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The honey bee genome has the capacity to produce three phenotypically distinct organisms (two diploid female castes: queen and worker, and a haploid male drone). Previous studies have implicated metabolic flux acting via epigenetic regulation in directing nutrition-driven phenotypic plasticity in the honey bee. However, the cis-acting DNA regulatory elements that establish tissue and polyphenism -specific epigenomes and gene expression programmes, remain unclear. Using a high resolution multiomic approach including assay for transposase-accessible chromatin by sequencing (ATAC-seq), RNA-seq and ChIP-seq, we produce the first genome-wide maps of the regulatory landscape across all three adult honey bee phenotypes identifying > 5000 regulatory regions in queen, 7500 in worker and 6500 in drone, with the vast majority of these sites located within intronic regions. These regions are defined by positive enrichment of H3K27ac and depletion of H3K4me3 and show a positive correlation with gene expression. Using ATAC-seq footprinting we determine queen, worker and drone -specific transcription factor occupancy and uncover novel phenotype-specific regulatory networks identifying two key nuclear receptors that have previously been implicated in caste-determination and adult behavioural maturation in honey bees; ecdysone receptor and ultraspiracle. Collectively, this study provides novel insights into key gene regulatory networks that are associated with these distinct polyphenisms in the honey bee.

Aims This study aimed to isolate and identify entomopathogenic fungi (EPF) from fungus-infected Ostrinia furnacalis larvae, screen their bio-efficacy against O. furnacalis, and select the most suitable virulent native EPF for biocontrol agent development. Methods and Results The occurrence of EPF isolated from various maize production regions in Xinjiang was investigated. Of 13,864 O. furnacalis cadavers surveyed, 536 were selected, and of 136 fungal specimens collected, 14 species were identified. Four fungal isolates were highly pathogenic to O. furnacalis: Aspergillus sp., Lecanicillium attenuatum, Beauveria bassiana and Penicillium polonicum. The Aspergillus sp. was the most abundant (42.25% distribution frequency). Bioassay results revealed that it was as pathogenic as B. bassiana (positive control), with 96.58% lethality against O. furnacalis (LC50: 1.40 × 104 conidia ml−1, LT50: 3.41 days). Through morphological examination and rDNA-benA and rDNA-CaM homogeneity analyses, the isolate was identified as Aspergillus nomius. Conclusions Four EPF species were highly pathogenic, with A. nomius being the most prevalent in Xinjiang. A. nomius is a potential biocontrol agent. Significance and Impact of Study For sustainable prevention and control of O. furnacalis infestation, identifying biocontrol agents with high virulence against O. furnacalis is crucial. The findings of this study support the development of EPF-based biocontrol approaches.

The flesh fly, Sarcophaga crassipalpis Macquart 1839 (Diptera: Sarcophagidae), colonizes estimation of the minimum post-mortem interval (min-PMI). This study examined the effects of different types of tissues and temperature on the development of S. crassipalpis. To the best of our knowledge, the present study is the first to investigate the effects of life-history information of S. crassipalpis. Larvae were reared on three tissue types, chicken heart, bovine minced meat, and bovine tongue, at five temperatures, 15, 20, 25, 30, and 32°C. Pupal and larval development time, adult weight, and pupal and larval survival differed significantly for tissue type and temperature. Temperature had a significant effect on pupal weight but tissue type did not significantly affect pupal weight. The duration of the larval and pupal stages (except of chicken heart, 30°C) decreased with increasing temperature, except for 32°C, in all tissue types. Larval survival was lowest at 32°C in bovine minced meat, bovine tongue, and chicken heart, and at 32°C, all pupae failed to reach the adult stage in minced meat. The results of this study highlight the necessity of considering larval diet and temperature in forensic investigations.

Zebrafish larvae have classically been used as a high-throughput model with which to test both the bioactivity and toxicity of known and novel compounds, making them a promising whole organism New Approach Method in the context of the international momentum to eliminate animal testing. Larvae are generally exposed to the chemicals being tested in a static environment and the concentration-response patterns are calculated based on the initial bath concentrations of the compounds. This approach rarely takes into account the absorption, distribution, metabolism, and excretion of the compounds being tested, which can have a significant effect on the toxicokinetic profiles of the compounds and thus impact the predictive ability of the model. In this study, we have evaluated the toxicokinetic profile of 5 known toxicants, 3 phenolic compounds, along with thiabendazole and 3,4-dicholoronalanine, at 6, 8, 24, 72, and 120 h postfertilization in order to match the exposure timelines of a standard in vitro fish embryo toxicity test. It was revealed that in addition to bioaccumulation effects, the compounds were all actively metabolized and excreted by the larvae. Importantly, comparisons between the toxicants revealed that the patterns of uptake and metabolism were varied and could often partially explain the differences in their concentration-response patterns. The findings of this study are significant as they highlight the requirement for an assessment of the stability and toxicokinetic profile of chemicals tested using standard zebrafish larval toxicity assays in order to better understand and compare their toxicity profiles.

Synopsis Temperature profoundly affects ectotherm physiology. Although differential thermal responses influence fitness, thus driving population dynamics and species distributions, our understanding of the molecular architecture underlying these responses is limited, especially during the critical larval stage. Here, using RNA-sequencing of laboratory-reared Atlantic cod (Gadus morhua) larvae of wild origin, we find changes in gene expression in thousands of transcripts consistent with a severe cellular stress response at both ambient and projected (+2°C and +4°C) temperatures. In addition, specific responses to stress, heat, and hypoxia were commonly identified in gene ontology enrichment analyses and 33 of the 44 genes comprising the minimum stress proteome of all organisms were upregulated. Earlier onset of the stress response was evident at higher temperatures; concomitant increased growth and mortality suggests a reduction in fitness. Temporal differences in gene expression levels do not correspond to differences in growing degree days, suggesting negative physiological consequences of warming beyond accelerated development. Because gene expression is costly, we infer that the upregulation of thousands of transcripts in response to warming in larval cod might act as an energetic drain. We hypothesize that the energetically costly stress response, coupled with increased growth rate at warmer temperatures, leads to faster depletion of energy reserves and increased risk of mortality in larval cod. As sea surface temperatures continue to rise over the next century, reduced fitness of Atlantic cod larvae might lead to population declines in this ecologically and socioeconomically important species. Further, our findings expand our understanding of transcriptomic responses to temperature by ectothermic vertebrate larvae beyond the critical first-feeding stage, a time when organisms begin balancing the energetic demands of growth, foraging, development, and maintenance. Linking the molecular basis of a thermal response to key fitness-related traits is fundamentally important to predicting how global warming will affect ectotherms.

Antheraea pernyi Guérin-Méneville (Lepidoptera: Saturniidae) is of high economic value as a source of silk, food, and bioactive substances with medicinal properties. A. pernyi larvae are prone to A. pernyi vomit disease (AVD), which results in substantial economic losses during cultivation; however, the relationship between AVD and A. pernyi gut microbiota remains unclear. Here, we investigated the bacterial community in the midgut and feces of A. pernyi larvae with and without AVD using 16S rRNA gene sequencing with Illumina MiSeq technology. Compared with healthy larvae, intestinal bacterial diversity and community richness increased and decreased in larvae with mild and severe AVD, respectively. In addition, the proportion of gut Enterobacter Hormaeche and Edwards(Enterobacteriales: Enterobacteriaceae) and Enterococcus Thiercelin and Jouhaud (Lactobacillales: Enterococcaceae) was higher and lower, respectively, in larvae with mild AVD than those in healthy larvae. A. pernyi vomit disease infection significantly increased the genera with abundance

Plutella xylostella (L.) (Lepidoptera: Plutellidae) is one of the most destructive pests of Brassicaceae vegetables. Cantharidin is an insect-derived defensive toxin, which has been reported to have toxicity to a variety of pests and especially lepidopteran pests. Although the toxicity of cantharidin on P. xylostella has been demonstrated, there is little information available on the specific detoxification response of P. xylostella against cantharidin. This study investigates the enzymatic response (including serine/threonine phosphatases [PSPs], carboxylesterases [CarEs], glutathione-S-transferases [GSTs], and cytochrome P450 monooxygenases [P450]) in P. xylostella to the sublethal and low lethal concentrations of cantharidin (LC10 and LC25). Results showed that the inhibitory activity of PSPs was increased and then decreased in vivo, while PSPs activity could be almost completely inhibited in vitro. Interestingly, the activities of detoxification enzymes (GST, CarE, and P450) in P. xylostella displayed a trend of decreasing and then increasing after exposure to the two concentrations of cantharidin. Notably, the increase in P450 enzyme activity was the most significant. The increasing trend of detoxification enzyme activity was congruent with the recovery trend of PSPs activity. This study contributes to our understanding of the detoxification mechanism of cantharidin in P. xylostella and helps in the further development of biogenic agents.

