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Insects, Volume 10, Issue 11 (November 2019)

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Cover Story (view full-size image) Ants deposit trail pheromones that guide nestmates to food sources. We predicted that ant community [...] Read more.
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Open AccessArticle
Comparative Proteomic Analysis Reveals Immune Competence in Hemolymph of Bombyx mori Pupa Parasitized by Silkworm Maggot Exorista sorbillans
Insects 2019, 10(11), 413; https://doi.org/10.3390/insects10110413 - 18 Nov 2019
Viewed by 272
Abstract
The silkworm maggot, Exorista sorbillans, is a well-known larval endoparasitoid of the silkworm Bombyx mori that causes considerable damage to the silkworm cocoon crop. To gain insights into the response mechanism of the silkworm at the protein level, we applied a comparative [...] Read more.
The silkworm maggot, Exorista sorbillans, is a well-known larval endoparasitoid of the silkworm Bombyx mori that causes considerable damage to the silkworm cocoon crop. To gain insights into the response mechanism of the silkworm at the protein level, we applied a comparative proteomic approach to investigate proteomic differences in the hemolymph of the female silkworm pupae parasitized by E. sorbillans. In total, 50 differentially expressed proteins (DEPs) were successfully identified, of which 36 proteins were upregulated and 14 proteins were downregulated in response to parasitoid infection. These proteins are mainly involved in disease, energy metabolism, signaling pathways, and amino acid metabolism. Eight innate immune proteins were distinctly upregulated to resist maggot parasitism. Apoptosis-related proteins of cathepsin B and 14-3-3 zeta were significantly downregulated in E. sorbillans-parasitized silkworm pupae; their downregulation induces apoptosis. Quantitative PCR was used to further verify gene transcription of five DEPs, and the results are consistent at the transcriptional and proteomic levels. This was the first report on identification of possible proteins from the E. bombycis-parasitized silkworms at the late stage of parasitism, which contributes to furthering our understanding of the response mechanism of silkworms to parasitism and dipteran parasitoid biology. Full article
(This article belongs to the Special Issue Insect Immunity and Pathology)
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Open AccessArticle
Olfactory Learning in the Stingless Bee Melipona eburnea Friese (Apidae: Meliponini)
Insects 2019, 10(11), 412; https://doi.org/10.3390/insects10110412 - 18 Nov 2019
Viewed by 264
Abstract
Olfactory learning and floral scents are co-adaptive traits in the plant–pollinator relationship. However, how scent relates to cognition and learning in the diverse group of Neotropical stingless bees is largely unknown. Here we evaluated the ability of Melipona eburnea to be conditioned to [...] Read more.
Olfactory learning and floral scents are co-adaptive traits in the plant–pollinator relationship. However, how scent relates to cognition and learning in the diverse group of Neotropical stingless bees is largely unknown. Here we evaluated the ability of Melipona eburnea to be conditioned to scent using the proboscis extension reflex (PER) protocol. Stingless bees did not show PER while harnessed but were able to be PER conditioned to scent when free-to-move in a mini-cage (fmPER). We evaluated the effect of: 1) unconditioned stimulus (US) reward, and 2) previous scent–reward associations on olfactory learning performance. When using unscented-US, PER-responses were low on day 1, but using scented-US reward the olfactory PER-response increased on day 1. On day 2 PER performance greatly increased in bees that previously had experienced the same odor and reward combination, while bees that experienced a different odor on day 2 showed poor olfactory learning. Bees showed higher olfactory PER conditioning to guava than to mango odor. The effect of the unconditioned stimulus reward was not a significant factor in the model on day 2. This indicates that olfactory learning performance can increase via either taste receptors or accumulated experience with the same odor. Our results have application in agriculture and pollination ecology. Full article
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Open AccessArticle
The Competitive Mating of Irradiated Brown Marmorated Stink Bugs, Halyomorpha halys, for the Sterile Insect Technique
Insects 2019, 10(11), 411; https://doi.org/10.3390/insects10110411 - 16 Nov 2019
Cited by 1 | Viewed by 427
Abstract
The sterility of eggs and nymphs from gamma-irradiated male Halyomorpha halys was investigated to determine the potential for the sterile insect technique (SIT). Males irradiated at 0, 16, 24 and 32 Gy were placed with untreated virgin females, and egg sterility was determined, [...] Read more.
The sterility of eggs and nymphs from gamma-irradiated male Halyomorpha halys was investigated to determine the potential for the sterile insect technique (SIT). Males irradiated at 0, 16, 24 and 32 Gy were placed with untreated virgin females, and egg sterility was determined, showing 54.3% at 16 Gy. The percentage of sterility from irradiation was 26 percent lower than previous results from the USA and the variance was very high. Competitive overflooding ratio trials between irradiated virgin males and fertile virgin males at a 5:1 ratio resulted in the expected egg sterility, indicating competitive performance by irradiated males. By July and August, older, irradiated overwintered males were significantly less competitive than similar, non-irradiated males. There is a need to revisit the irradiation delivery method to achieve proper precision around the paternal dose required for an expected >80% egg sterility and subsequent ~99% endpoint sterility estimated at adult emergence in the F1 phase. These results suggest that the mating competitiveness and competency of males after irradiation at 16 Gy is not limiting to the sterile insect technique for suppression. A wild harvest of overwintering males using the aggregation pheromone, followed by irradiation and male release, might replace rearing. Mass-collected, sterilized bugs could be transported from an area of high H. halys density and shipped for release to enable suppression or eradication elsewhere. This concept is under development but further work is needed now to understand the difference in results between the US and Italian irradiators and increase the reliability of dosimetry. Full article
(This article belongs to the Special Issue Sterile Insect Technique (SIT) and Its Applications)
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Open AccessArticle
Evidence of Leaf Consumption Rate Decrease in Fall Armyworm, Spodoptera frugiperda, Larvae Parasitized by Coccygidium luteum
Insects 2019, 10(11), 410; https://doi.org/10.3390/insects10110410 - 16 Nov 2019
Viewed by 311
Abstract
Biological control is one of the best options for the sustainable management of the invasive maize pest Spodoptera frugiperda in Africa. However, there is limited knowledge of the efficacy of native natural enemies of S. frugiperda and their potential use in integrated pest [...] Read more.
