evaluate the effects of T. notata and C. montrouzieri footprints on both their own and each other's foraging behavior and predatory potential. Arena bioassays were conducted to evaluate the effect of the footprints on conspecifics and heterospecifics. In addition, the chemical profiles of these footprints were analyzed by gas chromatography, flame ionization detection, and mass spectrometry (GC‐FID and GC–MS). For this, extracts of footprints were obtained from a glass Petri dish where 20 adults (males or females) were allowed to walk for 24 h. Behavioral bioassays showed that both species can recognize each other's footprints. Tenuisvalvae notata females avoided areas treated with footprints of heterospecific females, whereas C. montrouzieri females were arrested on areas with footprints of T. notata females. Tenuisvalvae notata adults did not change their predation rate when exposed to the footprints of heterospecifics, but second instars had a higher predation rate on areas treated with heterospecific footprints. Second instar C. montrouzieri also had an increase in predation when exposed to footprints of heterospecifics, and females captured significantly more prey when exposed to footprints of conspecifics, whereas males' predation rate decreased when exposed to footprints of both conspecifics and heterospecifics. The chemical analyses showed that the hexane extracts of footprints were composed mainly of linear hydrocarbons, saturated and non‐saturated, from C20 to C33. The composition (qualitative and quantitative) of footprint extracts is species‐ and sex‐specific. This is the first report on the effects of footprints on the behavior of these species.

Detrimental Effects of Metabolic Gas Accumulation within Termite Bioassay Arenas

Effects of metabolic gas accumulation on termite behavior in tightly sealed laboratory bioassay arenas were compared with arenas with partial air exchange, or open ventilation. Laboratory studies with termites are often conducted in glass or plastic arenas. These studies can last from a few days up to 12-16 weeks or longer. Based on personal observation and published laboratory studies, how tightly study arenas are sealed is not consistent. In some instances arenas are tightly sealed with impermeable rubber stoppers or tight-fitting lids. In other studies, arenas are covered with semi-air-permeable Parafilm®, or loose-fitting aluminum foil or ventilated lids to allow air exchange. Generally, arenas are covered and not completely open to the air, thereby maintaining high moisture requirements needed to avoid termite dehydration. In this study, to determine metabolic gas accumulation and gas effects on termite groups collected from two field colonies, bioassay arenas were capped with either tightly sealed metal lids, semi-air-permeable Parafilm, or stainless steel mesh to allow non-restricted ventilation. Gas concentrations in these arenas and termite behavior and mortality were determined over time. During the study no visible ataxic termite behavior or significant mortality was observed within Parafilm or stainless-steel-mesh capped arenas. In contrast, tightly sealed arenas quickly accumulated significantly more metabolic gases compared with the other arena configurations, causing obvious abnormal termite behavior within 96 hours after these arenas were sealed. Within 8 to 10 days 100% moribund or dead termites occurred in tightly sealed arenas, whereas termites in semi-permeable membrane or non-restricted ventilated arenas were behaving normally. Excess gas accumulation in tightly sealed arenas first elicited negative effects between 96 and 144 hours. Our results show that tightly sealed bioassay arenas must be avoided, even for short-term studies lasting only a few days, as metabolic gases accumulate rapidly, causing negative effects on termites beginning within 4 to 6 days.

Phonotactic responses of Brown Rats (Rattus norvegicus) to begging calls of Starling nestlings (Sturnus vulgaris)

Foraging behavior of Brown Rats (Rattus norvegicus) is commonly thought to be guided by olfactory cues. Here we tested the hypothesis that foraging Brown Rats eavesdrop on bird vocalizations to locate prey. We recorded calls of nestling Starlings (Sturnus vulgaris) with microphones sensitive in the sonic and ultrasonic range, respectively, and compiled clear begging calls into a single sound file (2-min long) which included the entire recorded sound range (0–100 kHz; Audio File 1). Sound analyses revealed a fundamental two-tone modulated sound centered around 3 kHz, a first and second harmonic at, respectively, 9 and 16 kHz, and bands of ultrasonic frequency components at 20, 25, and 37 kHz. We subjected Audio File 1 to low- and highpass-filtering, thereby producing a sonic-range only file (< 20 kHz; Audio File 2), and an ultrasonic-range only file (20–100 kHz; Audio File 3). In binary-choice large arena bioassays, we then tested the behavioral responses of single Brown Rats to paired trap boxes each fitted with a speaker, one of which emitting a white noise control and the other playing back Audio File 1 (Exp. 1), Audio File 2 (Exp. 2), or Audio File 3. In each of experiments 1–3, female and male rats (i) significantly more often entered first the trap box broadcasting an Audio File, and (ii) spent significantly more time in the arena quadrant with an Audio File trap box. Our data support the conclusion that foraging Brown Rats, as opportunistic omnivores, exploit begging calls of nestling Starlings, and possibly other birds, as cues to obtain a proteinaceous meal.

