Field-collected Insects Research Articles

Determinants of Choice of Marketing Outlet for Edible Insects among Smallholder Farmers and Traders in Western Kenya

The edible insect sector has, in recent times, gained significant prominence and attention. Particularly, the government of Kenya has made remarkable steps to achieve a potentially large and valuable edible insect market, with a significant milestone being the passing of regulations on edible insects as a new source of proteins. However, research on the edible insect marketing environment is still indistinct. Therefore, the study sought to evaluate the determinants of the market outlets preferred or used by smallholder farmers and traders of domesticated and field-collected edible insects (including cricket, bees, winged termites, lake flies, and dung beetle) in Siaya and Vihiga counties. This cross-sectional study was done among 188 edible insects’ farmers and traders. Purposive sampling identified the study area, while snowball sampling reached the study participants. Data was collected using structured questionnaires and analyzed using multinomial logit regression to assess independent-dependent variable relationships, yielding marginal effects. Study findings showed that at 95% confidence interval, the yield was significant to the three outlets used by the farmers. Gender was significant to both institution and open-air markets outlets but insignificant to selling at the farm gate. Age, education level, and experience in marketing were insignificant to all the three market outlets. Marketing training was significant to the institution and open-air markets. The study also showed that farmers and traders had limited choices to sell their edible insect produce, which was majorly affected by yield and age variables. In view of these findings, enhancing edible insect marketing and training through initiatives that would increase production among farmers and breaking the attitudes toward open-air marketing among male farmers is pivotal to the thriving of the novel food enterprise towards achieving food security in the region.

Extended stage duration and diminished fecundity in deltamethrin-resistant Triatoma infestans (Klug, 1834) of the Argentinean Chaco.

Pyrethroid-resistance is an emergent trait in populations of various insect species. For Triatoma infestans (Klug, 1834) (Heteroptera: Reduviidae), the major vector of Chagas disease in the southern part of South America, hotspot areas of pyrethroid-resistance have recently been found in the Chaco Province of Argentina. Resistant condition can reduce fitness of individuals in the absence of insecticide exposure, that is, fitness costs. We evaluated the existence of developmental and/or reproductive costs in T. infestans collected from two areas of pyrethroid-resistance in Chaco Province, Argentina. Three toxicological groups were defined from field-collected insects: susceptible (survival <20%), moderately resistant (survival between 20% and 80%) and highly resistant (survival >80%). Cohorts of the three toxicological groups were followed-up to study life cycle and reproductive parameters. Additionally, we parameterized matrix population growth models. First and IV nymphal stages of the resistant groups exhibited a longer stage duration than susceptible ones. The reproductive days and hatching success showed significant lower values revealing reproductive costs for the resistant groups. Matrix analysis showed lower population growth rates for the resistant groups. Our results support developmental and reproductive costs for pyrethroid-resistant individuals. This trait could be interpreted as lower population recovery ability for pyrethroid-resistant individuals compared to susceptible insects after alternative vector control actions.

Heterorhabditis amazonensis to Control Euschistus heros (Hemiptera: Pentatomidae) in Laboratory and Field Conditions.

This study evaluated the potential of entomopathogenic nematodes (EPNs) to control Euschistus heros (Fabricius) under laboratory and field conditions. Eight isolates of the species Heterorhabditis amazonensis were evaluated on field-collected and laboratory-reared insect populations. The isolates that performed best in the virulence test were evaluated for in vivo production on Galleria mellonella Fabricius larvae. Then, these were evaluated in field conditions on E. heros from the laboratory and field populations. The isolates IBCB-n46, NEPET11, and IBCB-n40 caused the highest mortality in adult E. heros from laboratory rearing (100, 94, and 80%, respectively), and IBCB-n46 and NEPET11 were the best against the field population (71 and 47% mortality, respectively). In the assay to evaluate production, the isolate IBCB-n46 achieved the best result (3.4 × 105 IJs/g per larvae), followed by isolates UEPN05 (1.4 × 105 IJs/g per larvae) finally NEPET11 (7.5 × 104 IJs/g per larvae). In the assay in field conditions, all treatments differed from the control treatment, no significant differences were observed in the evaluated factors (isolates and insect populations), and the highest percentage of mortality was obtained by the UEPN05 isolate when applied to insects from the laboratory (18.57% mortality).