Biological control is one of the best options for the sustainable management of the invasive maize pest Spodoptera frugiperda in Africa. However, there is limited knowledge of the efficacy of native natural enemies of S. frugiperda and their potential use in integrated pest management. The endoparasitoid wasp Coccygidium luteum is one of the natural enemies of S. frugiperda in Africa. This study assessed, under laboratory conditions, the effect of C. luteum on the leaf consumption rate of its host. Fifty first instar S. frugiperda larvae were exposed to C. luteum for oviposition and the maize leaf consumption rate of parasitized larvae was assessed and compared to 50 unparasitized larvae from the same cohort. Coccygidium luteum completed a generation, from egg to adult emergence, in 16.7 days. The leaf consumption rate of parasitized S. frugiperda larvae declined gradually compared to unparasitized larvae and the overall consumption reduction by parasitized S. frugiperda larvae was 89%. Our findings show that C. luteum could reduce damage caused by S. frugiperda to maize farms but, prior to its use in biological control programmes, further studies are needed to assess potential parasitism rates in field conditions and develop a cost-effective mass production system. Full article
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Open AccessArticle
Characterization of the Expression and Functions of Two Odorant-Binding Proteins of Sitophilus zeamais Motschulsky (Coleoptera: Curculionoidea)
Insects 2019, 10(11), 409; https://doi.org/10.3390/insects10110409 - 15 Nov 2019
Viewed by 312
Abstract
Odorant-binding proteins (OBPs) are important in insect chemical communication. The objective of this research was to identify the functions of two OBPs in Sitophilus zeamais. qRT-PCR and western blot (WB) were performed to investigate the expression profiles at the transcript and protein [...] Read more.
Odorant-binding proteins (OBPs) are important in insect chemical communication. The objective of this research was to identify the functions of two OBPs in Sitophilus zeamais. qRT-PCR and western blot (WB) were performed to investigate the expression profiles at the transcript and protein levels, respectively. Fluorescence competitive binding assays were used to measure the ability of the OBPs to bind to host volatiles, and a Y-tube olfactometer was used to verify the results (attraction/no response) via behavioral experiments. The RNAi was used to verify the function by knocking down the ability of proteins to bind odorants. qRT-PCR showed the highest expression SzeaOBP1 and SzeaOBP28 at the low-instar larva (LL) and eclosion adult (EA) stages, respectively. WB showed that both SzeaOBP1 and SzeaOBP28 were highly expressed in the EA stage. Fluorescence competitive binding assays indicated that SzeaOBP1 exhibited extremely high binding affinity with cetanol. SzeaOBP28 exhibited a pronounced binding affinity for 4-hydroxy-3-methoxybenzaldehyde. The behavioral experiment showed that the adult S. zeamais responded strongly to 4-hydroxy-3-methoxybenzaldehyde and valeraldehyde from Sorghum bicolor. The RNAi knockdown individuals displayed behavioral differences between normal insects and dsRNA (SzeaOBP1)-treated insects. We infer that they both have functions in perception and recognition of host volatiles, whereas SzeaOBP28 may also have other functions. Full article
(This article belongs to the Special Issue Semiochemicals and Insect Behavior)
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Open AccessArticle
What Can Integrated Analysis of Morphological and Genetic Data Still Reveal about the Anastrepha fraterculus (Diptera: Tephritidae) Cryptic Species Complex?
Insects 2019, 10(11), 408; https://doi.org/10.3390/insects10110408 - 15 Nov 2019
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Abstract
The South American fruit fly Anastrepha fraterculus (Wiedemann) is a complex of cryptic species, the so-called “Anastrepha fraterculus complex”, for which eight morphotypes are currently recognized. A previous analysis of ITS1 in samples of the Anastrepha fraterculus complex, while revealing high distinctiveness [...] Read more.
The South American fruit fly Anastrepha fraterculus (Wiedemann) is a complex of cryptic species, the so-called “Anastrepha fraterculus complex”, for which eight morphotypes are currently recognized. A previous analysis of ITS1 in samples of the Anastrepha fraterculus complex, while revealing high distinctiveness among samples from different localities of South America, Central America, and Mexico, no direct association was made between sequence type and morphotype. In the present report, a correlated analysis of morphometry and ITS1 data involved individuals belonging to the same population samples. Although showing a low level of intra-populational nucleotide variability, the ITS1 analysis indicated numerous inter-population sequence type variants. Morphotypes identified by morphometric analysis based on female wing shape were highly concordant with ITS1 genetic data. The correlation of genetic divergence and morphological differences among the tested samples gives strong evidence of a robust dataset, thereby indicating the existence of various taxonomic species within the A. fraterculus complex. However, the data revealed genetic and morphological variations in some regions, suggesting that further analysis is still required for some geographic regions. Full article
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Open AccessArticle
The Power of Electropenetrography in Enhancing Our Understanding of Host Plant-Vector Interactions
Insects 2019, 10(11), 407; https://doi.org/10.3390/insects10110407 - 15 Nov 2019
Viewed by 230
Abstract
The invasive Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is the primary vector of the phloem-infecting bacterium, Candidatus Liberibacter asiaticus. Candidatus L. asiaticus is the putative causal agent of Huanglongbing (HLB) disease, a destructive disease of Citrus. While many Citrus species are [...] Read more.
The invasive Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is the primary vector of the phloem-infecting bacterium, Candidatus Liberibacter asiaticus. Candidatus L. asiaticus is the putative causal agent of Huanglongbing (HLB) disease, a destructive disease of Citrus. While many Citrus species are susceptible to D. citri probing and HLB disease, there are marked behavioral differences in D. citri probing responses and Ca. Liberibacter asiaticus infection severity among Citrus species. Using four mandarin hybrid selections and pummelo plants variably resistant to D. citri probing, oviposition, and survival, we explored probing differences using electropenetrography (EPG), conducted an oviposition and survival study, and determined host plant metabolites using gas-chromatography mass-spectroscopy (GC-MS). We found thirty-seven D. citri probing variables to be significantly different among tested mandarin selections and pummelo, in addition to differential oviposition and survivorship abilities on tested plants. We found sixty-three leaf metabolites with eight being significantly different among tested mandarin selections and pummelo. Detailed analysis of probing behavior, oviposition, survivorship, and host plant metabolite concentrations reveals the complex, layered resistance mechanisms utilized by resistant Citrus against D. citri probing. EPG is a powerful technology for screening Asian citrus psyllid resistant Citrus to elucidate host plant-vector interactions, with an aim to minimize vector probing and eliminate the spread of the bacterial pathogen, Ca. L. asiaticus. Full article
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Open AccessCorrection
Correction: Wang, C.; et al. Changes in Indoor Insecticide Residue Levels after Adopting an Integrated Pest Management Program to Control German Cockroach Infestations in an Apartment Building. Insects 2019, 10, 304
Insects 2019, 10(11), 406; https://doi.org/10.3390/insects10110406 - 15 Nov 2019
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Abstract
Following the publication of our article [...] Full article
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Open AccessArticle
The Invasion of the Dwarf Honeybee, Apis florea, along the River Nile in Sudan
Insects 2019, 10(11), 405; https://doi.org/10.3390/insects10110405 - 15 Nov 2019
Viewed by 380
Abstract
The spread of the dwarf honeybee, Apis florea, in Sudan along the river Nile in a linear fashion provides a good model for studying the population dynamics and genetic effects of an invasion by a honeybee species. We use microsatellite DNA analyses [...] Read more.