Bioactivity of essential oil-based nano-biopesticides toward Rhyzopertha dominica (Coleoptera: Bostrichidae)

Essential oils (EOs) are promising active ingredients to produce biopesticides, although their physicochemical characteristics are a critical issue to develop commercial formulates. Repellent and toxic activity of EOs against crop and stored product pests has been widely investigated in the last decades; however, no information is available on the occurrence of adverse undesirable behavioral responses (i.e. habituation) toward these repellents in target pest species. In this study, stable EO-based nano-emulsions from commercial fennel (Foeniculum vulgare), mint (Mentha x piperita) and sweet orange (Citrus sinensis) EOs were developed, and their repellence was tested against a major stored product pest, the lesser grain borer Rhyzopetha dominica. Besides, the occurrence of habituation in R. dominica adults following successive exposure to the repellent formulations was evaluated, considering the main characteristics of this kind of non-associative learning. Nanometric droplet size was achieved for all developed nano-emulsions (<200 nm). All the tested EO-based nano-formulations were repellent to R. dominica, whereas F. vulgare-based nano-emulsions triggered lower repellence both in area choice and arena bioassays. The occurrence of habituation was validated for the strongest repellents, M. piperita and C. sinensis formulations and the decline of R. dominica responses was frequency-dependent. Furthermore, insects completely recovered their responsiveness toward the biopesticides just after 24 h from the end of exposure. The decline of the responsiveness in R. dominica adults was attributable to real learning process, since motor and sensory fatigue were excluded by testing stimulus specificity and dishabituation. The results demonstrated that habituation could occur for repellent EO-based formulations, thus this behavioral process can reduce the effectiveness of these kind of treatments against R. dominica and should be considered to articulate adequate IPM programs against stored product pests.

Necrobia rufipes (De Geer) Infestation in Pet Food Packaging and Setup of a Monitoring Trap.

Simple SummaryNecrobia rufipes (Coleoptera: Cleridae) is an emerging pest of pet food stores. Information on infestation modalities for this pest is absent and specific monitoring tools are missing. In this paper, the adults’ and larvae’s ability to enter into pet food packaging was evaluated. Furthermore, to set up of a monitoring trap behavioral bioassays were carried out: testing two adhesive surfaces, one generally used in mouse glue trap and the other used in cockroach trap, to evaluate their ability in avoiding insects’ escape; screening different molecules, as candidate food attractants: methyl cyclopentenolone, squalene and stearic acid. The results evidenced that N. rufipes enter in packaging through the air vent valves, suggesting that a way to prevent insect infestation would be to modify packaging. Tests showed that the glues have strong differences in the ability to retain the caught insects, with mouse glue more effective than cockroach glue. The behavioral bioassays indicated that methyl cyclopentenolone and squalene are able to attract N. rufipes adults in olfactometer. Finally, the dual-choice arena bioassays showed that a mixture of pet food and methyl cyclopentenolone elicited the strongest attraction in N. rufipes adults, suggesting that this mixture can be used as lure in monitoring traps.Necrobia rufipes (De Geer) (Coleoptera: Cleridae), also known as the red-legged ham beetle, is a newly emerging pest of pet food stores, causing apprehension among producers worldwide. Concerns about this pest are exacerbated by the lack of information about infestation modalities in pet food, while specific monitoring tools are missing. Considering that adequate pet food packaging could limit N. rufipes infestations, information about the penetration modalities in commonly used pet food packaging is needed. Moreover, the development of appropriate monitoring instruments is urgent to detect pest presence early and to reduce chemical treatments for its control. In this paper, the adults’ and larvae’s ability to enter into pet food packaging was evaluated. Furthermore, to develop monitoring traps, behavioral bioassays were done: (1) testing two different commercial adhesive surfaces, one generally used in mouse glue traps (MG), and the other used in cockroach glue traps (CG), to evaluate their different abilities in avoiding insects’ escape; (2) screening different molecules, typical of the substrates attacked by N. rufipes, as candidate food attractants for this pest: methyl cyclopentenolone (MCP), squalene (SQ), and stearic acid (SA). The results show that N. rufipes adults and larvae enter into packaging through the air vent valves on the bottom, suggesting that a way to improve the packaging to prevent insect infestation would be to modify these points of weakness. Laboratory tests show that the different bioassayed glues have strong differences in the ability to retain the caught insects, with MG being more effective than CG. The behavioral bioassay indicated that MCP and SQ attract N. rufipes adults in olfactometer. Finally, the results of dual-choice arena bioassays show that among the candidate attractant tested, a mixture of pet food (PF) and MCP elicited the strongest attraction in N. rufipes adults. These results encourage further experiments with the use of an MG adhesive trap loaded with a mixture of PF+MCP to test the effectiveness of such a tool for monitoring N. rufipes in pet food industries and warehouses.