Use of Pyriproxyfen to Induce Oogenesis in Diapausing Megacopta cribraria (Heteroptera: Plataspidae), and Evaluation of Pyriproxyfen-Induced Eggs for Rearing the Parasitoid Paratelenomus saccharalis (Hymenoptera: Scelionidae).

Simple SummaryRearing of many hymenopteran egg parasitoids requires a reliable supply of host eggs. The parasitoid Paratelenomus saccharalis can be reared on eggs produced from field collected kudzu bug, Megacopta cribraria, however field-collected hosts obtained during reproductive diapause do not readily produce eggs and must be reared under a long day-length photoperiod to terminate diapause. In this study we found that an exogenous application of pyriproxyfen was able to terminate diapause, leading to a significant increase in egg production. The eggs produced by pyriproxyfen-treated M. cribraria were accepted by the egg parasitoid Paratelenomus saccharalis, however parasitoid emergence was reduced when compared to eggs from untreated hosts. When the effects of pyriproxyfen treatment on egg production and parasitoid emergence were evaluated together, the net increase in parasitoid yield due to pyriproxyfen treatment was approximately 87%. This method has the potential to increase parasitoid yield and reduce production costs in egg parasitoid rearing programs.The mass rearing of hymenopteran egg parasitoids requires an abundant supply of host eggs. The onset of reproductive diapause and subsequent decline in egg production poses a challenge for parasitoid rearing when using host colonies augmented by field-collected insects. We investigated the application of pyriproxyfen, a juvenile hormone analog, to induce oviposition in diapausing adult kudzu bugs, Megacopta cribraria (Fabricius) (Heteroptera: Plataspidae), and the use of eggs produced by pyriproxyfen-treated kudzu bugs to rear the egg parasitoid, Paratelenomus saccharalis (Dodd) (Hymenoptera: Scelionidae). The effects of pyriproxyfen and photoperiod treatments on host mortality, egg production, and rates of parasitoid eclosion from the eggs were used to calculate the parasitoid yield for the different treatment regimes. A combination of pyriproxyfen and a long-day photoperiod increased the parasitoid yield by 87% compared to acetone and a long-day photoperiod. The general applicability of JH-analog mediated egg production for parasitoid rearing is discussed.

Rapid Molecular Diagnostics in the Field and Laboratory to Detect Plant Pathogen DNA in Potential Insect Vectors.

A variety of sensitive and specific molecular diagnostic assays has been described for detecting nucleic acids in biological samples that may harbor pathogens of interest. These methods include very rapid, isothermal nucleic acid amplification methods that can be deployed outside of the laboratory environment, such as loop-mediated isothermal DNA amplification (LAMP) and recombinase-polymerase amplification (RPA). However, all molecular diagnostic assays must be preceded by nucleic acid extraction from the biological samples of interest, which provides suitable template molecules for the assays. To exploit the features of the amplification assays and be utilized outside of the lab, these methods must be rapid and avoid the need for typical laboratory chemicals and equipment. We describe a protocol for the extraction of DNA from field-collected insects that can be implemented at the point of collection and used to detect the presence of DNA sequences from potential plant pathogens that may be vectored by the insects. This protocol provides template DNA that is suitable for PCR, LAMP, and RPA. The FTA PlantSaver card-based DNA extraction product was also confirmed to amplify the mitochondrial cytochrome oxidase 1 (CO1) universal barcode that could later be sequenced to identify any insect. Lastly, we provide an example using field-collected insects, Neokolla (Graphocephala) heiroglyphica, and demonstrate the detection of the plant pathogen Xylella fastidiosa in carrier insects using PCR, RPA, and LAMP.

Comparison of Diet Preferences of Laboratory-Reared and Apartment-Collected German Cockroaches.