The spread of the dwarf honeybee, Apis florea, in Sudan along the river Nile in a linear fashion provides a good model for studying the population dynamics and genetic effects of an invasion by a honeybee species. We use microsatellite DNA analyses to assess the population structure of both invasive A. florea and native Apis mellifera along the river Nile. The invasive A. florea had significantly higher population densities than the wild, native A. mellifera. Nevertheless, we found no indication of competitive displacement, suggesting that although A. florea had a high invasive potential, it coexisted with the native A. mellifera along the river Nile. The genetic data indicated that the invasion of A. florea was established by a single colony. Full article
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Open AccessCommunication
First Report of Cattle Tick Rhipicephalus (Boophilus) annulatus in Egypt Resistant to Ivermectin
Insects 2019, 10(11), 404; https://doi.org/10.3390/insects10110404 - 15 Nov 2019
Viewed by 248
Abstract
Tick control is mainly dependent on the application of acaricides, but resistance has developed to almost all classes of acaricides, including macrolactones. Therefore, we aimed to investigate ivermectin resistance among tick populations in middle Egypt. The larval immersion test was conducted using a [...] Read more.
Tick control is mainly dependent on the application of acaricides, but resistance has developed to almost all classes of acaricides, including macrolactones. Therefore, we aimed to investigate ivermectin resistance among tick populations in middle Egypt. The larval immersion test was conducted using a commercial formulation of ivermectin (1%). Different concentrations of the immersion solution (0.0000625% (625 × 10−7%), 0.000125% (125 × 10−6%), 0.0005% (5 × 10−4%), 0.001% (1 × 10−3%), 0.0025% (2.5 × 10−3%), 0.005% (5 × 10−3), and 0.01% (1 × 10−2%)) were prepared by diluting a commercial ivermectin (1%) with distilled water containing 1% (v/v) ethanol and 2% (v/v) TritonX-100. Field populations of Rhipicephalus annulatus were collected from five different localities in Beni-Suef province, Egypt. Adult engorged female ticks were collected and assessed for oviposition and egg fertility. Eggs were collected, and hatched larvae were then used in the experiment. Application of acaricides was conducted on 10-day-old larvae. There was a significant difference in the LC50 (50% lethal concentration) among the examined localities on the log dose-response plot, where, the LC50 of tick populations from two localities (Emin elaros and Aldiabia) was higher than the other localities (Alhalabia, Alkom, and Beshnna). Besides, tick populations from Emin elaros and Aldiabia showed higher LC90 values with lower slope values compared to those from Alhalabia, Alkom, and Beshnna. According to these values (LC50, LC90, and slope values), as well as a history of acaricide failure to ticks in these areas, R. annulatus developed resistance to ivermectin. This study documents the first report of field populations of R. annulatus resistant to ivermectin in Egypt. Full article
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Open AccessArticle
Electrophysiological Responses of Eighteen Species of Insects to Fire Ant Alarm Pheromone
Insects 2019, 10(11), 403; https://doi.org/10.3390/insects10110403 - 14 Nov 2019
Viewed by 267
Abstract
Olfaction plays a dominant role in insect communication. Alarm pheromones, which alert other insects of the same species of impending danger, are a major class of releaser pheromones. The major components of alarm pheromones in red imported fire ants, honeybees and aphids have [...] Read more.
Olfaction plays a dominant role in insect communication. Alarm pheromones, which alert other insects of the same species of impending danger, are a major class of releaser pheromones. The major components of alarm pheromones in red imported fire ants, honeybees and aphids have been identified as 2-ethyl-3,6-dimethylpyrazine (2E-3,6-DP), isopentyl acetate (IPA), and E-β-farnesene (EβF), respectively. In this study, electroantennography (EAG) responses to EDP (a mixture of 2-ethyl-3,6-dimethylpyrazine and 2-ethyl-3,5-dimethylpyrazine), IPA and EβF were investigated in a wide range of insect species. Beside imported fire ants, the EDP (2-ethyl-3,6(5)-dimethylpyrazine) elicited significant EAG response from all other tested insects, including six ant species and one hybrid ant, honeybee, bagrada bug, lady beetle, housefly, small hive beetle, yellow fever mosquito, termite, bedbug, water hyacinth weevil, southern green stink bug and two aphid species. In contrast, IPA elicited significant EAG response only in the honeybee, red imported fire ant, an Aphaenogaster ant, and the water hyacinth weevil. The EβF only elicited EAG responses in two aphids, small hive beetle and housefly. The results clearly indicate that EDP can be detected by widespread insect species that did not coevolve with S. invicta and further suggested alkylpyrazine may activate multiple generally tuned olfactory receptors (ORs) across a wide number of insect species. Full article
(This article belongs to the Special Issue Semiochemicals and Insect Behavior)
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Open AccessCommunication
Descriptive Bacterial and Fungal Characterization of Propolis Using Ultra-High-Throughput Marker Gene Sequencing
Insects 2019, 10(11), 402; https://doi.org/10.3390/insects10110402 - 12 Nov 2019
Viewed by 258
Abstract
Bees harbor microorganisms that are important for host health, physiology, and survival. Propolis helps modulate the immune system and health of the colony, but little information is available about its microbial constituents. Total genomic DNA from samples of natural propolis from Apis mellifera [...] Read more.