Efficacy Determination of Commercial Deltamethrin-Treated Storage Bags on Trogoderma granarium Everts Adults and Larvae

Trogoderma granarium Everts, the khapra beetle, is a serious stored product pest known to feed on &gt;100 different products worldwide and is a major threat to global food security. Deltamethrin-treated storage bags are a resource that could be used to limit infestations during storage of grain in bags. We investigated the efficacy of deltamethrin-treated bags against T. granarium adults and larvae. Deltamethrin-treated and untreated packaging materials were affixed into the bottom of plastic Petri dishes (62 or 137 cm2) to create a bioassay arena. Adult T. granarium were exposed and observed to determine the time to knockdown and the subsequent mortality rate within 24 h. Adult T. granarium were knocked down in &lt;60 min, and 100% of adults were knocked down or dead after 24 h. Trogoderma granarium larvae were exposed for 0.33, 1, 2, 3, or 4 d or continually exposed and monitored for larval death and adult emergence. Larvae exposed for 4 d had 50% mortality versus 97% if continually exposed. Utilizing this deltamethrin-treated packaging could cause disruptions in natural populations of T. granarium found in storage facilities, and the treated packaging is an effective tool that could be implemented into an integrated pest management program for bagged grain.

A blend of formic acid, benzoic acid, and aliphatic alkanes mediates alarm recruitment responses in western carpenter ants, Camponotus modoc

AbstractFormicine ants in distress spray alarm pheromone which typically recruits nestmates for help. Studying the western carpenter ant, Camponotus modoc Wheeler (Hymenoptera: Formicidae), our objectives were to (1) determine the exocrine glands that contain alarm recruitment pheromone, (2) identify the key alarm recruitment pheromone components, and (3) ascertain the pheromone components that are discharged by distressed ants. In Y‐tube olfactometer experiments, extracts of poison glands, but not of Dufour’s glands, elicited anemotactic responses from worker ants. Gas chromatographic‐mass spectrometric analyses of poison gland extracts revealed the presence of (1) aliphatic alkanes (undecane, tridecane, pentadecane, heptadecane), (2) aliphatic alkenes [(Z)‐7‐pentadecene, (Z)‐7‐ and (Z)‐8‐heptadecene], (3) two acids (formic, benzoic), and (4) other oxygenated compounds (hexadecan‐1‐ol, hexadecyl formate, hexadecyl acetate). Testing the responses of worker ants in Y‐tube olfactometers to complete and partial synthetic blends of these compounds revealed that the acids and the alkanes are essential alarm pheromone components. In two‐choice arena bioassays, micro‐locations treated with synthetic alarm pheromone recruited worker ants. Acids and alkanes were abundant in the poison gland and the Dufour’s gland, respectively, suggesting that the alarm pheromone components originate from both glands. Moreover, alarm pheromone sprays of ants differed in that all sprays contained formic acid but only some also contained alkanes, implying that ants can independently discharge the content of either one or both glands in accordance with the type of distress incident they experience.

Plant secondary metabolites and low temperature are the major limiting factors for Beauveria bassiana (Bals.-Criv.) Vuill. (Ascomycota: Hypocreales) growth and virulence in a bark beetle system

Beauveria bassiana is a ubiquitous entomopathogen and widely used as a biological control agent for a variety of arthropod pests, including bark beetles. The North American spruce beetle (Dendroctonus rufipennis) is a major pest in forest landscapes and recent studies show that B. bassiana is pathogenic to beetles in the lab but successful field applications have been limited by abiotic and biotic factors within the study system. To understand how habitat conditions impact fungal ecology, we evaluated variation in B. bassiana radial growth across conditions representative of the spruce beetle habitat, incorporating a range of temperatures, competition with spruce beetle symbiotic fungi, as well as exposure to Engelmann spruce tree secondary metabolites, nutrient limitations, osmotic potentials, and sunlight. Pathogenicity to spruce beetle was subsequently quantified in tests varying beetle origin, temperature, and bioassay arena substrate. Three major findings emerged: (1) growth of genetically similar B. bassiana isolates varied considerably in response to abiotic conditions, suggesting significant within-haplotype phenotypic diversity; (2) low ambient temperatures and exposure to Engelmann spruce tree secondary metabolites, two conditions which are prevalent in spruce beetle habitat, strongly inhibit B. bassiana growth; and (3) pathogenicity varied across environments: all isolates appeared pathogenic under room conditions, but when beetles inhabited in planta bioassays under in natura conditions most isolates were not pathogenic. This is the first study to evaluate the inhibitory effects that a series of tree secondary metabolites have on B. bassiana growth, virulence and pathogenicity. These collective findings have implications for field applications of B. bassiana but also the interpretation of entomopathogenicity, and could help to explain discrepancies between laboratory and field tests.