The German cockroach, Blattella germanica (L.) (Blattodea: Ectobiidae), is a common pest of human-built structures worldwide. German cockroaches are generalist omnivores able to survive on a wide variety of foods. A number of studies have concluded that laboratory-reared B. germanica self-select diets with an approximate 1P:3C (protein-to-carbohydrate) ratio. We predicted that field-collected insects would exhibit more variable dietary preferences, related to the wide-ranging quality, quantity, and patchiness of foods available to them. We compared diet self-selection of B. germanica within apartments and in the laboratory by offering them a choice of two complementary diets with 1P:1C and 1P:11C ratios. We observed high variation in the population-level self-selection of these diets among individual apartment sites as well as among various life stages tested in laboratory-based assays. Significant differences between populations in various apartments as well as between populations maintained in the laboratory suggested that factors beyond temporary food scarcity influence diet choice. Nevertheless, we found significant correlations between the amounts of diets ingested by cockroaches in apartments and cockroaches from the same populations assayed in the laboratory, as well as between males, females, and nymphs from these populations. These findings suggest that females, males, and nymphs within apartments adapt to the local conditions and convergently prefer similar amounts of food of similar dietary protein content.

Epicuticular hydrocarbons of the redbanded stink bug Piezodorus guildinii (Heteroptera: Pentatomidae): sexual dimorphism and alterations in insects collected in insecticide-treated soybean crops.

The redbanded stink bug Piezodorus guildinii (Heteroptera: Pentatomidae) is one of the most important species affecting soybean crops in southern South America. Capillary gas chromatography coupled to mass spectrometry was used to characterize the epicuticular hydrocarbon profiles of field-collected insects, and to identify differences in their composition between fifth-instar nymphs and adults, males and females, and between bugs collected in insecticide-treated and insecticide-free soybean crops. Straight chain saturated n-C27 and n-C29, and monomethyl and dimethyl chains of C31 and C33 were the most abundant compounds. A group of volatile hydrocarbons with n-C13 and n-C15 as the predominant compounds were also detected. The hydrocarbon pattern was different between nymphs and adults, either males or females. Heneicosene was almost exclusively detected in adult males and was the most important component to differentiate between both sexes, followed by tricosadiene. The total hydrocarbon amount was significantly higher in nymphs, males and females collected in insecticide-treated fields compared with insects obtained from untreated fields. Differences were found in the epicuticular hydrocarbon pattern among nymphs and adults, as well as sexual dimorphism in adult stink bugs. Interestingly, an alteration was also found in the hydrocarbon profile of insects collected in insecticide-treated soybean crops and its relevance is discussed within a pest management context.

The Detection of Homopterous Phytoplasma Vectors Infecting Some Ornamental and Medicinal Plants In Egypt الکشف عن نواقل الفيتوبلازما متجانسة الاجنحة التى تصيب بعض نباتات الزينة والنباتات الطبية في مصر

This work is the first record of a phytoplasma associated with homopterous phytoplasma vectors infecting some ornamental and medicinal plants in Egypt. The field study was conducted during two successive seasons 2009 and 2011 in four different locations in Giza and Cairo governorates of Egypt, one was chosen and located inside Rose garden, Fac. of Agri., Cairo Univ., and the three others were clollected from different fields located outside, in A-zohreiya gardens, Cairo governorate, Orman garden, Giza governorate and 6-October city. The infected sample of (rose, jasmine, rue, Egyptian henbane and periwinkle) plants were showing the symptom infection (phyllody, whitches-broom, little leaf and yellowing and revealed that the percentage of natural infection were (22.2, 82.7, 18.9 and 22.2%) while was 25% of Egyptian henbance virescence disease. Ten different identificated species of field-collected insects (Cicadulina bipunctella, Exitiaus taeniaticeps, Neolimus aegyptiacus, toya sp., Empoasca sp; Metatua sp; Sogatella vibix; Chiasmus sp; Balclutha sp; and Chlorotettix sp.) determined whether they can play a role in phytoplasma transmission or not. However, Sogatella vibix; Chiasmus sp; Balclutha sp . and Chlorotettix sp. were not vectors of phytoplasma. With note, the presence of the psyllid (jumping plant-lice); infesting the ornamental/medicinal crops was detected, and that is the first record in Egypt

Environmental Acquisition of Gut Symbiotic Bacteria in the Saw-Toothed Stinkbug, Megymenum gracilicorne (Hemiptera: Pentatomoidea: Dinidoridae).