Bees harbor microorganisms that are important for host health, physiology, and survival. Propolis helps modulate the immune system and health of the colony, but little information is available about its microbial constituents. Total genomic DNA from samples of natural propolis from Apis mellifera production hives from four locations in Mexico were used to amplify a region of the 16S rRNA gene (bacteria) and the internal transcriber spacer (fungi), using PCR. The Illumina MiSeq platform was used to sequence PCR amplicons. Extensive variation in microbial composition was observed between the propolis samples. The most abundant bacterial group was Rhodopila spp. (median: 14%; range: 0.1%–27%), a group with one of the highest redox potential in the microbial world. Other high abundant groups include Corynebacterium spp. (median: 8.4%; 1.6%–19.5%) and Sphingomonas spp. (median: 5.9%; 0.03%–14.3%), a group that has been used for numerous biotechnological applications because of its biodegradative capabilities. Bacillus and Prevotella spp. alone comprised as much as 88% (53% and 35%, respectively) of all bacterial microbiota in one sample. Candida (2%–43%), Acremonium (0.03%–25.2%), and Aspergillus (0.1%–43%) were among the most abundant fungi. The results contribute to a better understanding of the factors associated with the health of Apis mellifera production hives. Full article
(This article belongs to the Special Issue New Perspectives on Honeybee Health)
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Open AccessReview
Towards Precision Nutrition: A Novel Concept Linking Phytochemicals, Immune Response and Honey Bee Health
Insects 2019, 10(11), 401; https://doi.org/10.3390/insects10110401 - 12 Nov 2019
Viewed by 311
Abstract
The high annual losses of managed honey bees (Apis mellifera) has attracted intensive attention, and scientists have dedicated much effort trying to identify the stresses affecting bees. There are, however, no simple answers; rather, research suggests multifactorial effects. Several works have [...] Read more.
The high annual losses of managed honey bees (Apis mellifera) has attracted intensive attention, and scientists have dedicated much effort trying to identify the stresses affecting bees. There are, however, no simple answers; rather, research suggests multifactorial effects. Several works have been reported highlighting the relationship between bees’ immunosuppression and the effects of malnutrition, parasites, pathogens, agrochemical and beekeeping pesticides exposure, forage dearth and cold stress. Here we analyze a possible connection between immunity-related signaling pathways that could be involved in the response to the stress resulted from Varroa-virus association and cold stress during winter. The analysis was made understanding the honey bee as a superorganism, where individuals are integrated and interacting within the colony, going from social to individual immune responses. We propose the term “Precision Nutrition” as a way to think and study bees’ nutrition in the search for key molecules which would be able to strengthen colonies’ responses to any or all of those stresses combined. Full article
(This article belongs to the Special Issue Biology of Social Insect Diseases)
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Open AccessArticle
The Influence of New Surveillance Data on Predictive Species Distribution Modeling of Aedes aegypti and Aedes albopictus in the United States
Insects 2019, 10(11), 400; https://doi.org/10.3390/insects10110400 - 12 Nov 2019
Viewed by 305
Abstract
The recent emergence or reemergence of various vector-borne diseases makes the knowledge of disease vectors’ presence and distribution of paramount concern for protecting national human and animal health. While several studies have modeled Aedes aegypti or Aedes albopictus distributions in the past five [...] Read more.
The recent emergence or reemergence of various vector-borne diseases makes the knowledge of disease vectors’ presence and distribution of paramount concern for protecting national human and animal health. While several studies have modeled Aedes aegypti or Aedes albopictus distributions in the past five years, studies at a large scale can miss the complexities that contribute to a species’ distribution. Many localities in the United States have lacked or had sporadic surveillance conducted for these two species. To address these gaps in the current knowledge of Ae. aegypti and Ae. albopictus distributions in the United States, surveillance was focused on areas in Texas at the margins of their known ranges and in localities that had little or no surveillance conducted in the past. This information was used with a global database of occurrence records to create a predictive model of these two species’ distributions in the United States. Additionally, the surveillance data from Texas was used to determine the influence of new data from the margins of a species’ known range on predicted species’ suitability maps. This information is critical in determining where to focus resources for the future and continued surveillance for these two species of medical concern. Full article
(This article belongs to the Special Issue Vectors and Vector-borne Diseases)
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Open AccessArticle
Bmserpin2 Is Involved in BmNPV Infection by Suppressing Melanization in Bombyx mori
Insects 2019, 10(11), 399; https://doi.org/10.3390/insects10110399 - 11 Nov 2019
Viewed by 270
Abstract
Melanization, an important defense response, plays a vital role in arthropod immunity. It is mediated by serine proteases (SPs) that convert the inactive prophenoloxidase (PPO) to active phenoloxidase (PO) and is tightly regulated by serine protease inhibitors (serpins) which belong to a well [...] Read more.
Melanization, an important defense response, plays a vital role in arthropod immunity. It is mediated by serine proteases (SPs) that convert the inactive prophenoloxidase (PPO) to active phenoloxidase (PO) and is tightly regulated by serine protease inhibitors (serpins) which belong to a well distributed superfamily in invertebrates, participating in immune mechanisms and other important physiological processes. Here, we investigated the Bmserpin2 gene which was identified from a transcriptome database in response to Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that Bmserpin2 was expressed in all tissues, with maximum expression in fat body. Upon BmNPV infection, the expression of Bmserpin2 was up-regulated in P50 (susceptible strain) and BC9 (resistant strain) in haemocytes, fat body and the midgut. However, up-regulation was delayed in BC9 (48 or 72 h), in contrast to P50 (24 h), after BmNPV infection. Meanwhile, Bmserpin2 could delay or inhibit melanization in silkworm haemolymph. Significant increased PO activity can be observed in Bmserpin2-depleted haemolymph under NPV infection. Furthermore, the viral genomic DNA copy number was decreased in Bmserpin2-depleted haemolymph. We conclude that Bmserpin2 is an inducible gene which might be involved in the regulation of PPO activation and suppressed melanization, and have a potential role in the innate immune system of B. mori. Full article
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Open AccessArticle
Effects of Dual Exposure to the Herbicides Atrazine and Paraquat on Adult Climbing Ability and Longevity in Drosophila melanogaster
Insects 2019, 10(11), 398; https://doi.org/10.3390/insects10110398 - 10 Nov 2019
Viewed by 335
Abstract
Anthropomorphic effects are changing the planet, and therefore, organisms are being exposed to many new biotic and abiotic stressors. Exposure to multiple stressors can affect organisms in ways that are different than the sum of their individual effects, and these interactions are often [...] Read more.