The parasitoid Cotesia glomerata L. (Hymenoptera: Braconidae) oviposits in larvae of the large cabbage white butterfly (Pieris brassicae L.). Many parasitoids are attracted by volatile organic compounds (VOCs) emitted by the plant that the host insects feed on. The objectives of the study were to identify the VOCs in leaves of two varieties of cabbage (white cabbage, Brassica oleracea var. capitata f. alba and cauliflower, B. oleracea var. botrytis) damaged by P. brassicae caterpillars which elicit antennal responses of C. glomerata, and characterize the olfactory spectra of females and males. Leaf extracts were analyzed by using gas chromatography equipped with an electroantennographic detector (GC–EAD) and GC–mass spectrometry. In total, 32 olfactory-active compounds for C. glomerata in cauliflower and 24 in white cabbage were revealed. The females perceived more compounds than males. Hexan-1-ol, (E)-3-hexen-1-ol, (E)-2-octenal, benzylcianide, tetradecanal, and two unidentified compounds elicited EAG responses in females but not in males. Females were more sensitive to (E)-3-hexenal, (Z)-3-hexenal, and pentadecenal, whereas males showed higher sensitivity to (Z)-3-hexenyl butanoate, heptanal, (Δ)-2-pentenal, (E)-2-hexenol, and octanal. The olfactory spectrum of C. glomerata was expanded from 18 to 41 VOCs emitted by different varieties of cabbage damaged by P. brassicae caterpillars. Eight EAG-active VOCs were common for all cabbage varieties. In tritrophic interactions, benzylcyanide can serve as an important signal for C. glomerata females indicating damage of cabbage caused by P. brassicae caterpillars. The data are useful for development of a push–pull strategy for P. brassicae control, based on parasitoid behavior regulation by VOCs.

Aedes aegypti mosquitoes are capable of vectoring a wide range of diseases including dengue, yellow fever, and Zika viruses, with approximately half of the worlds’ population at risk from such diseases. Development of combined predator–parasite treatments for the control of larvae consistently demonstrates increased efficacy over single-agent treatments, however, the mechanism behind the interaction remains unknown. Treatments using the natural predator Toxorhynchites brevipalpis and the entomopathogenic fungus Metarhizium brunneum were applied in the laboratory against Ae. aegypti larvae as both individual and combined treatments to determine the levels of interaction between control strategies. Parallel experiments involved the removal of larvae from test arenas at set intervals during the course of the trial to record whole body caspase and phenoloxidase activities. This was measured via luminometric assay to measure larval stress factors underlying the interactions. Combined Metarhizium and Toxorhynchites treatments were seen to drastically reduce lethal times as compared to individual treatments. This was accompanied by increased phenoloxidase and caspase activities in combination treatments after 18 h (p < 0.001). The sharp increases in caspase and phenoloxidase activities suggest that combined treatments act to increase stress factor responses in the larvae that result in rapid mortality above that of either control agent individually. This work concludes that the underlying mechanism for increased lethality in combined parasite–predator treatments may be related to additive stress factors induced within the target host larvae.

The characterization of the Bacillus thuringiensis (Berliner) LBIT-418 strain was based on a previous work which indicated its high insecticidal potential. Therefore, toxicological, molecular, and biochemical characterizations were conducted in this work to identify its unique features and its potential to be developed as a bioinsecticide. This strain, originally isolated from a healthy mosquito larva, was identified within the subspecies kenyae by sequencing of the hag gene and by the multilocus sequence typing (MLST) technique. Genes cry1Ac2, cry1Ea3, cry2Aa1 and cry2Ab4, and a cry1Ia were detected in its genome, in addition to a vip3Aa gene. In this research, the latter protein was successfully cloned, expressed, and purified and showed high toxicity towards the fall armyworm, Spodoptera frugiperda (J.E. Smith), fourth instar larvae in bioassays using the microdroplet ingestion technique, estimating an LD50 of 21.38 ng/larva. Additional bioassays were performed using the diet surface inoculation technique of the strain’s spore-crystal complex against diamondback moth larvae, Plutella xylostella (Linnaeus), estimating an LC50 of 10.22 ng/cm2. Its inability to produce β-exotoxin was demonstrated by bioassays against the nematode Caenorhabditis elegans Maupas and by HPLC analysis. These results support the high potential of this strain to be developed as a bioinsecticide.

Aims Bacillus cereus is often responsible for foodborne diseases and both local and systemic infections in humans. Cases of infection in other mammals are rather rare. In this study, we report a B. cereus feed-related outbreak that caused the death of 6234 pigs in Italy. Methods and Results Massive doses of a Gram-positive, spore-forming bacterium were recovered from the animal feed, faeces of survived pigs and intestinal content of dead ones. The B. cereus MM1 strain was identified by MALDI-TOF MS and typified by RAPD-PCR. The isolate was tested for the production of PC-PLC, proteases, hemolysins and biofilm, for motility, as well as for the presence of genes encoding tissue-degrading enzymes and toxins. Antimicrobial resistance and pathogenicity in Galleria mellonella larvae were also investigated. Our results show that the isolated B. cereus strain is swimming-proficient, produces PC-PLC, proteases, hemolysins, biofilm and carries many virulence genes. The strain shows high pathogenicity in G. mellonella larvae. Conclusions The isolated B. cereus strain demonstrates an aggressive profile of pathogenicity and virulence, being able to produce a wide range of determinants potentially hazardous to pigs' health. Significance and Impact of Study This study highlights the proficiency of B. cereus to behave as a devastating pathogen in swine if ingested at high doses and underlines that more stringent quality controls are needed for livestock feeds and supplements.

Black fungus gnat larvae are one of the primary insect pests in greenhouse and nursery crops, and Bradysia ocellaris (Comstock) (Diptera: Sciaridae) is one common pest species. This pest is difficult to control in Brazil because of the absence of registered insecticides. The aim of this work was to evaluate the effects of some insecticides on B. ocellaris larvae. We also verified that the insect growth regulator novaluron caused the deformation of B. ocellaris. Of the insecticides evaluated, malationa, and thiamethoxam showed high mortality rate (96 and 86 % respectively). Further, bioassays with acetamiprid (78 %) and novaluron (44 %) showed that the lethal concentrations (LC50) were 19.18 mg a.i.L-1 at 48h to acetamiprid and 1.24 mg a.i.L-1 at 120 h to novaluron. When larvae were fed on potato pieces treated with novaluron, independently of the dose, the mortality rate was 100 %, since no larvae could complete the development cycle. Among all evaluated insecticides, only acetamiprid and novaluron were considered effective tools for control of B. ocellaris larvae under laboratory conditions.