Many plant-sucking stinkbugs possess a specialized symbiotic organ with numerous crypts in a posterior region of the midgut. In stinkbugs of the superfamily Pentatomoidea, specific γ-proteobacteria are hosted in the crypt cavities, which are vertically transmitted through host generations and essential for normal growth and survival of the host insects. Here we report the discovery of an exceptional gut symbiotic association in the saw-toothed stinkbug, Megymenum gracilicorne (Hemiptera: Pentatomoidea: Dinidoridae), in which specific γ-proteobacterial symbionts are not transmitted vertically but acquired environmentally. Histological inspection identified a very thin and long midgut symbiotic organ with two rows of tiny crypts whose cavities harbor rod-shaped bacterial cells. Molecular phylogenetic analyses of bacterial 16S rRNA gene sequences from the symbiotic organs of field-collected insects revealed that (i) M. gracilicorne is stably associated with Pantoea-allied γ-proteobacteria within the midgut crypts, (ii) the symbiotic bacteria exhibit a considerable level of diversity across host individuals and populations, (iii) the major symbiotic bacteria represent an environmental bacterial lineage that was reported to be capable of symbiosis with the stinkbug Plautia stali, and (iv) the minor symbiotic bacteria also represent several bacterial lineages that were reported as cultivable symbionts of P. stali and other stinkbugs. The symbiotic bacteria were shown to be generally cultivable. Microbial inspection of ovipositing adult females and their eggs and nymphs uncovered the absence of stable vertical transmission of the symbiotic bacteria. Rearing experiments showed that symbiont-supplemented newborn nymphs exhibit improved survival, suggesting the beneficial nature of the symbiotic association.

Bacteria cultured from the gut of Meccus pallidipennis (Hemiptera: Reduviidae), a triatomine species endemic to Mexico.

The study of intestinal microbiota in vector insects like triatomines is paramount in parasitology because many parasitic species inhabit the vector's gut. Although knowledge on the gut microbiota in various vectors of the parasitic flagellate Trypanosoma cruzi has grown, research efforts have focused on South American triatomines. This study reports the isolation of bacterial microbiota in the anterior and posterior gut of Meccus pallidipennis (a triatomine species endemic to Mexico) by culture, as well as its identification by phenotypic and biochemical tests and its quantification by counting colony-forming units. The study was performed on fifth-instar nymph and adult specimens of M. pallidipennis, either laboratory-bred or collected in the field and either infected or not with T. cruzi. Overall, 17 bacterial species were identified, with the genera Bacillus and Staphylococcus being the most prevalent regardless of the origin of the insects. No differences were observed in the number of bacterial species in the gut of laboratory-bred and field-collected insects, neither with respect to life stage or infection status. In general, the Shannon-Weaver diversity index was higher in non-infected insects than in infected ones. Further studies using non-culture methods are required to determine whether bacterial species diversity is modified by laboratory breeding.

Gut microbiota degrades toxic isothiocyanates in a flea beetle pest.

Microbial symbionts of herbivorous insects have been suggested to aid in the detoxification of plant defense compounds; however, quantitative studies on microbial contribution to plant toxin degradation remain scarce. Here, we demonstrate microbiome-mediated degradation of plant-derived toxic isothiocyanates in the cabbage stem flea beetle Psylliodes chrysocephala, a major pest of oilseed rape. Suppression of microbiota in antibiotic-fed beetles resulted in up to 11.3-fold higher levels of unmetabolized isothiocyanates compared to control beetles but did not affect other known detoxification pathways in P.chrysocephala. We characterized the microbiome of laboratory-reared and field-collected insects using 16S rRNA amplicon sequencing and isolated bacteria belonging to the three core genera Pantoea, Acinetobacter and Pseudomonas. Only Pantoea isolates rapidly degraded isothiocyanates in vitro, and restored isothiocyanate degradation in vivo when reintroduced in antibiotic-fed beetles. Pantoea was consistently present across beetle life stages and in field and lab populations. In addition, Pantoea was detected in undamaged tissues of the host plant Brassica rapa, indicating that P.chrysocephala could possibly acquire an isothiocyanate detoxifying bacterium through their diet. Our results demonstrate that both insect endogenous mechanisms and the microbiota can contribute to the detoxification of plant defense compounds and together they can better account for the fate of ingested plant metabolites.