Anthropomorphic effects are changing the planet, and therefore, organisms are being exposed to many new biotic and abiotic stressors. Exposure to multiple stressors can affect organisms in ways that are different than the sum of their individual effects, and these interactions are often difficult to predict. Atrazine and paraquat are two of the most widely used herbicides in the United States, and are individually known to increase oxidative damage, affect dopaminergic functioning, reduce longevity, and alter motor ability in non-target organisms. We measured the effects of individual and combined exposure to low doses of atrazine and paraquat on climbing ability and longevity of Drosophila melanogaster. Atrazine and paraquat interact to affect D. melanogaster climbing ability and longevity in different ways. Atrazine appeared to have a weak mitigative effect against the decrease in climbing ability caused by paraquat. In contrast, combined exposure to atrazine and paraquat had detrimental synergistic effects on female longevity. Overall, this study shows that atrazine and paraquat can interact and that it is important to measure several traits when assessing the consequences of exposure to multiple stressors. Future studies should continue to assess the impacts of stressor interactions on organisms, as many combinations have never been examined. Full article
(This article belongs to the Special Issue Ecology and Evolutionary Biology of Drosophila )
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Open AccessArticle
Identification and Expression Profiling of Peripheral Olfactory Genes in the Parasitoid Wasp Aphidius ervi (Hymenoptera: Braconidae) Reared on Different Aphid Hosts
Insects 2019, 10(11), 397; https://doi.org/10.3390/insects10110397 - 08 Nov 2019
Viewed by 251
Abstract
Generalist parasitoids of aphids, such as the wasp Aphidius ervi, display significant differences in terms of host preference and host acceptance, depending on the host on which they developed (natal host), which is preferred over a non-natal host, a trait known as [...] Read more.
Generalist parasitoids of aphids, such as the wasp Aphidius ervi, display significant differences in terms of host preference and host acceptance, depending on the host on which they developed (natal host), which is preferred over a non-natal host, a trait known as host fidelity. This trait allows females to quickly find hosts in heterogeneous environments, a process mediated by chemosensory/olfactory mechanisms, as parasitoids rely on olfaction and chemical cues during host selection. Thus, it is expected that proteins participating in chemosensory recognition, such as odorant-binding proteins (OBPs) and odorant receptors (ORs) would play a key role in host preference. In this study, we addressed the effect of parasitoid reciprocal host switching between two aphid hosts (Sitobion avenae and Acyrthosiphon pisum) on the expression patterns of chemosensory genes in the wasp A. ervi. First, by using a transcriptomic approach based on RNAseq of A. ervi females reared on S. avenae and A. pisum, we were able to annotate a total of 91 transcripts related to chemoperception. We also performed an in-silico expression analysis and found three OBPs and five ORs displaying different expression levels. Then, by using qRT-PCR amplification, we found significant differences in the expression levels of these eight genes when the parasitoids were reciprocally transplanted from S. avenae onto A. pisum and vice versa. This suggests that the expression levels of genes coding for odorant receptors and odorant-binding proteins would be regulated by the specific plant–aphid host complex where the parasitoids develop (maternal previous experience) and that chemosensory genes coding for olfactory mechanisms would play a crucial role on host preference and host acceptance, ultimately leading to the establishment of host fidelity in A. ervi parasitoids. Full article
(This article belongs to the Special Issue Insect Parasites: A Focus on Wasps and Nematodes)
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Open AccessArticle
Knockdown of β-N-acetylglucosaminidase 2 Impairs Molting and Wing Development in Lasioderma serricorne (Fabricius)
Insects 2019, 10(11), 396; https://doi.org/10.3390/insects10110396 - 08 Nov 2019
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Abstract
β-N-acetylglucosaminidases (NAGs) are carbohydrate enzymes that degrade chitin oligosaccharides into N-acetylglucosamine monomers. This process is important for chitin degradation during insect development and metamorphosis. We identified and evaluated a β-N-acetylglucosaminidase 2 gene (LsNAG2) from [...] Read more.
β-N-acetylglucosaminidases (NAGs) are carbohydrate enzymes that degrade chitin oligosaccharides into N-acetylglucosamine monomers. This process is important for chitin degradation during insect development and metamorphosis. We identified and evaluated a β-N-acetylglucosaminidase 2 gene (LsNAG2) from the cigarette beetle, Lasioderma serricorne (Fabricius). The full-length open reading frame of LsNAG2 was 1776 bp and encoded a 591 amino acid protein. The glycoside hydrolase family 20 (GH20) catalytic domain and an additional GH20b domain of the LsNAG2 protein were highly conserved. Phylogenetic analysis revealed that LsNAG2 clustered with the group II NAGs. Quantitative real-time PCR analyses showed that LsNAG2 was expressed in all developmental stages and was most highly expressed in the late larval and late pupal stages. In the larval stage, LsNAG2 was predominantly expressed in the integument. Knockdown of LsNAG2 in fifth instar larvae disrupted larval–pupal molting and reduced the expression of four chitin synthesis genes (trehalase 1 (LsTRE1), UDP-N-acetylglucosamine pyrophosphorylase 1 and 2 (LsUAP1 and LsUAP2), and chitin synthase 1 (LsCHS1)). In late pupae, LsNAG2 depletion resulted in abnormal adult eclosion and wing deformities. The expression of five wing development-related genes (teashirt (LsTSH), vestigial (LsVG), wingless (LsWG), ventral veins lacking (LsVVL), and distal-less (LsDLL)) significantly declined in the LsNAG2-depleted beetles. These findings suggest that LsNAG2 is important for successful molting and wing development of L. serricorne. Full article
(This article belongs to the Special Issue RNAi in Insect Pest Control)
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Open AccessArticle
Recent Advances in Management by Pheromones of Thaumetopoea Moths in Urban Parks and Woodland Recreational Areas
Insects 2019, 10(11), 395; https://doi.org/10.3390/insects10110395 - 08 Nov 2019
Viewed by 233
Abstract
Caterpillars of the pine processionary moths, Thaumetopoea complex, cause serious defoliation to Cedrus, Pinus, and Pseudotsuga trees. Thaumetopoea caterpillars also have fine hairs on their abdominal tergites that contain a protein that can severely irritate and cause dermatitis in humans and domestic [...] Read more.