The insecticide sensitivity and resistance status of both adults and larvae from six Aedes albopictus samples collected in Tuskegee, Tuscaloosa, Birmingham, Dothan, Mobile, and Montgomery, Alabama, were evaluated for the levels of sensitivity and resistance to eight insecticides: β-cyfluthrin, chlorpyrifos, deltamethrin, etofenprox, fenitrothion, permethrin, resmethrin, and malathion. Adult Ae. albopictus from all locations showed similar results for the difference between the time to 100% mortality and the diagnostic time in the CDC bottle bioassay, although Ae. albopictus survive longer than the diagnostic time to permethrin, fenitrothion, and resmethrin treatments. The larval bioassay indicated that malathion was the least toxic to Ae. albopictus from all locations (LC50: ranging from 0.1 ppm to 1.2 ppm), followed by resmethrin and etofenprox (LC50: 0.05 ppm–0.4 ppm), and deltamethrin and fenitrothion (LC50: 0.01 ppm–0.06 ppm). Chlorpyrifos exhibited the highest larval toxicity (LC50: 0.003 ppm–0.05 ppm). The resistance status of Ae. albopictus from all six locations was similar to the resistance levels found in a previous survey in 2004, indicating that in Alabama the development of resistance is slow in this strain, although comparing the resistance of Ae. albopictus from Tuskegee to that of a susceptible strain showed that it is resistant to chlorpyrifos. The slopes of the dose–response curves to most of the insecticides tested for these field populations of Ae. albopictus were generally similar to or slightly higher than those measured eighteen years previously, indicating that these populations are relatively homozygous in response to all the insecticides tested.

A predator, Laricobius osakensis Montgomery and Shiyake (Coleoptera: Derodontidae), is being mass-produced and released for the biological control of the invasive hemlock woolly adelgid (HWA), Adelges tsugae Annand (Hemiptera: Adelgidae). To better understand and predict the seasonality of this predator in North America, the development and reproduction of L. osakensis were evaluated at constant temperatures ranging from 5 to 22°C. The predicted seasonal biology was compared with data from field collections. L. osakensis did not complete development from egg to adult at the two lowest temperatures tested, 5 and 8°C, but did so at the highest temperature of 22°C. The minimum development thresholds were estimated for eggs (4.2°C), first (1.8°C), second (5.5°C), third (4.6°C), and fourth instar (4.1°C), prepupa (3.6°C), and pupa (7.5°C). Oviposition rates were significantly greater at 5 and 10°C than at 20 and 25°C. Head capsule width significantly increased for each of the four larval instars with a mean of 0.19, 0.26, 0.35, and 0.44 mm, respectively. Laboratory and field data were used to develop a phenology forecasting model to predict the occurrence of all developmental stages of L. osakensis. This model will allow land managers to more accurately predict the optimal timing for L. osakensis larval sampling throughout its established range.

Habitat diversification has been shown to positively influence a variety of ecosystem services to agriculture, including biological control of arthropod pests. The impact of increased biodiversity tends to be species specific though, and practices therefore need to be developed on a case-by-case basis for each cropping system. In perennial systems, numerous studies have demonstrated that cover crops can have positive impacts on soil quality and other ecosystem services, such as pollination and pest management. However, few studies have focused on the use of cover crops to enhance pest control in almond orchards, especially winter cover crops. The primary pest of almonds in North America is navel orangeworm, Amyelois transitella Walker, which overwinter as larva or pupa on remnant nuts, many of which remain on the orchard soil surface. In the spring, first flight adults subsequently use these remnant nuts as reproductive substrate. An experiment was conducted to evaluate the influence of two distinct winter cover crop mixtures on overwintering mortality and spring egg deposition of A. transitella. Remnant nuts placed into cover crop plots produced fewer adult A. transitella in the spring, suggesting increased overwintering mortality. Additionally, spring egg deposition was reduced on remnant nuts in the cover crops, possibly due to the ground covers interfering with host location and access. In this way, winter cover crops appear to contribute to the reduction of A. transitella populations in the orchard by altering abiotic and physical conditions, although studies to document specific mechanisms are still needed.

The larval ecology of Culicoides (Diptera: Ceratopogonidae) influences their spatial distributions and the pathogens they transmit. These features are of special concern for deer farmers in Florida where epizootic hemorrhagic disease virus (EHDV) is a major source of mortality in captive herds. Rarity of larval morphological expertise leads many researchers to study larval ecology by quantifying emergence, either with field emergence traps or removing substrate from the field for observation under laboratory conditions. We investigated the comparability of these methods in Florida seepages where two recently implicated EHDV vectors, Culicoides stellifer Coquillett and Culicoides venustus Hoffman, are common. We compared the abundance and composition of emerging Culicoides collected from emergence traps with removed substrate samples (soil plugs) at three seepages. Soil plugs were sampled adjacent to the emergence trap and from underneath the trap footprint, and then monitored under laboratory conditions for 11–13 wk to compare the methods and to assess the role of incubation period for removed substrate samples. Emergence traps and removed substrate sampling largely agreed on community compositions and trends within different seepages. However, comparatively large numbers of C. stellifer emerged later than expected and well into the incubation period with emergence still occurring after 13 wk (90 d). Removed substrate samples were more similar to emergence traps at shorter incubation times. The importance of time for the capture of Culicoides in removed substrate sampling was more pronounced than we anticipated and is important from both a methodological and biological perspective.

Lymantria dispar L. is a serious invasive defoliator of broadleaf trees in both North America and Eurasia. When rearing L. dispar for research, the artificial diet must have a source of iron that is bioavailable and in the quantity needed to ensure normal development and survival. The standard iron supplement in L. dispar diet, amorphous ferric phosphate, is no longer commercially available, which motivated studies on alternative iron sources and concentrations. The responses of three L. dispar populations (including an L. dispar asiatica Vnukovskij population) to seven different iron doses were determined over two generations. The response to eight iron compounds and no added iron was also assessed over two generations for an L. dispar dispar population. The optimal levels of iron were 100, 150, and 198 mg/l of available iron for the New Jersey Standard Strain, a near-wild West Virginia population, and a near-wild Russian Far East population, respectively. All three populations showed symptoms of insufficient iron when reared on the lowest dose of iron and signs of excess iron at the highest two doses. All of the alternate iron sources evaluated provided adequate available iron, although three sources had some issues that may not make them the best options. These results reveal differences in nutritional requirements among different populations of L. dispar and verify the existence of viable replacement sources of iron for the standard amorphous ferric phosphate long used in L. dispar rearing.

Entomopathogenic fungi allow chemical-free and environmentally safe vector management. Beauveria bassiana (Balsamo-Crivelli) Vuillemin is a promising biological control agent and an important component of integrated vector management. We investigated the mortality of Aedes albopictus (Skuse) larvae exposed to five concentrations of B. bassiana using Mycotrol ESO and adult oviposition behavior to analyze the egg-laying preferences of wild Ae. albopictus in response to different fungal concentrations. We examined the mortality of mid-instars exposed to B. bassiana concentrations of 1 × 104, 1 × 105, 1 × 106, 1 × 107, and 1 × 108 conidia/ml every 24 h for 12 d. In the oviposition behavior study, the fungus was applied to wooden paddles at 1 × 105, 1 × 107, and 1 × 109 conidia/ml, and the paddles were individually placed into quad-ovitraps. Both experiments contained control groups without B. bassiana. Kaplan–Meier survival analysis revealed that larval mortality was concentration dependent. The median lethal concentration was 2.43 × 105 conidia/ml on d 12. The median lethal time was 3.68 d at 1 × 106 conidia/ml. Oviposition monitoring revealed no significant difference in egg count between the control and treatment paddles. We observed an inverse relationship between the concentration of B. bassiana and the percentage of paddles with eggs. We concluded that concentrations above 1 × 106 conidia/ml are larvicidal, and Ae. albopictus laid similar numbers of eggs on fungus-impregnated and control wooden substrates; however, they were more likely to oviposit on substrates without B. bassiana. With these findings, we suggest that B. bassiana-infused ovitraps can be used for mosquito population monitoring while also delivering mycopesticides to adult mosquitoes.