Potential of entomopathogenic nematode isolates from Rwanda to control the tomato leaf miner, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

Tomato leaf miner, Tuta absoluta (Meyrick), is a major threat to tomato production as it can cause up to 100% yield loss under both greenhouse and open-field conditions. Chemical control, which is associated with several undesirable effects, remains the only option readily available for this pest since its invasion of Rwanda in the year 2015. This study assessed the potential of using local isolates of entomopathogenic nematodes (EPNs) in management of T. absoluta in Rwanda. Six EPNs including four locally isolated strains: Steinernema sp. RW14-M-C2a-3, Steinernema sp. RW14-M-C2b-1, S. carpocapsae RW14-G-R3a-2 and Heterorhabditis bacteriophora RW14-N-C4a, and two exotic species: S. carpocapsae All and H. bacteriophora H06 were evaluated. Three bioassays were conducted in the laboratory, using a tomato leaflet with third instar T. absoluta larva in gallery and 9-cm Petri dishes as bioassay arenas in a completely randomized design with three replications. The EPNs were applied at a volume of 1 ml containing 500 infective juveniles per leaflet, while sterile tap water was used as negative control. Larval mortality was checked continuously for 96 h at 24 h interval. The results revealed that all the tested EPNs were able to find and kill T. absoluta larvae inside the leaf galleries; and their efficacy increased with exposure time. The pathogenicity effects were significantly different (p < 0.05) among EPNs. In the first 24 h after inoculation, the efficacy of local EPN isolates (53.3–96.7%) was significantly higher than the one of exotic species (0.0–26.7%). The efficacy of three Rwandan EPN isolates, Steinernema sp. RW14-M-C2a-3, Steinernema sp. RW14-M-C2b-1, and S. carpocapsae RW14-G-R3a-2 was not significantly different from 24 to 96 h after inoculation, except for S. carpocapsae RW14-G-R3a-2 during 24 h after inoculation in bioassay 3. There was insignificant difference among all the EPN isolates after 96 h of exposure. This is the first study carried out in Rwanda that investigated the potential of locally isolated EPNs against T. absoluta. Field experiments should be conducted to fully explore the possibilities of using local EPN isolates in integrated pest management of T. absoluta in Rwanda.

Interplay between Selenium, selenoprotein genes, and oxidative stress in honey bee Apis mellifera L.

The honey bee, Apis mellifera L., is a major pollinator insect that lacks novel “selenoprotein genes”, rendering it susceptible to elevated levels of Selenium (Se) occurring naturally in the environment. We investigated the effects of two inorganic forms of Se on biological traits, oxidative stress, and gene regulation. Using bioassay arenas in the laboratory, one-day old sister bees were fed ad libitum 4 different concentrations of selenate and selenite, two common inorganic forms of Se. The transcription levels of 4 honey bee antioxidant genes were evaluated, and three putative selenoprotein-like genes (SELENOT, SELENOK, SELENOF) were characterized as well as Sbp2, a Selenium binding protein required for the translation of selenoproteins mRNA. Oxidative stress and Se residues were subsequently quantified in honey bee bodies throughout the experiment.Se induced higher oxidative stress in treated honey bees leading to a significantly elevated protein carbonyl content, particularly at the highest studied concentrations. Early upregulations of Spb2 and MsrA were identified at day 2 of the treatment while all genes except SELENOT were upregulated substantially at day 8 to alleviate the Se-induced oxidative stress levels. We determined that doses between 60 and 600 mg.Se.L−1 were acutely toxic to bees (<48 h) while doses between 0.6 and 6 mg.Se.L−1 led to much lower mortality (7–16)%. Furthermore, when fed ad libitum, Se residue data indicated that bees tolerated accumulation up to 0.12 µg Se bee−1 for at least 8 days with a Se LC50 of ∼6 mg/L, a field realistic concentration found in pollen of certain plants in a high Se soil environment.