Caterpillars of the pine processionary moths, Thaumetopoea complex, cause serious defoliation to Cedrus, Pinus, and Pseudotsuga trees. Thaumetopoea caterpillars also have fine hairs on their abdominal tergites that contain a protein that can severely irritate and cause dermatitis in humans and domestic animals. The control of the T. pityocampa pine processionary moth has become necessary in many European countries because of the sanitary risks that are related to larval urtication and the defoliation threat to pine forests and plantations. New research activities have been aimed at the development of eco-friendly, innovative technologies for Integrated Pest Management (IPM) of these moths, particularly in urban parks and woodland recreational areas. This paper describes the recent advances in the use of pheromones in monitoring, mass trapping, and mating disruption related to management of processionary moths T. hellenica and T. pityocampa. According to the results, the use of pheromones may provide a practical alternative to insecticide sprays, as they can be safe and simple as compared to other control methods. Full article
(This article belongs to the Special Issue Semiochemicals and Insect Behavior)
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Open AccessArticle
Foodborne Transmission of Deformed Wing Virus to Ants (Myrmica rubra)
Insects 2019, 10(11), 394; https://doi.org/10.3390/insects10110394 - 07 Nov 2019
Viewed by 316
Abstract
Virus host shifts occur frequently, but the whole range of host species and the actual transmission pathways are often poorly understood. Deformed wing virus (DWV), an RNA virus described from honeybees (Apis mellifera), has been shown to have a broad host [...] Read more.
Virus host shifts occur frequently, but the whole range of host species and the actual transmission pathways are often poorly understood. Deformed wing virus (DWV), an RNA virus described from honeybees (Apis mellifera), has been shown to have a broad host range. Since ants are often scavenging on dead honeybees, foodborne transmission of these viruses may occur. However, the role of the ant Myrmica rubra as an alternative host is not known and foodborne transmission to ants has not been experimentally addressed yet. Here, we show with a 16-week feeding experiment that foodborne transmission enables DWV type-A and -B to infect M. rubra and that these ants may serve as a virus reservoir. However, the titers of both plus- and minus-sense viral RNA strands decreased over time. Since the ants were fed with highly virus-saturated honeybee pupae, this probably resulted in initial viral peaks, then approaching lower equilibrium titers in infected individuals later. Since DWV infections were also found in untreated field-collected M. rubra colonies, our results support the wide host range of DWV and further suggest foodborne transmission as a so far underestimated spread mechanism. Full article
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Open AccessArticle
Modeling Temperature Effects on Population Density of the Dengue Mosquito Aedes aegypti
Insects 2019, 10(11), 393; https://doi.org/10.3390/insects10110393 - 07 Nov 2019
Viewed by 287
Abstract
Mosquito density plays an important role in the spread of mosquito-borne diseases such as dengue and Zika. While it remains very challenging to estimate the density of mosquitoes, modelers have tried different methods to represent it in mathematical models. The goal of this [...] Read more.
Mosquito density plays an important role in the spread of mosquito-borne diseases such as dengue and Zika. While it remains very challenging to estimate the density of mosquitoes, modelers have tried different methods to represent it in mathematical models. The goal of this paper is to investigate the various ways mosquito density has been quantified, as well as to propose a dynamical system model that includes the details of mosquito life stages leading to the adult population. We first discuss the mosquito traits involved in determining mosquito density, focusing on those that are temperature dependent. We evaluate different forms of models for mosquito densities based on these traits and explore their dynamics as temperature varies. Finally, we compare the predictions of the models to observations of Aedes aegypti abundances over time in Vitòria, Brazil. Our results indicate that the four models exhibit qualitatively and quantitatively different behaviors when forced by temperature, but that all seem reasonably consistent with observed abundance data. Full article
(This article belongs to the Special Issue Vector Biology and Ecology)
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Open AccessFeature PaperArticle
Gall-Colonizing Ants and Their Role as Plant Defenders: From ’Bad Job’ to ’Useful Service’
Insects 2019, 10(11), 392; https://doi.org/10.3390/insects10110392 - 06 Nov 2019
Viewed by 365
Abstract
Galls are neoformed structures on host plant tissues caused by the attack of insects or other organisms. They support different communities of specialized parasitic insects (the gall inducers), and can also provide refuge to other insects, such as moths, beetles and ants, referred [...] Read more.
Galls are neoformed structures on host plant tissues caused by the attack of insects or other organisms. They support different communities of specialized parasitic insects (the gall inducers), and can also provide refuge to other insects, such as moths, beetles and ants, referred to as secondary occupants. This study focuses on galls induced by the oak gall wasp Andricus quercustozae and secondarily colonized by ants in a mixed oak forest. A field survey and two experiments were carried out to a) study ant (species-specific) preferences for different features of the galls, b) describe differences in gall architecture due to ant activity, c) analyse the effects of the presence of gall-dwelling ants on plant health. The results show that there are differences between ant species in gall colonization and in the alteration of gall opening and inner structure. We verified that gall-dwelling ants protect their host plants efficiently, offering them an indirect defence mechanism against enemies (predators and pathogens). The data suggest a new paradigm in ant–plant relationships mediated by the presence of galls on the plants whose ecological and evolutionary implications are discussed. Full article
(This article belongs to the Special Issue Ants as Partners and Hosts)
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Open AccessArticle
Do Host Plant and Associated Ant Species Affect Microbial Communities in Myrmecophytes?
Insects 2019, 10(11), 391; https://doi.org/10.3390/insects10110391 - 06 Nov 2019
Viewed by 299
Abstract
Ant-associated microorganisms can play crucial and often overlooked roles, and given the diversity of interactions that ants have developed, the study of the associated microbiomes is of interest. We focused here on specialist plant-ant species of the genus Allomerus that grow a fungus [...] Read more.