Spodoptera frugiperda (J.E. Smith) is a severe and fast-spreading pest of numerous agro-economic crops, including miscellaneous, vegetables, and green manure crops. Understanding pest ecology represents a core component in integrated pest management decision-making. In Taiwan, peanut (Arachis hypogaea L.) is an important miscellaneous crop, whereas sesbania (Sesbania roxburghii Merr.) is the most frequently used green manure crop. To improve the S. frugiperda management in Taiwan, the demographic characteristics and population simulation of this pest reared on peanut and sesbania leaves were analyzed using the age-stage, two-sex life table theory. The intrinsic rate of increase, finite rate of increase, and net reproductive rate of S. frugiperda were higher when reared on peanut (0.1625 d−1, 1.1764 d−1, 264.9 offspring) than on sesbania (0.0951 d−1, 1.0997 d−1, and 30.3 offspring). Population projection of S. frugiperda on peanut demonstrated that this crop is a more suitable host plant than sesbania. Yet, this suboptimal host still assures an increasing trend of more than 357-fold individuals in 75 d, from the initial cohort of 10 eggs. Our data suggest that green manure plants in fallowing fields may support the pest’s survival all year round, and may be responsible for a successful establishment and unexpected outbreaks of this invasive pest on the neighboring crops in Taiwan. Our study thus highlights the importance of assessing the population dynamics and areawide pest management of an invasive polyphagous pest on a noneconomic crop to mitigate the potential risk of reinfestation and thus outbreaks.

Parasitoid wasps are invaluable agents in pest biological control. Early detection and identification of parasitoid immatures are vital in characterizing parasitoid–host interactions and for evaluating parasitism rates accurately in the field. Trichogramma is the most widely used parasitoid wasp, and several studies have been performed for its molecular identification. However, those studies were mainly focused on Trichogramma adults and rarely on immatures. Here, we report a method to detect and identify Trichogramma larvae in their host eggs. We designed a pair of Trichogramma-specific primers that amplified Trichogramma mtCOI sequences from Corcyra cephalonica (Stainton) eggs parasitized by any of eight Trichogramma species tested but not from nonparasitized eggs of four lepidopteran hosts. This PCR method reliably detected Trichogramma immatures in parasitized eggs as early as 1 h after parasitism. We further developed an RFLP (restriction fragment length polymorphism) assay using restriction enzymes SspI and VspI to differentiate eight Trichogramma species at their immature stage. Overall, we developed a sensitive and reliable PCR–RFLP method to detect and identify immature-stage Trichogramma in their lepidopteran hosts. This method shows promise for conveniently identifying Trichogramma in insectaries and accurately evaluating parasitism rates in the field.

Research indicates that nanoparticles can be an effective agricultural pest management tool, though unintended effects on the insect must be evaluated before their use in agroecosystems. Chrysodeixis includens (Walker) was used as a model to evaluate chronic parental and generational exposure to empty, positively charged zein nanoparticles ((+)ZNP) and methoxyfenozide-loaded zein nanoparticles (+)ZNP(MFZ) at low-lethal concentrations. To determine concentration limits, an acute toxic response test on meridic diet evaluated (+)ZNP(MFZ) and technical grade methoxyfenozide using two diet assay techniques. No differences in acute toxicity were observed between the two treatments within their respective bioassays. With these results, population dynamics following chronic exposure to low-lethal concentrations were evaluated. Parental lifetables evaluated cohorts of C. includens reared on diet treated with LC5 equivalents of (+)ZNP, (+)ZNP(MFZ), or technical grade methoxyfenozide. Compared to technical grade methoxyfenozide, (+)ZNP(MFZ) lowered both the net reproductive rate and intrinsic rate of increase, and was more deleterious to C. includens throughout its lifespan. This was contrasted to (+)ZNP, which showed no differences in population dynamics when compared with the control. To evaluate chronic exposure to (+)ZNP, generational lifetables reared cohorts of C. includens on LC5 equivalent values of (+)ZNP and then took the resulting offspring to be reared on either (+)ZNP or untreated diet. No differences in lifetable statistics were observed between the two treatments, suggesting that (+)ZNP at low ppm do not induce toxic generational effects. This study provides evidence into the effects of nanodelivered methoxyfenozide and the generational impact of (+)ZNP.

Chalcid Brachymeria podagrica (Fabricius), formerly known as Brachymeria fonscolombei (Dufour), is a parasitoid wasp that infests dipteran larvae of fly, Sarcophaga dux (Thomson), (Diptera: Sarcophagidae) recovered from the dead body of sheep in Bisha City, Asir region–Saudi Arabia. S. dux larvae were infested by B. podagrica. The first instar larvae and pupae of B. podagrica were found inside the larvae and pupae of S. dux. The adults of the parasitic wasp emerged from the pupae of the host. Adults and larvae of the parasitoid wasp were observed under a dissecting microscope. Identification was made by following the identification keys. It was confirmed that it was B. podagrica, which is herewith reported for the first time in Saudi Arabia.

To glean more information on mosquito diversity and distribution in Auroville, a cross-sectional study was carried out by mapping the distribution of water bodies and habitats supporting immature stages on the one hand and the distribution of water bodies/habitats supporting mosquito immature stages on the other. A satellite image covering an area of 8.08 km2 was overlaid with a grid of 500 × 500 m. Fifteen modules were selected and the area of each module served as the sampling site for the entomological survey. Adult and larval stages were sampled. Diversity indices were analyzed to compare mosquito diversity. Rarefaction estimations were used to compare abundance and richness of the mosquito species between different zones. In total, 750 mosquito larvae and 84 resting adults were sampled. Eighteen species of mosquitoes belonging to 11 subgenera and 7 genera were documented. Genera included Aedes (Johann Wilhelm Meigen 1818, Diptera, Culicidae), Anopheles (Johann Wilhelm Meigen 1818, Diptera, Culicidae), Armigeres (Theobald 1901, Diptera, Culicidae), Culex (Carl Linnaeus 1758, Diptera, Culicidae), Lutzia (Theobald 1903, Diptera, Culicidae), and Mimomyia (Theobald 1903, Diptera, Culicidae). Of the 18 mosquito species identified, 8 species are new records for Auroville. The Alpha (α) biodiversity indices show that the mosquito fauna is diverse (S = 18; DMg = 2.732 [95% CI: 2.732–2.732]). The Shannon-Weiner (Hʹ = 2.199 [95% CI: 2.133–2.276]) and Simpson indices (λ = 0.8619 [95% CI: 0.8496–0.8723]) measured species richness, evenness, and dominance. The values of these indices suggest high species richness, evenness, and dominance. Prevailing conditions can provide suitable environment for establishment of different mosquito species in this ecosystem. Given the sociodemographic characteristics of this area, research on mosquito diversity and risk of vector-borne diseases will be of great use.

The purpose of this research was to document the incidence of Japanese beetle, Popillia japonica Newman (Coleoptera: Scarabaeidae), infection by the microsporidian pathogen, Ovavesicula popilliae Andreadis & Hanula (Microsporida: Pleistophoridae) while monitoring Japanese beetle populations at nine golf courses in Michigan from 1999 to 2018. We also compared the current distribution of the pathogen in Michigan to the known distribution in 1999. Beetles were sampled a total of seven different years, in three time periods: 1999–2000, 2005–2007, and 2015–2018. At these nine golf courses, O. popilliae infection rates of adults increased from 0.39% in 2000 to 29.5% in 2018, while Japanese beetle adult populations declined 73.2%, and Japanese beetle larval density declined 78.6% (from 1999 to 2018). In previous research, larval infection rates averaged at least twice the infection rates of adults, because all infections originate in the larval stage, and at least 75% of infected larvae die before pupation. This means that an adult infection rate of 29.5%, combined with previously observed 50% reductions in egg production by infected females, are consistent with the observed population declines. The current distribution of O. popilliae in southern Michigan was determined by sampling Japanese beetle larvae and adults at 38 sites in 2017 and 2018. Ovavesicula popilliae was found at 21 of 38 sites and in 16 of 21 counties, compared with two sites in one of 16 counties when it was first detected in Michigan in 1999 and 2000.

This paper deals with species A, C, D, and E of the Lindesayi Complex of Anopheles subgenus Anopheles (Diptera: Culicidae) that were recently recorded from Bhutan. Species B is not included due to insufficient data. Species A is Anopheles lindesayi sensu stricto, and species C, D, and E are new species that are formally described and named herein as Anopheles druki Somboon, Namgay & Harbach, Anopheles himalayensis Somboon, Namgay & Harbach and Anopheles thimphuensis Somboon, Namgay & Harbach, respectively. Morphological characteristics of the adults, larval and pupal stages of each species are provided and compared with An. lindesayi from Bhutan.

