Probing behavior of the leafhopper analyzed through DC electropenetrography and microscopy.

Yamatotettix flavovittatus Matsumara is a new leafhopper species vector of sugarcane white leaf (SCWL) phytoplasma that causes sugarcane chlorosis symptoms. The effects of probing behavior of Y. flavovittatus on sugarcane and its implication for SCWL phytoplasma transmission are yet to be studied. In this research, we used DC electropenetrography (EPG) to define waveforms produced by adult and fifth-instar nymphal Y. flavovittatus on sugarcane and correlated them with salivary sheath termini (likely stylet tip locations) via light and scanning electron microscopy. The following six waveforms and associated activities are described: (NP) non-probing, (Yf1) stylet probing into epidermal cells, (Yf2) stylet probing through mesophyll/parenchyma, (Yf3) stylet contact with phloem and likely watery salivation, (Yf4) active ingestion of sap from phloem, probably sieve elements, and (Yf5) unknown stylet activity in multiple cell types. Study findings reveal that the Y. flavovittatus vector ingests sieve tube element more frequently and for longer durations than any other cell type, supporting that Y. flavovittatus is primarily a phloem feeder. Adult Y. flavovittatus show a longer total probing duration and produces a high density of puncture holes on sugarcane leaves. Moreover, probing behaviors revealed that adults typically ingest phloem sap more frequently and for longer durations than fifth-instar nymphs, enhancing sap ingestion. Furthermore, we propose that adults are more likely to acquire (during Yf4) and inoculate (during Yf3) higher amounts of phytoplasma than fifth-instar nymphs. This information on the penetration behavior of leafhopper Y. flavovittatus serves as a basis for advanced studies on the transmission mechanism of SCWL phytoplasma.

Evaluation of the Physical Characteristics and Chemical Properties of Black Soldier Fly (Hermetia illucens) Larvae as a Potential Protein Source for Poultry Feed.

The aim of this study was to investigate and compare the effects of different drying methods on the physical and chemical properties of black soldier fly larvae (BSFL) to determine their potential as an alternative protein source in animal feed. The experimental design was a 2 × 3 factorial arrangement in a completely randomized design (BSFL type × drying method), with five replications. The influence of post-harvest procedures was studied, including the different BSFL types (non-defatted and defatted) and drying methods (parabola dome, hot air oven, and microwave). The results showed that the types of BSFL, drying methods, and their interaction significantly (p < 0.001) influenced the feed's physical properties; these included the brightness of color (L* 29.74-54.07; a* 0.40-5.95; b* 9.04-25.57), medium bulk density (381.54-494.58 g/L), free flow with an angle of repose (41.30-45.40°), and small particle size. They significantly (p < 0.001) influenced the nutritive value of BSFL, which contained 42-59% crude protein, 7-14% crude fiber, 9-30% ether extract, and 5035-5861 kcal/kg of energy. Overall, both BSFL types and all the drying methods resulted in a slight variation in the proximate composition. However, a microwave and a hot-air oven were considered the most suitable methods for producing BSFL powder because of the high levels of nutrients retained and the improved physical parameters when compared to a parabola dome. This characterization of the physical and chemical composition of BSFL represents a preliminary methodology that could be used to initially preprocess larvae for use as an alternative protein source in animal feed and for other applications.

Indoor resting behavior of Aedes aegypti (Diptera: Culicidae) in northeastern Thailand.

BACKGROUND: Aedes aegypti is a vector of several arboviruses, notably dengue virus (DENV), which causes dengue fever and is often found resting indoors. Culex spp. are largely nuisance mosquitoes but can include species that are vectors of zoonotic pathogens. Vector control is currently the main method to control dengue outbreaks. Indoor residual spraying can be part of an effective vector control strategy but requires an understanding of the resting behavior. Here we focus on the indoor-resting behavior of Ae. aegypti and Culex spp. in northeastern Thailand. METHODS: Mosquitoes were collected in 240 houses in rural and urban settings from May to August 2019 at two collection times (morning/afternoon), in four room types (bedroom, bathroom, living room and kitchen) in each house and at three wall heights (< 0.75 m, 0.75-1.5 m, > 1.5 m) using a battery-driven aspirator and sticky traps. Household characteristics were ascertained. Mosquitoes were identified as Ae. aegypti, Aedes albopictus and Culex spp. Dengue virus was detected in Ae. aegypti. Association analyses between urban/rural and within-house location (wall height, room), household variables, geckos and mosquito abundance were performed. RESULTS: A total of 2874 mosquitoes were collected using aspirators and 1830 using sticky traps. Aedes aegypti and Culex spp. accounted for 44.78% and 53.17% of the specimens, respectively. Only 2.05% were Ae. albopictus. Aedes aegypti and Culex spp. rested most abundantly at intermediate and low heights in bedrooms or bathrooms (96.6% and 85.2% for each taxon of the total, respectively). Clothes hanging at intermediate heights were associated with higher mean numbers of Ae. aegypti in rural settings (0.81 [SEM: 0.08] vs. low: 0.61 [0.08] and high: 0.32 [0.09]). Use of larval control was associated with lower numbers of Ae. aegypti (yes: 0.61 [0.08]; no: 0.70 [0.07]). All DENV-positive Ae. aegypti (1.7%, 5 of 422) were collected in the rural areas and included specimens with single, double and even triple serotype infections. CONCLUSIONS: Knowledge of the indoor resting behavior of adult mosquitoes and associated environmental factors can guide the choice of the most appropriate and effective vector control method. Our work suggests that vector control using targeted indoor residual spraying and/or potentially spatial repellents focusing on walls at heights lower than 1.5 m in bedrooms and bathrooms could be part of an integrated effective strategy for dengue vector control.