Ant-associated microorganisms can play crucial and often overlooked roles, and given the diversity of interactions that ants have developed, the study of the associated microbiomes is of interest. We focused here on specialist plant-ant species of the genus Allomerus that grow a fungus to build galleries on their host-plant stems. Allomerus-inhabited domatia, thus, might be a rich arena for microbes associated with the ants, the plant, and the fungus. We investigated the microbial communities present in domatia colonised by four arboreal ants: Allomerus decemarticulatus, A. octoarticulatus, A. octoarticulatus var. demerarae, and the non-fungus growing plant-ant Azteca sp. cf. depilis, inhabiting Hirtella physophora or Cordia nodosa in French Guiana. We hypothesized that the microbial community will differ among these species. We isolated microorganisms from five colonies of each species, sequenced the 16S rRNA or Internal TranscribedSpacer (ITS) regions, and described both the alpha and beta diversities. We identified 69 microbial taxa, which belong to five bacterial and two fungal phyla. The most diverse phyla were Proteobacteria and Actinobacteria. The microbial community of Azteca cf. depilis and Allomerus spp. differed in composition and richness. Geographical distance affected microbial communities and richness but plant species did not. Actinobacteria were only associated with Allomerus spp. Full article
(This article belongs to the Special Issue Ants as Partners and Hosts)
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Open AccessArticle
Insulin Receptor Substrate Gene Knockdown Accelerates Behavioural Maturation and Shortens Lifespan in Honeybee Workers
Insects 2019, 10(11), 390; https://doi.org/10.3390/insects10110390 - 05 Nov 2019
Viewed by 318
Abstract
In animals, dietary restriction or suppression of genes involved in nutrient sensing tends to increase lifespan. In contrast, food restriction in honeybees (Apis mellifera) shortens lifespan by accelerating a behavioural maturation program that culminates in leaving the nest as a forager. [...] Read more.
In animals, dietary restriction or suppression of genes involved in nutrient sensing tends to increase lifespan. In contrast, food restriction in honeybees (Apis mellifera) shortens lifespan by accelerating a behavioural maturation program that culminates in leaving the nest as a forager. Foraging is metabolically demanding and risky, and foragers experience increased rates of aging and mortality. Food-deprived worker bees forage at younger ages and are expected to live shorter lives. We tested whether suppression of a molecular nutrient sensing pathway is sufficient to accelerate the behavioural transition to foraging and shorten worker life. To achieve this, we reduced expression of the insulin receptor substrate (irs) gene via RNA interference in two selected lines of honeybees used to control for behavioural and genetic variation. irs encodes a membrane-associated protein in the insulin/insulin-like signalling (IIS) pathway that is central to nutrient sensing in animals. We measured foraging onset and lifespan and found that suppression of irs reduced worker bee lifespan in both genotypes, and that this effect was largely driven by an earlier onset of foraging behaviour in a genotype-conditional manner. Our results provide the first direct evidence that an IIS pathway gene influences behavioural maturation and lifespan in honeybees and highlight the importance of considering social environments and behaviours when investigating the regulation of aging and lifespan in social animals. Full article
(This article belongs to the Special Issue Mechanisms of Nutritional Resource Exploitation by Insects)
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Open AccessArticle
Overwintering States of the Pale Grass Blue Butterfly Zizeeria maha (Lepidoptera: Lycaenidae) at the Time of the Fukushima Nuclear Accident in March 2011
Insects 2019, 10(11), 389; https://doi.org/10.3390/insects10110389 - 04 Nov 2019
Viewed by 338
Abstract
The Fukushima nuclear accident in March 2011 caused the massive release of anthropogenic radioactive materials from the Fukushima Dai-ichi Nuclear Power Plant to its surrounding environment. Its biological effects have been studied using the pale grass blue butterfly, Zizeeria maha (Lepidoptera: Lycaenidae), but [...] Read more.
The Fukushima nuclear accident in March 2011 caused the massive release of anthropogenic radioactive materials from the Fukushima Dai-ichi Nuclear Power Plant to its surrounding environment. Its biological effects have been studied using the pale grass blue butterfly, Zizeeria maha (Lepidoptera: Lycaenidae), but the overwintering states of this butterfly remain elusive. Here, we conducted a series of field surveys in March 2018, March 2019, and April 2019 in Fukushima and its vicinity to clarify the overwintering states of this butterfly at the time of the Fukushima nuclear accident. We discovered overwintering individuals in situ associated with the host plant Oxalis corniculata under natural straw mulch as first-instar to fourth-instar larvae in March 2018 and 2019. No other developmental stages were found. The body length and width were reasonably correlated with the accumulated temperature. On the basis of a linear regression equation between body size and accumulated temperature, together with other data, we deduced that the pale grass blue butterfly occurred as fourth-instar larvae in Fukushima and its vicinity at the time of the accident. This study paves the way for subsequent dosimetric analyses that determine the radiation doses absorbed by the butterfly after the accident. Full article
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Open AccessArticle
Transgenic Winter Wheat Expressing the Sucrose Transporter HvSUT1 from Barley does not Affect Aphid Performance
Insects 2019, 10(11), 388; https://doi.org/10.3390/insects10110388 - 04 Nov 2019
Viewed by 396
Abstract
Winter wheat expressing the sucrose transporter HvSUT1 from barley (HOSUT) has an increased yield potential. Genetic engineering should improve cultivars without increasing susceptibility to biotic stresses or causing negative impacts on ecosystem services. We studied the effects of HOSUT wheat on cereal aphids [...] Read more.
Winter wheat expressing the sucrose transporter HvSUT1 from barley (HOSUT) has an increased yield potential. Genetic engineering should improve cultivars without increasing susceptibility to biotic stresses or causing negative impacts on ecosystem services. We studied the effects of HOSUT wheat on cereal aphids that feed on the sugar-rich phloem sap. Three HOSUT winter wheat lines, their conventional parental cultivar Certo, and three conventional cultivars were used. Clip cage experiments in the greenhouse showed no differences in life-table parameters of Rhopalosiphum padi and Sitobion avenae (Hemiptera: Aphididae) on transgenic lines compared to Certo, except higher fecundity of S. avenae on one HOSUT line. Population development of both aphid species over three weeks on caged flowering tillers did not reveal differences between the HOSUT lines and Certo. When aphids were monitored in a Swiss field study over two years, no differences between HOSUT lines and Certo were observed. We conclude that HOSUT wheat did not have consistent effects on aphids compared to the parental cultivar and measured parameters were generally in the range observed for the conventional winter wheat cultivars. Thus, HOSUT wheat is unlikely to suffer from increased aphid damage. Full article
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Open AccessArticle
Effect of Diets and Low Temperature Storage on Adult Performance and Immature Development of Phyllonorycter ringoniella in Laboratory
Insects 2019, 10(11), 387; https://doi.org/10.3390/insects10110387 - 04 Nov 2019
Viewed by 264
Abstract
The Asiatic apple leaf miner, Phyllonorycter ringoniella (Matsumura), is an important insect pest to apples. We developed a rearing method for P. ringoniella in the laboratory. Overwintering pupae were collected from our apple orchard, and crabapple seedlings were selected as oviposition substrate and [...] Read more.