Since many noctuid moth species are highly destructive crop pests, it is essential to establish proper management strategies, which primarily require accurate and rapid species identification. However, diagnosis of noctuid species in the field, particularly at the larval stage, is very difficult due to their morphological similarity and individual color variation. In particular, caterpillars of Spodoptera exigua (Hübner), Spodoptera litura (Fabricius), Spodoptera frugiperda (Smith), and Mamestra brassicae (L.) (Lepidoptera: Noctuidae) are hard to be identified by morphology and frequently found on the same host crops in the same season, thus requiring a reliable species diagnosis method. To efficiently diagnose these species, we identified species-specific internal transcribed spacer 1 (ITS1) sequences and developed two molecular species diagnosis protocols using ITS1 markers. The first protocol was multiplex conventional PCR in conjunction with subsequent gel electrophoresis for species identification based on amplicon size. The second protocol was based on multiplex real-time PCR using fluorescent dye-labeled primers for single-step diagnosis. Template genomic DNA (gDNA) prepared by the DNA release method was also suitable for both protocols as the template prepared by DNA extraction. The two protocols enabled rapid and robust species diagnosis using a single multiplex PCR step. Depending on laboratory instrumentation, one of the two protocols can be easily adapted for species diagnosis of the four noctuid caterpillars in the field, which is essential for establishing proper management strategies. The multiplex real-time PCR protocol, in particular, will facilitate accurate diagnosis of the four species in a single step regardless of template gDNA quality.

Despite a robust biocontrol program against emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), parasitoid populations cannot build quickly enough to save mature ash trees. The future of ash as a viable component of North American forests depends on survival of immature ash that were too small to be attacked during the initial outbreak. This study was designed to quantify impacts of established introduced parasitoids and native woodpeckers on the population growth of emerald ash borer infesting regenerating ash saplings in white ash forests. Most emerald ash borer larvae were killed by Tetrastichus planipennisi Yang and woodpeckers in the fourth instar. Life-table analyses revealed that the estimated net reproductive rate of emerald ash borer was R0 = 4.2 in eastern New York and R0 = 0.0 in western New York. Without mortality from parasitoids and woodpeckers, R0 values would have been 16.4 and 7.9 in eastern and western New York, respectively. We monitored the health of mature and sapling ash trees from 2012 to 2017 and found that large trees were significantly more likely to be infested with emerald ash borer and to die than smaller trees. Fifty-four percent to 81% of ash saplings contained no living emerald ash borer, and the density of emerald ash borer at all sites was very low (< 4/m2). This study adds to the growing body of evidence that emerald ash borer biocontrol is contributing to population control and can help promote survival of young ash trees as forests recover from emerald ash borer outbreaks.

The alfalfa leafcutting bee Megachile rotundata (Fabricius) (Hymenoptera: Megachilidae) is the primary pollinator for the alfalfa seed industry. It is a solitary cavity nesting bee that utilizes leaf lined brood cells provisioned with pollen for larval development and pupation into the adult stage. During development, multiple pathogens, parasitoids, and predators can prey upon or use the larvae as a host, resulting in the loss of the future adult bee. As such, the presence of invertebrate pests and fungal pathogens presents a major concern for commercial alfalfa seed growers. In the present study, we used historic data from the Parma Cocoon Diagnostic Laboratory to determine baseline rates of pathogens, parasitoids, and predators of Megachile rotundata brood cells and used this analysis to determine cutoffs for management practices to inform growers when the purchase of new bee stocks should be considered. Additionally, we compared the presence of chalkbrood, predators, and parasitoids in samples collected from both grower-produced stocks and newly purchased Canadian bees. The results of the investigation provide historic averages of the presence of chalkbrood, predators, and parasitoids, show a significant increase in chalkbrood and predators in 2007–2011, and find a significant difference in rates of chalkbrood and predators between samples from Canadian suppliers and grower stocks. We speculate that these differences may have resulted from economic conditions that increased the cost of Canadian Megachile rotundata cells and likely resulted in increased reliance on 2nd-year U.S. grower stocks and subsequently increased infection rates during this time period.

We report the multi-year collection of the Gulf Coast tick, Amblyomma maculatum Koch (Acaridae: Ixodida: Ixodidae) in Staten Island, New York City (NYC) as well as their detection in Brooklyn, NYC, and in Atlantic and Cumberland counties in southern New Jersey, USA. The first and most common detections were of adults, however in Freshkills Park on Staten Island larvae were also collected in a following year. The presence of larvae indicates that adults are successfully finding hosts in Staten Island. While it is still unknown how A. americanum reached Staten Island, immatures of this species often parasitize migratory birds, which are now often seen in Freshkills Park. We describe the landscape features of the area in Staten Island where populations were highest and larvae were detected, which could have facilitated the establishment of A. maculatum. Notably, we also report the presence of human pathogens Rickettsia parkeri in 5/10 (50%) of adults tested and R. felis in 1/24 (4.17%) of larvae tested. In addition to established populations in Staten Island we found evidence of A. maculatum in NJ and other NYC boroughs, suggesting current or future establishment is possible. The failure thus far to detect established populations in these areas may be due to inherent difficulties in detecting low density, spatially heterogeneous incipient populations, which could require targeted surveillance efforts for this species. We discuss the consequences to public health of the establishment of A. maculatum and detection of two additional rickettsial pathogens in the densely populated northeastern United States.

Asian chestnut gall wasp (ACGW) (Dryocosmus kuriphilus Yasumatsu), an invasive pest native to China, was detected in Michigan, the largest chestnut producer in North America, in 2015. Along with quantifying gall densities, we tracked dates and cumulative growing degree days corresponding to ACGW life stages in five, seven, and nine orchards in 2017–2019, respectively. Gall formation, triggered by the onset of feeding by overwintered larvae, began soon after bud break. Most adult wasps emerged in mid summer, after pollen production peaked. Density of ACGW in all sites dropped sharply in 2019, probably as a result of larval mortality caused by severely cold temperatures in late January. Gall density was generally lower on Chinese chestnut (Castanea mollisima Blume) trees and C. mollisima hybrids, which share some coevolutionary history with ACGW, than on cultivars of European chestnut (C. sativa Miller) and Japanese-European (C. crenata Sieb. & Zucc. x C. sativa) trees, including the popular Colossal cultivar. Torymus sinensis Kamijo (Hymenoptera: Torymidae), a larval parasitoid previously introduced into the U.S. and several countries in Asia and Europe for ACGW biocontrol, appears to be spreading with ACGW in Michigan. Parasitoid larvae were recorded in four, six, and eight of the infested sites we sampled in 2017–2019, respectively, and parasitism rates ranged from 5 to70% of galls.

A new species of the subgenus Pseudoficalbia of the genus Uranotaenia is described from larvae found in a hole in a log at high altitude in montane forest in the Mongar District of Bhutan. Based on morphology, the larvae are sufficiently distinct from other species of the subgenus to be described and formally named here as Uranotaenia bhutanensis Somboon, Namgay & Harbach, n. sp. The species is a member of the Bimaculata Series. The chaetotaxy of the fourth-instar larva is provided.

While traits of plant resistance to herbivory often change during ontogeny, it is unknown whether the primability of this resistance depends on the plant’s developmental stage. Resistance in non-flowering Arabidopsis thaliana against Pieris brassicae larvae is known to be primable by prior egg deposition on leaves. We investigated whether this priming effect is maintained in plants at the flowering stage. Larval performance assays revealed that flowering plants’ resistance to herbivory was not primable by egg deposition. Accordingly, transcriptomes of flowering plants showed almost no response to eggs. In contrast, egg deposition on non-flowering plants enhanced the expression of genes induced by subsequent larval feeding. Strikingly, flowering plants showed constitutively high expression levels of these genes. Larvae performed generally worse on flowering than on non-flowering plants, indicating that flowering plants constitutively resist herbivory. Furthermore, we determined the seed weight in regrown plants that had been exposed to eggs and larvae during the non-flowering or flowering stage. Non-flowering plants benefitted from egg priming with a smaller loss in seed yield. The seed yield of flowering plants was unaffected by the treatments, indicating tolerance towards the larvae. Our results show that the primability of anti-herbivore defences in Arabidopsis depends on the plant’s developmental stage.