Xylella fastidiosa inoculation behaviors (EPG X wave) are performed differently by blue-green sharpshooters based on infection status of prior probing host.

Does Xylella fastidiosa, a bacterial plant pathogen with noncirculative foregut-borne transmission, manipulate behavior of its sharpshooter vector to facilitate its own inoculation? To answer this question, blue-green sharpshooters, Graphocephala atropunctata (Signoret), were reared on basil to clean their foreguts, then removed from the colony and given one of four pre-electropenetrography (EPG) treatments: i) old colony adults on basil, ii) young colony adults on basil, iii) young colony adults held on healthy grapevine for 4 days, and iv) young colony adults held on Xf-infected (symptomatic) grapevine for 4 days. After treatments, stylet probing behaviors were recorded on healthy grapevine via AC-DC electropenetrography. Waveforms representing putative Xf inoculation (XB1 [salivation and rinsing egestion] and XC1 [discharging egestion]) and other behaviors were statistically compared among treatments. Mean number of events per insect and 'total' duration per insect of XB1 and XC1 were highest for insects from healthy grape, lowest for basil (regardless of insect age), and intermediate for Xf-infected grape. The surprising results showed that prior exposure to healthy grapevines had a stronger effect on subsequent performance of inoculation behaviors on healthy grapevine than did prior exposure to Xf-infected grapevine. It is hypothesized that non-Xf microbes were acquired from healthy grapevine, causing greater clogging of the precibarium, leading to more performance of inoculation behaviors. This study shows for the first time that presence of noncirculative, foregut-borne microbes can directly manipulate a vector's behavior to increase inoculation. Also, EPG can uniquely visualize the dynamic interactions between vectors and the microbes they carry.

Selective Elimination of Wolbachia from the Leafhopper Yamatotettix flavovittatus Matsumura.

Wolbachia infections affect the reproductive system and various biological traits of the host insect. There is a high frequency of Wolbachia infection in the leafhopper Yamatotettix flavovittatus Matsumura. To investigate the potential roles of Wolbachia in the host, it is important to generate a non-Wolbachia-infected line. The efficacy of antibiotics in eliminating Wolbachia from Y. flavovittatus remains unknown. This leafhopper harbors the mutualistic bacterium Candidatus Sulcia muelleri, which has an important function in the biological traits. The presence of Ca. S. muelleri raises a major concern regarding the use of antibiotics. We selectively eliminated Wolbachia, considering the influence of antibiotics on leafhopper survival and Ca. S. muelleri prevalence. The effect of artificial diets containing different doses of tetracycline and rifampicin on survival was optimized; high dose (0.5 mg/ml) of antibiotics induces a high mortality. A concentration of 0.2 mg/ml was chosen for the subsequent experiments. Antibiotic treatments significantly reduced the Wolbachia infection, and the Wolbachia density in the treated leafhoppers sharply declined. Wolbachia recurred in tetracycline-treated offspring, regardless of antibiotic exposure. However, Wolbachia is unable to be transmitted and restored in rifampicin-treated offspring. The dose and treatment duration had no significant effect on the infection and density of Ca. S. muelleri in the antibiotic-treated offspring. In conclusion, Wolbachia in Y. flavovittatus was stably eliminated using rifampicin, and the Wolbachia-free line was generated at least two generations after treatment. This report provides additional experimental procedures for removing Wolbachia from insects, particularly in host species with the coexistence of Ca. S. muelleri.

Nutritional composition of various insects and potential uses as alternative protein sources in animal diets.

The aim of the present investigation is to determine the nutritional composition of various insects and their potential uses as alternative protein sources in animal diets. The feeding industry requires production systems that use accessible resources, such as feed resources, and concentrates on the potential impacts on production yield and nutritional quality. Invertebrate insects, such as black soldier flies, grasshoppers, mealworms, housefly larvae, and crickets, have been used as human food and as feed for nonruminants and aqua culture while for ruminants their use has been limited. Insects can be mass-produced, participating in a circular economy that minimizes or eliminates food- and feed-waste through bioconversion. Although the model for formula-scale production of insects as feed for domestic animals has been explored for a number of years, significant production and transformation to being a conventional protein resource remains to be deeply investigated. This review will focus on the nutritional composition of various insects and their potential use as alternative protein sources, as well as their potential use to promote and support sustainable animal production. Furthermore, nutritional compositions, such as high protein, lauric acid omega 6, and omega 3, and bioactive compounds, such as chitin, are of great potential use for animal feeding.