Identification and genome sequencing of RNA viruses in the eucalyptus snout beetle Gonipterus spp. (Coleoptera: Curculionidae).

The genomes of two putative new RNA viruses (macula-like virus and bunya-like virus) were identified in total RNA extracted from dead eucalyptus snout beetles (Gonipterus spp.) from a laboratory colony. However, only bunya-like virus was detected in field-collected insects. The macula-like virus has a monopartite single-stranded RNA genome that contains three open reading frames (ORFs) encoding an RNA-dependent RNA polymerase (RdRp), a capsid protein (CP), protein with unknown function. The bunya-like virus genome was predicted to consist of two RNA segments: a large segment (L) encoding a single protein (RdRp) and a small segment (S) encoding a putative nucleocapsid protein.

Protein-carbohydrate regulation and nutritionally mediated responses to Bt are affected by caterpillar population history.

The widespread adoption of genetically modified crops, including Bacillius thuringensis (Bt) crops that target chewing insects, has transformed agricultural pest management. This increased use of Bt has raised concerns about the onset of resistance amongst target pests. Recent studies have shown that for some caterpillars, nutritional foraging (e.g. the ratio of proteins and carbohydrates consumed) can affect the insect susceptibility to the Bt toxin Cry1Ac. However, studies on both nutritional foraging and Bt susceptibility tend to rely on laboratory colonies without specifically addressing physiological differences that may occur between populations of the same species. Here, we used choice assays, no choice assays and dose response assays to address two overarching questions: Do populations of Spodoptera frugiperda (J.E. Smith) vary in their protein-carbohydrate foraging behavior? and Does protein-carbohydrate intake impact S. frugiperda's susceptibility to the Bt toxin Cry1F? All three of our S. frugiperda populations actively regulated their protein-carbohydrate intake, but we observed significant differences between populations with respect to their self-selected protein-carbohydrate intake. We also found that feeding at the protein-carbohydrate intake target slightly increased Cry1F susceptibility for one S. frugiperda population, but had no effect on the other two populations. Our findings indicate that inherent differences exist in the nutritional physiology of three S. frugiperda populations, possibly related to the time spent in culture. This suggests that population-level differences are an important consideration when drawing parallels between field-collected and laboratory-reared insects.

It’s all about the fluxes: Temperature influences ion transport and toxicity in aquatic insects

Many freshwater ecosystems are becoming saltier and/or warmer, but our understanding of how these factors interact and affect the physiology and life history outcomes of most aquatic species remain unknown. We hypothesize that temperature modulates ion transport rates. Since ion transport is energetically expensive, increases in salinity and/or temperature may influence ion flux rates and ultimately, organismal performance. Radiotracer (22Na+, 35SO4−2, and 45Ca2+) experiments with lab-reared mayflies (N. triangulifer) and other field-collected insects showed that increasing temperature generally increased ion transport rates. For example, increasing temperature from 15 °C to 25 °C, increased 22Na+ uptake rates by two-fold (p < 0.0001) and 35SO4−2 uptake rates by four-fold (p < 0.0001) in the caddisfly, Hydropsyche sparna. Smaller changes in 22Na+ and 35SO4−2 uptake rates were observed in the mayflies, Isonychia sayi and Maccaffertium sp., suggesting species-specific differences in the thermal sensitivity of ion transport. Finally, we demonstrated that the toxicity of SO4 was influenced by temperature profoundly in a 96-h bioassay. Under the saltiest conditions (1500 mg L−1 SO4), mayfly survival was 78 % at 15 °C, but only 44 % at 25 °C (p < 0.0036). Conceivably, the energetic cost of osmoregulation in warmer, saltier environments may cause significant major ion toxicity in certain freshwater insects.