Soybean gall midge, Resseliella maxima Gagné (Diptera: Cecidomyiidae), is a newly identified pest confirmed on soybean, Glycine max (L.) Merr. (Fabales: Fabaceae). To date, soybean gall midge has been found in Nebraska, Iowa, South Dakota, Minnesota, and Missouri, and has caused severe economic loss to commercial fields since 2018. Much is still unknown about this pest, so research efforts have been focused on biology and management. Larvae feed on the inside of the stem just above the soil line and are difficult to access and time-consuming to sample. In order to accelerate nondestructive sampling efforts, we developed an injury rating system to quantify the severity of plant injury from soybean gall midge larvae. Research plots from 2019 and 2020 in Iowa and Nebraska were evaluated for injury throughout the growing season and yield was measured. Our objective was to describe the relationship between injury severity and yield loss caused from soybean gall midge. A nonlinear regression model was developed to validate our injury rating system and to express the relationship between season long injury severity and yield loss. Results from our analysis indicate the injury rating system we developed correlates well with yield loss caused by larvae and may be an important tool for understanding the economic impact of this emergent pest of soybeans.

The armyworm, Mythimna separata (Walker) (Lepidoptera: Noctuidae), is an important polyphagous pest with a strong migratory ability. Recently, third-generation larvae have become an increasingly serious pest threat in Jilin Province of northeast China. To investigate the population source of this species, scanning entomological radar observations and insect mitochondrial cytochrome oxidase I (COI) genes were used in this study. Five main results were found: (1) The peak period in captured second-generation moths was from mid to late July. The temperature and wind speeds were optimum for the moths to have migrated. Strong southwesterly winds occurred during the peak migration period. (2) Radar observations indicated that most of the moths’ migration took place at a height of 600 m, often in a dense layer which formed at heights of 350–800 m. (3) Analyses of adult ovarian development and larval haplotypes showed third-generation larvae were progeny of both locally produced progeny and immigrant moths. (4) Based on our back-tracking and haplotype analyses, immigration led to an outbreak originated in the same source area to the southwest. (5) Emigration of second-generation moths was confirmed by both radar observation and mtDNA analysis. Forward trajectories indicated that the moths were capable of immigrating far from their overwintering range. These results are useful for improving the forecasting systems of this insect pest species.

Background Anopheles stephensi is a newly invaded vector in Sri Lanka. It has been identified in coastal areas in the northern and eastern parts of the country and evidences the ability to breed in brackish water environments. Methods Laboratory investigations were conducted with batches of 100 first and third instar larvae exposed to a salinity gradient (0–40 ppt). Survival rates at 1 h, 24 h and until pupation were recorded for first and third instar larvae at different salinity levels. The experiment was repeated four times for both instars. Data were analysed using the general linear model and probit analysis. Results Significant variations in adult emergence were observed from both larval stages at different salinity levels (p80% up to 12.5 ppt, while 100% mortality was observed for from the ≥25 ppt level of salinity. More than 90% of the third instar larvae pupated from salinity levels Conclusions This indicates a high potential of increasing density of A. stephensi in coastal ecosystems in lagoons and other saline water bodies. Hence it is high time to redesign vector control interventions for vector breeding in coastal ecosystems.

The control of medically important container-inhabiting mosquitoes is an ongoing challenge for mosquito control operations. Truck-mounted application equipment is a common option for rapid areawide larvicide deployment utilized by mosquito control operations. We tested the effectiveness of two truck-mounted sprayers (A1 Super Duty + Buffalo Turbine CSM3), for the deployment of water-dispersible biopesticides (VectoBac WDG:VectoLex WDG 50:50). Sixty residences within four residential neighborhoods in New Jersey were treated in 2019 and 2020. Three empty bioassay cups were placed in specific locations on each property (front yard/ back yard/ side of house), with an additional cup placed in an adjacent catch basin. This approach was replicated in two untreated control neighborhoods. Following larvicide application, cups were subjected to bioassays wherein larval mortality was tracked through adult eclosion. Overall, average larval mortality rates were 56% higher in treated cups compared against untreated controls. Mortality rates were affected by cup location, with 39% mortality in bioassay cups from back yards, 54% in those from the sides of houses, 73% in front yards, and 76% from cups in catch basins. Mortality did not differ significantly between the four treated neighborhoods, nor by the type of sprayer used. Our research shows that truck-mounted sprayers can be an effective method for larvicide deployment in residential neighborhoods, but effectiveness may depend upon the location of the target treatment area in relation to residences and other geographic obstacles.

Iodothyronine deiodinases (DIO) are key enzymes that influence tissue-specific thyroid hormone levels during thyroid-mediated amphibian metamorphosis. Within the larger context of evaluating chemicals for thyroid system disrupting potential, chemical activity toward DIOs is being evaluated using high-throughput in vitro screening assays as part of U.S. EPA’s ToxCast program. However, existing data gaps preclude any inferences between in vitro chemical inhibition of DIOs and in vivo outcomes relevant to ecological risk assessment. This study aimed to generate targeted data in a laboratory model species (Xenopus laevis) using a model DIO inhibitor, iopanoic acid (IOP), to characterize linkages between in vitro potency, in vivo biochemical responses, and adverse organismal outcomes. In vitro potency of IOP toward DIOs was evaluated using previously developed in vitro screening assays, which showed concentration-dependent inhibition of human DIO1 (IC50: 97 µM) and DIO2 (IC50: 231 µM) but did not inhibit human or X. laevis DIO3 under the assay conditions. In vivo exposure of larval X. laevis to 0, 2.6, 5.3, and 10.5 µM IOP caused thyroid-related biochemical profiles in the thyroid gland and plasma consistent with hyperthyroxinemia but resulted in delayed metamorphosis and significantly reduced growth in the highest 2 exposure concentrations. Independent evaluations of dio gene expression ontogeny, together with existing literature, supported interpretation of IOP-mediated effects resulting in a proposed adverse outcome pathway for DIO2 inhibition leading to altered amphibian metamorphosis. This study highlights the types of mechanistic data needed to move toward predicting in vivo outcomes of regulatory concern from in vitro bioactivity data.

Plant resistance is a key strategy for the management of Oryzophagus oryzae (Costa Lima) (Coleoptera: Curculionidae), an important pest in South American rice paddies. The present study investigated the resistance of rice cultivars in terms of feeding and oviposition preference, growth, development, and biological performance of O. oryzae under natural conditions of field infestation during two consecutive rice seasons. There were no effects of the six cultivars on the feeding and oviposition preferences of O. oryzae as evaluated 5, 8, and 11 d After Flooding (DAF) of the plots, indicating the absence of antixenosis. Cultivars did not differ in terms of egg viability and larval density of first instars on the roots at 15 DAF. Significant differences were found 25 and 35 DAF when larval density per sample was high on ‘BRS Pampa CL’ (up to 24.5), intermediate on ‘BRS Querência’ and ‘BRS Ligeirinho’ (up to 16.1), and low on ‘BRS Atalanta’, ‘BRS Firmeza’, and ‘Dawn’ (up to 8.8). The cultivars ‘BRS Atalanta’, ‘BRS Firmeza’, and ‘Dawn’ caused malnutrition and inhibition of larval growth. These effects, typical of antibiosis, resulted in delayed pupation and emergence of adults; in addition, emerged females had body weight decreased strongly. The cultivars BRS Pampa CL, BRS Querência, and BRS Ligeirinho are susceptible, resulting in high larval populations and more suitable development of O. oryzae; antibiosis, as indicated for ‘BRS Atalanta’, ‘BRS Firmeza’, and ‘Dawn’, probably is the key mechanism of rice resistance to O. oryzae.