Key Elements of Photo Attraction Bioassay for Insect Studies or Monitoring Programs.

Optimized visual attractants will increase insect trapping efficiency by using the target insect's innate behaviors (positive photo-taxis) as a means to lure the insect into a population control or monitoring trap. Light emitting diodes (LEDs) have created customizable lighting options with specific wavelengths (colors), intensities, and bandwidths, all of which can be customized to the target insects. Photo-attraction behavioral bioassays can use LEDs to optimize the attractive color(s) for an insect species down to specific life history stages or behaviors (mating, feeding, or seeking shelter). Researchers must then confirm the bioassay results in the field and understand the limited attractive distance of the visual attractants. The cloverleaf bioassay arena is a flexible method to assess photo attraction while also assessing a range of natural insect behaviors such as escape and feeding responses. The arena can be used for terrestrial or aerial insect experiments, as well as diurnal, and nocturnal insects. Data collection techniques with the arena are videotaping, counting contact with the lights, or physically collecting the insects as they are attracted towards the lights. The assay accounts for insects that make no-choice and the arenas can be single (noncompetitive) color or multiple (competitive) colors. The cloverleaf design causes insects with strong thigmotaxis to return to the center of the arena where they can view all the options in a competitive LED tests. The cloverleaf arena presented here has been used with mosquitoes, bed bugs, Hessian fly, house flies, biting midges, red flour beetles, and psocids. Bioassays are used to develop accurate and effective insect traps to guide the development and optimization of insect traps used to monitor pest population fluctuations for disease vector risk assessments, the introduction of invasive species, and/or be used for population suppression.

Aerosol insecticide distribution inside a flour mill: Assessment using droplet measurements and bioassays

The distribution of aerosol applications of pyrethrin + methoprene, generated from a mechanical fogger, and pyrethrin + pyriproxyfen, dispensed from a pressurized cylinder, were characterized inside a pilot-scale flour mill using measurements of particle size and concentration and effects on adult confused flour beetles, Tribolium confusum Jacqueline duVal, in bioassay arenas. Particle measurements were made using TSI Aerodynamic Particle Sizer (APS) units placed in an open straight line at distances of 4.3, 8.9, and 13.5 m from the aerosol release point (open configuration). Measurements were also made using a second configuration (termed obstructed), which was done by moving the APS unit at 8.9 m underneath a piece of equipment, and moving the APS unit at 13.5 m behind a support beam. Actual concentration and calculated deposition were about 4× greater for the pyrethrin + methoprene aerosol compared to the pyrethrin + pyriproxyfen aerosol. However, efficacy was similar since bioassays using adult T. confusum showed no difference in recovery after exposure to either insecticide. Concentration and calculated deposition of both aerosols decreased with increasing distance from the spray release point and when the APS units were in the obstructed configuration, and recovery of bioassay insects after exposure increased with increasing distance from the spray release point. Results from this field trial show how efficacy of aerosol applications is impacted by distance and obstacles, and how use of equipment that measures droplet size and concentration can help quantify the dispersion and spread of insecticidal aerosols. Results also provide guidance to develop relationships between aerosol deposition and efficacy and thus improve pest management programs for structural management of stored product insects.

Insect pathogenic fungi and bed bugs: behaviour, horizontal transfer and the potential contribution to IPM solutions

The increasing problem of bed bugs requires the development of new control strategies, and insect pathogenic fungi can contribute towards management. We used laboratory bioassays with Isaria fumosoroseus, Lecanicillium muscarium and Beauveria bassiana to evaluate their virulence to the bed bug. Only B. bassiana significantly affected bed bug survival and was dependent on dose and formulation. A 2% B. bassiana oil formulation induced horizontal transfer to elevate mortality in a 10-day arena bioassay. Temporal distribution of contagious individuals and increasing the dose from 2 to 4% did not increase mortality. Horizontal transfer mainly occurred between adults, and only partly between adults and nymphs. Bed bugs showed activity peaks during the night, and activity was increased by elevated levels of CO2. Distribution between harbourages was not affected by CO2 activation, level of infection or the bio-pesticide, and horizontal transfer was not dependent on the degree of aggregation. Movement in the arenas negatively affected horizontal transfer when the number of susceptible individuals was large. Level of infection also influenced behaviour as the bed bug movement increased with elevated disease burden. The use of fungi as a part of an integrated pest management strategy seems to be an interesting option that should be investigated further. B. bassiana kills bed bugs and can be carried to harbourages to target hidden individuals.