The comet assay in Ceraeochrysa claveri (Neuroptera: Chrysopidae): A suitable approach for detecting somatic and germ cell genotoxicity induced by agrochemicals

Some agrochemicals are genotoxic to several organisms. Nevertheless, few protocols are currently available for measuring the toxicogenetic effects of these compounds in target and non-target field-collected species of insects important to agriculture. Herein, we used the species Ceraeochrysa claveri (Neuroptera: Chrysopidae), a non-target predator insect, to investigate the ability of an azadirachtin-based biopesticide (Azamax™) to induce DNA damage. The alkaline version of the comet assay was standardized to evaluate genetic instability caused by the toxicant in somatic (gut) and germ (nurse cells and oocytes) cells of C. claveri. For this, C. claveri larvae were distributed into three groups (10/each) and treated with Azamax™ at 0, 0.3% or 0.5% throughout the larval stage. DNA damage (tail intensity) was measured in adult insects, four days after emerged. The data showed that both doses of Azamax™ (0.3% and 0.5%) were able to significantly (p < 0.05) increase DNA damage in somatic and germ cells of C. claveri. In conclusion, C. claveri (intestinal and ovarian cells) was a sensitive bioindicator for identifying Azamax™ genotoxic potential, whereas the comet assay was a useful tool for detecting the genotoxic hazard of the pesticide in the field-collected insect species. Given that estimation of adverse effects of pollutants on ecosystems is an essential component of environmental risk assessment, the approach used can be recommended to estimate the ecotoxicity of agricultural chemicals.

Efficacy of larvicides for the control of dengue, Zika, and chikungunya vectors in an urban cemetery in southern Mexico.

Many countries in Latin America have recently experienced outbreaks of Zika and chikungunya fever, in additional to the usual burden imposed by dengue, all of which are transmitted by Aedes aegypti in this region. To identify potential larvicides, we determined the toxicity of eight modern insecticides to A. aegypti larvae from a colony that originated from field-collected insects in southern Mexico. The most toxic compounds were pyriproxyfen (which prevented adult emergence) and λ-cyhalothrin, followed by spinetoram, imidacloprid, thiamethoxam, and acetamiprid, with chlorantraniliprole and spiromesifen the least toxic products. Field trails performed in an urban cemetery during a chikungunya epidemic revealed that insecticide-treated ovitraps were completely protected from the presence of Aedes larvae and pupae for 6 and 7weeks in spinosad (Natular G30) and λ-cyhalothrin-treated traps in both seasons, respectively, compared to 5-6weeks for temephos granule-treated ovitraps, but was variable for pyriproxyfen-treated ovitraps with and 1 and 5weeks of absolute control in the dry and rainy seasons, respectively. Insecticide treatments influenced the mean numbers of Aedes larvae + pupae in each ovitrap, mean numbers of eggs laid, and percentage of egg hatch over time in both trials. The dominant species was A. aegypti in both seasons, although the invasive vector Aedes albopictus was more prevalent in the rainy season (26.7%) compared to the dry season (10.2%). We conclude that the granular formulation of spinosad (Natular G30) and a suspension concentrate formulation of λ-cyhalothrin proved highly effective against Aedes spp. in both the dry and rainy seasons in the cemetery habitat in this region.

Chagas disease vector blood meal sources identified by protein mass spectrometry.

Chagas disease is a complex vector borne parasitic disease involving blood feeding Triatominae (Hemiptera: Reduviidae) insects, also known as kissing bugs, and the vertebrates they feed on. This disease has tremendous impacts on millions of people and is a global health problem. The etiological agent of Chagas disease, Trypanosoma cruzi (Kinetoplastea: Trypanosomatida: Trypanosomatidae), is deposited on the mammalian host in the insect’s feces during a blood meal, and enters the host’s blood stream through mucous membranes or a break in the skin. Identifying the blood meal sources of triatomine vectors is critical in understanding Chagas disease transmission dynamics, can lead to identification of other vertebrates important in the transmission cycle, and aids management decisions. The latter is particularly important as there is little in the way of effective therapeutics for Chagas disease. Several techniques, mostly DNA-based, are available for blood meal identification. However, further methods are needed, particularly when sample conditions lead to low-quality DNA or to assess the risk of human cross-contamination. We demonstrate a proteomics-based approach, using liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify host-specific hemoglobin peptides for blood meal identification in mouse blood control samples and apply LC-MS/MS for the first time to Triatoma dimidiata insect vectors, tracing blood sources to species. In contrast to most proteins, hemoglobin, stabilized by iron, is incredibly stable even being preserved through geologic time. We compared blood stored with and without an anticoagulant and examined field-collected insect specimens stored in suboptimal conditions such as at room temperature for long periods of time. To our knowledge, this is the first study using LC-MS/MS on field-collected arthropod disease vectors to identify blood meal composition, and where blood meal identification was confirmed with more traditional DNA-based methods. We also demonstrate the potential of synthetic peptide standards to estimate relative amounts of hemoglobin acquired when insects feed on multiple blood sources. These LC-MS/MS methods can contribute to developing Ecohealth control strategies for Chagas disease transmission and can be applied to other arthropod disease vectors.