Seasonal temperatures select for eclosion timing of temperate insects and their parasitoids. In western North America, the fruit fly Rhagoletis zephyria Snow (Diptera: Tephritidae) is parasitized by the hymenopterous wasps Utetes lectoides (Gahan), an egg parasite, and Opius downesi Gahan, a larval parasite (both Braconidae). Eclosion of wasps should be timed with the presence of susceptible fly stages, but reports indicate U. lectoides ecloses in the absence of flies under no-chill conditions. Based on this, we tested the hypotheses that chill durations and no-chill temperatures both differentially regulate eclosion times of R. zephyria and its parasitic wasps. When fly puparia were chilled at ~3°C for 130–180 d, U. lectoides and O. downesi always eclosed on average later than flies. However, after 180-d chill, flies eclosed on average earlier than after 130- and 150-d chill, whereas eclosion times of U. lectoides and O. downesi were less or not affected by chill duration. When fly puparia were exposed to 20–22°C (no chill), U. lectoides eclosed before flies, with 88.9% of U. lectoides versus only 0.61% of flies eclosing. Taken together, findings show that eclosion times of flies are more sensitive to changes in chill duration than those of wasps. Flies are less sensitive than wasps to no-chill in that most flies do not respond by eclosing after no-chill while most wasps do. Our results suggest that shorter winters and longer summers due to climate change could cause mismatches in eclosion times of flies and wasps, with potentially significant evolutionary consequences.

Hormozgan Province is one of the important foci of malaria in Iran. In addition to malaria pathogens, mosquitoes also transmit the pathogens that cause dirofilariasis and West Nile fever in the province. Also, the threat of emerging aedine-borne viruses that cause infections, such as Chikungunya, dengue, and Zika, is noticeable. There is little information about the fauna and bionomics of Culicinae in the province. The present investigation aimed to study the fauna, diversity, and bionomics of culicines. The study was conducted from September 2016 to April 2017 in four counties of Bandar Abbas, Bandar Khamir, Bashagard, and Jask. In total, 3,236 larvae and 1,901 adults including 16 culicine species were collected. The larvae of Culiseta longiareolata (Macquart) (25.65%), Culex pipiens Linnaeus (16.62%), and Cx. quinquefasciatus Say (16.16%) were most abundant and Cx. hortensis Ficalbi (0.09%) was least abundant. Among adults, Cx. laticinctus Edwards (33.19%), Cx. quinquefasciatus (31.09%), and Cx. pipiens (11.99%) were the most prevalent species and Cs. longiareolata (0.47%), Aedes caballus (Theobald) (0.90%), and Cx. bitaeniorhynchus Giles (0.90%) were the least prevalent species. The pairwise similarities of fauna of the counties and different collecting methods and diversity indices were investigated. More ecological data, especially on host preference, seasonality, and larval habitat characteristics, are needed as a basic knowledge for any intervention measures using integrated vector management.

Organic yard waste from western Washington, U.S. that may contain puparia of apple maggot fly, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), had been moved to central Washington for composting, threatening the $3 billion apple industry concentrated in that region. Heating waste to kill fly puparia before it is transported could be a solution to this problem. Here, we report results of studies in 2016–2021 that sought to identify a minimum heat treatment simulating that obtained using a low-pressure steam generator for maximizing kill of R. pomonella puparia. In two experiments, puparia were exposed to temperatures ramped linearly over 6 h from 21°C to 47.8, 51.1, 55.0, or 60.0°C in an oven. The 47.8, 51.1, and 55°C treatments did not achieve 100% mortality, although only one adult fly from 4,000 puparia was found in the 55°C treatment, while no puparia survived the 60°C treatment. In a third, similar experiment, no puparia out of 2,400 exposed to 55°C survived. In a fourth and final experiment conducted over 3 years, no puparia out of 61,223 exposed to a 6-h ramp from 21°C to 55°C followed by a 1-h hold time at 55°C produced flies. In addition, all puparia in this treatment died. Based on 42.3 to 69.8% control survival, 31,217 puparia were killed by this treatment with no survivors, for a probit 8.7190 level of security. Results suggest that the 55°C and 1-h hold time treatment here is close to the minimum heat regime needed for disinfesting organic waste of R. pomonella puparia.

Calliphora grahami (Aldrich, 1930) (Diptera: calliphoridae) is a forensically important blow fly that is widely distributed across Asia, North America, Russia, and Mexico. Calliphora grahami is frequently found on corpses during the spring, autumn, and winter seasons. It is among the early colonizers of cadavers during the cold season, and sometimes, the only necrophagous blow fly on cadavers. Therefore, this species is of forensic significance, although very few studies have explored the application of its intrapuparial age for PMI estimation. This study aimed to examine the intrapuparial development of C. grahami and establish a method for estimating its intrapuparial age. Herein, the C. grahami puparia were studied under six different temperatures (13, 16, 19, 22, 25, and 28°C) for the intrapuparial age estimation, and a total of 5776 puparia were sampled. The morphological changes were divided into 11 stages based on the 1) development of legs and wings, 2) differentiation of the head, thorax, and abdomen, 3) growth and color of the bristles, and 4) color changes of the compound eyes. The corresponding time of each stage was determined. Moreover, the observation and classification of individual morphological features, including compound eyes, antennae, mouthparts, thorax, abdomen, legs, and wings was used to improve the precision of intrapuparial age estimation. The findings of this study provide important information on the use of C. grahami intrapuparia to estimate the minimum postmortem interval (PMImin).

The apple maggot, Rhagoletis pomonella (Walsh), was introduced into the apple-growing regions of the Pacific Northwest in the U.S.A. during the past 60–100 yr. Apple maggot (larvae, puparia, and adults) is difficult to distinguish from its morphologically similar sister species, Rhagoletis zephyria Snow, which is native and abundant in the Pacific Northwest. While morphological identifications are common practice, a simple, inexpensive assay based on genetic differences would be very useful when morphological traits are unclear. Here we report nucleotide substitution and insertion–deletion mutations in the nontranscribed spacer (NTS) of the ribosomal RNA gene cistron of R. pomonella and R. zephyria that appear to be diagnostic for these two fly species. Insertion–deletion variation is substantial and results in a 49 base-pair difference in PCR amplicon size between R. zephyria and R. pomonella that can be scored using agarose gel electrophoresis. PCR amplification and DNA sequencing of 766 bp of the NTS region from 38 R. pomonella individuals and 35 R. zephyria individuals from across their geographic ranges led to the expected PCR fragments of approx. 840 bp and 790 bp, respectively, as did amplification and sequencing of a smaller set of 26 R. pomonella and 16 R. zephyria flies from a sympatric site in Washington State. Conversely, 633 bp mitochondrial COI barcode sequences from this set of flies were polyphyletic with respect to R. pomonella and R. zephyria. Thus, differences in NTS PCR products on agarose gels potentially provide a simple way to distinguish between R. pomonella and R. zephyria.

The present study aimed to determine the effects of the soil type and the moisture contents on the some life-history parameters of Lucilia sericata (Meigen, 1826) (Diptera: Calliphoridae). The larval and pupal survival, duration of development periods, and the weight of pupae and adult of L. sericata were examined at three different types of soil (clay, loamy, and sandy) with five moisture contents (0, 25, 50, 75, 100%). The post feeding L. sericata larvae were transferred to a plastic cup filled with soils with different moisture content, and all cups were kept at 27°C, 65% RH, and a photoperiod of 12:12 (L:D) h. In this article, the effects on some life-history parameters were compared with a two-way analysis of variance (ANOVA). Accordingly, the findings of this article indicate that moisture contents of soils and soil type have a significant effect on the development time of L. sericata. Furthermore, it has been found that larvae can survive on extremely wet substrates (75–100% humidity), although their development time is longer than other moisture content. Pupal and adult weight was significantly different among soil type and soil moisture. Our results provide a reference information for sufficient estimation of the effects of changes in moisture-related to soil type or climate on studies of forensic and biological control of L. sericata.