The Asiatic apple leaf miner, Phyllonorycter ringoniella (Matsumura), is an important insect pest to apples. We developed a rearing method for P. ringoniella in the laboratory. Overwintering pupae were collected from our apple orchard, and crabapple seedlings were selected as oviposition substrate and food source for the larval development. The mean developmental period was 25.9 ± 0.49 days from egg to adult and the survival rate was 0.387 under 25 ± 1 °C, 65 ± 5% RH and a photoperiod of 14:10 (L:D) h. Mean egg length and egg width were 0.336 ± 0.0043 and 0.259 ± 0.0046 mm, respectively. Mean body length and head width increased from 1.070 ± 0.0245 and 0.180 ± 0.0021 mm in first instar larvae to 5.027 ± 0.0718 and 0.321 ± 0.0021 mm in fifth instar larvae, respectively. The mean weight of the pupae was 0.946 ± 0.0132 mg. The wingspan of male adult (6.280 ± 0.0639 mm) was significantly larger than female (6.040 ± 0.0753 mm). The mean fecundity (56.9 ± 8.58 eggs per female) and longevity (8.2 ± 0.55 days) of females was highest when they were provided with 10% honey solution, followed by 10% sugar, water, and control. The females preferred to lay eggs (56.5 ± 3.84%) near the secondary leaf vein in a leaf. The larval mortality increased linearly (R2 = 0.94) with the number of larvae per leaf. The mortality of pupae increased from 5.6 ± 4.01 to 51.1 ± 9.88% as storage periods at 4 °C increased from 0 to 105 days. The rearing method and its biological characteristics presented in this study will contribute to further studies on this pest insect. Full article
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Open AccessArticle
Density-Dependent Growth and Fitness in Dastarcus helophoroides (Coleoptera: Bothrideridae)
Insects 2019, 10(11), 386; https://doi.org/10.3390/insects10110386 - 04 Nov 2019
Viewed by 244
Abstract
The ectoparasitoid Dastarcus helophoroides Fairmaire (Coleoptera: Bothrideridae) is an important natural enemy insect, which is artificially mass-reared and released into woodland to control medium and large longhorn beetle species. This study examined the developmental duration (days) of larvae and adult fitness (including numbers [...] Read more.
The ectoparasitoid Dastarcus helophoroides Fairmaire (Coleoptera: Bothrideridae) is an important natural enemy insect, which is artificially mass-reared and released into woodland to control medium and large longhorn beetle species. This study examined the developmental duration (days) of larvae and adult fitness (including numbers of adults emerging per host and mean body size) by exposing a single substitute host, a pupa of Zophobas morio (Coleoptera: Tenebrionidae), to different densities of D. helophoroides larvae. We showed that there was no significant effect on the rate of successful parasitism and cocoon formation, but emergence success and measures of individual adult body size (length, width, and weight) declined with increasing larval density. Larval period and cocoon period increased with larval density, while total weight of adults emerging per host increased initially before reaching a plateau. Our results suggest that a pupa of Z. morio could be successfully parasitized by a single D. helophoroides larva, but multiple D. helophoroides larvae can share one host. Excessive larval density caused intraspecific competition among D. helophoroides larvae, manifesting in extended developmental duration of immature stage and reduced fitness of adults. Furthermore, the tradeoff between the numbers of adults and body size may stabilize the population dynamics with detectable mutual interference, particularly in competing for limited host resources. These findings suggest six larvae per host would achieve the highest adult fitness and would enhance mass-rearing techniques as part of IPM strategies for longhorn beetles. Full article
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Open AccessReview
Current Knowledge of the Entomopathogenic Fungal Species Metarhizium flavoviride Sensu Lato and Its Potential in Sustainable Pest Control
Insects 2019, 10(11), 385; https://doi.org/10.3390/insects10110385 - 02 Nov 2019
Viewed by 315
Abstract
Fungal entomopathogens are gaining increasing attention as alternatives to chemical control of arthropod pests, and the literature on their use under different conditions and against different species keeps expanding. Our review compiles information regarding the entomopathogenic fungal species Metarhizium flavoviride (Gams and Rozsypal [...] Read more.
Fungal entomopathogens are gaining increasing attention as alternatives to chemical control of arthropod pests, and the literature on their use under different conditions and against different species keeps expanding. Our review compiles information regarding the entomopathogenic fungal species Metarhizium flavoviride (Gams and Rozsypal 1956) (Hypocreales: Clavicipitaceae) and gives account of the natural occurrences and target arthropods that can be controlled using M. flavoviride. Taxonomic problems around M. flavoviride species sensu lato are explained. Bioassays, laboratory and field studies examining the effect of fermentation, culture regimes and formulation are compiled along with studies on the effect of the fungus on target and non-target organisms and presenting the effect of management practices on the use of the fungus. Altogether, we provide information to help conducting basic studies, and by pointing out relatively uncharted territories, help to set new research areas. Full article
(This article belongs to the Special Issue Pest Management in Sustainable Farming Systems)
Open AccessArticle
A Comparison of Three Approaches for Larval Instar Separation in Insects—A Case Study of Dendrolimus pini
Insects 2019, 10(11), 384; https://doi.org/10.3390/insects10110384 - 02 Nov 2019
Viewed by 201
Abstract
The number of larval instars is important from both theoretical and practical perspectives. Three previous studies based on head capsule widths (HCWs) have suggested that Dendrolimus pini larvae pass through seven instars, but the estimated HCW means differed greatly. Various methods are available [...] Read more.
The number of larval instars is important from both theoretical and practical perspectives. Three previous studies based on head capsule widths (HCWs) have suggested that Dendrolimus pini larvae pass through seven instars, but the estimated HCW means differed greatly. Various methods are available for determining the number of instars; however, these methods have not been compared on the same dataset. Therefore, the main goal of this study was to compare three approaches for instar separation in D. pini larvae: visual approach followed by non-linear least squares (NLLS) estimation, kernel density estimation (KDE) followed by NLLS, and model-based clustering. Two criteria were used to assess whether the resulting instar separations adhered to Brooks-Dyar’s rule: Crosby’s growth rule and a coefficient of determination indicating the goodness of fit of a straight line to the ln-transformed mean HCW of the respective instars. Our results showing that D. pini larvae pass through eight instars differed greatly from reports in the literature. The best results were obtained by KDE followed by NLLS. For proper instar separation, both criteria of Brooks-Dyar’s rule must be met. Full article
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