Characterization of Cry toxins from autochthonous Bacillus thuringiensis isolates from Mexico

BackgroundChemical pesticides, widely used in agriculture and vector-borne disease control, have shown toxic effects on the environment and the people in contact with them. Bacillus thuringiensis is a widely used bacterium for alternative and safer control of insect pests. Its toxins are specific for insects but innocuous for mammals and may be used as powerful adjuvants when applied with vaccines. The objective of this work was to characterize some autochthonous B. thuringiensis strains, which could be used for the control of a local pest (Diatraea considerata Heinrich) that affects sugar cane crops in Sinaloa, Mexico. Also, to evaluate these strains as a source of Cry toxins, which may be used in the future as adjuvants for some vaccines. MethodsEight strains from field-collected dead insects were isolated. These were microbiologically identified as B. thuringiensis and confirmed by amplification and sequencing of 16S rDNA. Bioassays were performed to evaluate their pathogenicity against D. considerata, and Cry toxins were identified by proteomic analyses. ResultsAn increased mortality among larvae infected with strain Bt-D was observed, and its toxin was identified as Cry1Ac. ConclusionsThe observed data showed that the selected strain was pathogenic to D. considerata and seemed to produce Cry1Ac protein, which has been reported as an adjuvant in different types of immunization.

Application of an embryonated chicken egg model to assess the vector competence of Australian Culicoides midges for bluetongue viruses.

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of a number of globally important arboviruses that affect livestock, including bluetongue virus (BTV), African horse sickness virus and the recently emerged Schmallenberg virus. In this study, a model using embryonated chicken eggs (ECEs) was utilized to undertake vector competence studies of Australian Culicoides spp. for 13 laboratory-adapted or wild-type virus strains of BTV. A total of 7393 Culicoides brevitarsis were reared from bovine dung, and 3364 Culicoides were induced to feed from ECEs infected with different strains of BTV. Of those, 911 (27%) survived the putative extrinsic incubation period of 9-12 days. In some trials, virus was also transmitted onward to uninfected ECEs, completing the transmission cycle. This model does not rely on the use of colonized midges and has the capacity to assess the vector competence of field-collected insects with strains of virus that have not previously been passaged in laboratory culture systems. There is also potential for this model to be used in investigations of the competence of Culicoides spp. for other arboviruses.

Sequence polymorphism in an insect RNA virus field population: A snapshot from a single point in space and time reveals stochastic differences among and within individual hosts

Population structure of Homalodisca coagulata Virus-1 (HoCV-1) among and within field-collected insects sampled from a single point in space and time was examined. Polymorphism in complete consensus sequences among single-insect isolates was dominated by synonymous substitutions. The mutant spectrum of the C2 helicase region within each single-insect isolate was unique and dominated by nonsynonymous singletons. Bootstrapping was used to correct the within-isolate nonsynonymous:synonymous arithmetic ratio (N:S) for RT-PCR error, yielding an N:S value ~one log-unit greater than that of consensus sequences. Probability of all possible single-base substitutions for the C2 region predicted N:S values within 95% confidence limits of the corrected within-isolate N:S when the only constraint imposed was viral polymerase error bias for transitions over transversions. These results indicate that bottlenecks coupled with strong negative/purifying selection drive consensus sequences toward neutral sequence space, and that most polymorphism within single-insect isolates is composed of newly-minted mutations sampled prior to selection.