High abundance of hematophagous mosquitoes of the genus Mansonia Blanchard, 1901 (Diptera: Culicidae) threatens human and domestic animal health and well-being. Knowledge of the biology of nuisance mosquito species is necessary to understand specific ecological and biological factors to enable rapid and effective monitoring measures for sustainable control programs. The establishment and dispersion of Mansonia species are associated with the occurrence of aquatic macrophytes species, which are indispensable for the development of larvae and pupae. To increase knowledge of the host plants for Mansonia immature stages in Porto Velho, Rondonia State, Brazil, specimens of four plant species, which occur across the tributaries of the Madeira River were sampled and inspected for the presence of egg batches, larvae, and pupae. A total of 1,386 larvae and pupae of Mansonia spp. were collected attached to the roots of Eichhornia crassipes (Mart.) Solms (Commelinales: Pontederiaceae), Pistia stratiotes L. (Alismatales: Araceae), and Limnobium laevigatum (Humb. and Bonpl. Ex Willd.) Heine (Alismatales: Hydrocharitaceae). The novel association of Mansonia species with L. laevigatum is presented. Egg batches of Mansonia spp. were found only on Salvinia molesta D.S. Mitch. (Salviniales: Salviniaceae). Possible differences in the roles played by E. crassipes and S. molesta in the reproductive cycle of Mansonia spp. in the surveyed area are discussed. All species of host plants including E. crassipes, P. stratiotes, S. molesta, and L. laevigatum should be considered when planning macrophyte management for the control of Mansonia species.

Methyl benzoate (MBe) is a volatile organic molecule found in various plants; it is used as an insect semiochemical. MBe also has a biorational insecticidal effect against various agricultural and urban arthropod pests. The present study was the first to assess the larvicidal potential of MBe against fourth-instar larvae of the mosquitoes Aedes albopictus (Skuse) and Culex pipiens (L.). A positive association was observed between MBe concentrations and larval mortality in both the species. The highest mortality recorded was 100% for Ae. albopictus and 56% for Cx. pipiens after 24 h of exposure to 200 ppm MBe. The lethal median concentration (LC50) values of MBe against fourth-instar larvae of Ae. albopictus and Cx. pipiens were 61 ppm and 185 ppm, respectively. These results suggest that MBe has great potential for use as an environmentally friendly larvicidal agent for mosquito control.

The interspecific variation in susceptibility to insecticides by lepidopteran species of soybean [Glycine max L. (Merr.)], cotton (Gossypium hirsutum L.), and maize (Zea mays L.) crops from Brazil were evaluated. Populations of Anticarsia gemmatalis (Hübner) (Lepidoptera: Erebidae), Chrysodeixis includens (Walker), Helicoverpa armigera (Hübner), Spodoptera frugiperda (Smith), Spodoptera eridania (Stoll), Spodoptera cosmioides (Walker), and Spodoptera albula (Walker) (Lepidoptera: Noctuidae) were collected from 2019 to 2021. Early L3 larvae (F2 generation) were exposed to the formulated insecticides methoxyfenozide, indoxacarb, spinetoram, flubendiamide, and chlorfenapyr in diet-overlay bioassays. The median lethal concentrations (LC50) were used to calculate tolerance ratios (TR) of each species in relation to the most susceptible species to each insecticide. The lowest LC50 values were verified for A. gemmatalis to all insecticides tested. Chrysodeixis includens and most of the Spodoptera species were moderately tolerant to methoxyfenozide (TR < 8.0-fold) and indoxacarb (TR < 39.4-fold), whereas H. armigera was the most tolerant species to methoxyfenozide (TR = 21.5-fold), and indoxacarb (TR = 106.4-fold). Spodoptera cosmioides, S. eridania, and S. albula showed highest tolerance to spinetoram (TR > 1270-fold), S. eridania, S. frugiperda, and S. albula to flubendiamide (TR from 38- to 547-fold), and S. albula to indoxacarb (TR = 138.6-fold). A small variation in susceptibility to chlorfenapyr (TR < 4.4-fold) was found among the lepidopteran evaluated. Our findings indicate a large variation in susceptibility to indoxacarb, spinetoram, and flubendiamide and a relatively low variation in susceptibility to methoxyfenozide and chlorfenapyr by lepidopteran species of soybean, cotton, and maize from Brazil.

Emerald ash borer (Agrilus planipennis Fairmaire (Coleoptera: Buprestidae)), an invasive phloem-feeding beetle native to Asia, has devastated North American ash forests since its detection in Michigan, United States in 2002. As the emerald ash borer has continued to spread, the potential for successful long-term management hinges upon the release, establishment, and spread of introduced larval and egg parasitoids for biological control. Here, we focus on the establishment and evidence for spatial spread of introduced larval parasitoid, Spathius agrili Yang and Spathius galinae Belokobylskij & Strazanac (Hymenoptera: Braconidae) in the state of Maryland. To assess each species, we analyzed historical release and recovery data and resampled previous release sites and nonrelease sites for establishment. We found little evidence of establishment or spread for S. agrili, despite a comparatively large number of release locations, events, and individuals. By contrast, despite much lower propagule pressure and shorter history of releases, we detected multiple established populations of S. galinae at release sites and at sites up to 90 km from the nearest release point approximately 3 yr after its most current release. Our findings show that S. galinae has established and spread rapidly following field releases whereas its congener, S. agrili has not. Although it may still be too early to evaluate the level of population control and ash protection afforded by S. galinae, these findings indicate the need for continued investment in S. galinae for emerald ash borer classical biological control efforts.

The fall armyworm, Spodoptera frugiperda (Smith), is an invasive pest of cereal crops that now inhabits southern China year-round. Cultivation of crops unsuitable as host plants has been an effective pest management strategy for some insect pests, but the effects of green manure crops on S. frugiperda have not been investigated. An age-stage two-sex life table and tethered flight performance of S. frugiperda reared on different green manure species were obtained, and a population dynamics model established. Developmental durations of stages, survival rates, and fecundities of S. frugiperda differed significantly depending on host plant. Larvae fed Astragalus sinicus L. did not complete development. Although some larvae fed Vicia villosa Roth and Vicia sativa L. completed development, generation time was significantly prolonged, egg production was halved, and net reproductive rate decreased to 31% and 3% of those reared on corn, respectively. Survival rates of early-instars fed V. villosa and V. sativa were significantly lower than those fed corn. Population dynamics projections over 90 d showed the number of generations of S. frugiperda fed on V. villosa and V. sativa was reduced compared to those reared on corn. Flight performance of S. frugiperda reared on V. villosa decreased significantly compared to corn. Our results show that the three green manure species are unsuitable host plants for S. frugiperda. Therefore, reduction of corn production in southern China through rotation with these green manure crops may be a feasible method of ecological management of this major corn pest in China.

Dendroctonus armandi (Tsai and Li) (Coleoptera: Curculionidae: Scolytinae) is considered to be the most destructive forest pest in the Qinling and Bashan Mountains of China. Low winter temperatures limit insect’s populations, distribution, activity, and development. Insects have developed different strategies such as freeze-tolerance and freeze-avoidance to survive in low temperature conditions. In the present study, we used gene cloning, real-time polymerase chain reaction (PCR), RNA interference (RNAi), and heterologous expression to study the function of the D. armandi antifreeze protein gene (DaAFP). We cloned the 800 bp full-length cDNA encoding 228 amino acids of DaAFP and analyzed its structure using bioinformatics analysis. The DaAFP amino acid sequence exhibited 24–86% similarity with other insect species. The expression of DaAFP was high in January and in the larvae, head, and midgut of D. armandi. In addition, the expression of DaAFP increased with decreasing temperature and increasing exposure time. RNAi analysis also demonstrated that AFP plays an important role in the cold tolerance of overwintering larvae. The thermal hysteresis and antifreeze activity assay of DaAFP and its mutants indicated that the more regular the DaAFP threonine-cystine-threonine (TXT) motif, the stronger the antifreeze activity. These results suggest that DaAFP plays an essential role as a biological cryoprotectant in overwintering D. armandi larvae and provides a theoretical basis for new pest control methods.