Population Dynamics of Wolbachia in the Leafhopper Vector Yamatotettix flavovittatus (Hemiptera: Cicadellidae).

Wolbachia (Rickettsiales: Alphaproteobacteria) infections induce abnormalities in the reproductive system and affect various biological traits of the host insects. The density of Wolbachia is one of the major parameters that influence induced phenotypes and interactions with the hosts. Wolbachia occurs naturally in populations of the leafhopper Yamatotettix flavovittatus Matsumura (Hemiptera: Cicadellidae), which transmits phytoplasma that cause white leaf disease in sugarcane. However, the quantity and dynamics of Wolbachia in this leafhopper are not well understood. In the current study, we estimated the number of Wolbachia by absolute quantification of the copy number of wsp, which encodes the outer surface protein, using real-time quantitative polymerase chain reaction (PCR). This investigation was performed using natural populations and laboratory colonies from three lineages of leafhoppers (designated as UD, KK, and SK). There was no significant difference in the number of wsp copies in most of field-collected adults. During the immature developmental stages, there were differences in the dynamics of Wolbachia infection between the UD lineage and the other two lineages. However, the number of wsp copies increased in the early instar and plateaued in the later nymphal instars. Sex had no influence on the number of Wolbachia within the same lineages. The number of Wolbachia was relatively constant during the adult stage in the UD lineage but fluctuated in the other two lineages. In conclusion, the present data provide a framework for exploring the relationship between Wolbachia and the leafhopper and could facilitate future research into management strategies using Wolbachia.

Bacterial challenge-associated metabolic phenotypes in Hermetia illucens defining nutritional and functional benefits.

Black soldier fly (BSF, Hermetia illucens) is popular for its applications in animal feed, waste management and antimicrobial peptide source. The major advantages of BSF larva include their robust immune system and high nutritional content that can be further developed into more potential agricultural and medical applications. Several strategies are now being developed to exploit their fullest capabilities and one of these is the immunity modulation using bacterial challenges. The mechanism underlying metabolic responses of BSF to different bacteria has, however, remained unclear. In the current study, entometabolomics was employed to investigate the metabolic phenoconversion in response to either Escherichia coli, Staphylococcus aureus, or combined challenges in BSF larva. We have, thus far, characterised 37 metabolites in BSF larva challenged with different bacteria with the major biochemical groups consisting of amino acids, organic acids, and sugars. The distinct defense mechanism-specific metabolic phenotypes were clearly observed. The combined challenge contributed to the most significant metabolic phenoconversion in BSF larva with the dominant metabolic phenotypes induced by S. aureus. Our study suggested that the accumulation of energy-related metabolites provided by amino acid catabolism is the principal metabolic pathway regulating the defense mechanism. Therefore, combined challenge is strongly recommended for raising BSF immunity as it remarkably triggered amino acid metabolisms including arginine and proline metabolism and alanine, aspartate and glutamate metabolism along with purine metabolism and pyruvate metabolism that potentially result in the production of various nutritional and functional metabolites.

Global population genetic structure and demographic trajectories of the black soldier fly, Hermetia illucens.

BACKGROUND: The black soldier fly (Hermetia illucens) is the most promising insect candidate for nutrient-recycling through bioconversion of organic waste into biomass, thereby improving sustainability of protein supplies for animal feed and facilitating transition to a circular economy. Contrary to conventional livestock, genetic resources of farmed insects remain poorly characterised. We present the first comprehensive population genetic characterisation of H. illucens. Based on 15 novel microsatellite markers, we genotyped and analysed 2862 individuals from 150 wild and captive populations originating from 57 countries on seven subcontinents. RESULTS: We identified 16 well-distinguished genetic clusters indicating substantial global population structure. The data revealed genetic hotspots in central South America and successive northwards range expansions within the indigenous ranges of the Americas. Colonisations and naturalisations of largely unique genetic profiles occurred on all non-native continents, either preceded by demographically independent founder events from various single sources or involving admixture scenarios. A decisive primarily admixed Polynesian bridgehead population serially colonised the entire Australasian region and its secondarily admixed descendants successively mediated invasions into Africa and Europe. Conversely, captive populations from several continents traced back to a single North American origin and exhibit considerably reduced genetic diversity, although some farmed strains carry distinct genetic signatures. We highlight genetic footprints characteristic of progressing domestication due to increasing socio-economic importance of H. illucens, and ongoing introgression between domesticated strains globally traded for large-scale farming and wild populations in some regions. CONCLUSIONS: We document the dynamic population genetic history of a cosmopolitan dipteran of South American origin shaped by striking geographic patterns. These reflect both ancient dispersal routes, and stochastic and heterogeneous anthropogenic introductions during the last century leading to pronounced diversification of worldwide structure of H. illucens. Upon the recent advent of its agronomic commercialisation, however, current human-mediated translocations of the black soldier fly largely involve genetically highly uniform domesticated strains, which meanwhile threaten the genetic integrity of differentiated unique local resources through introgression. Our in-depth reconstruction of the contemporary and historical demographic trajectories of H. illucens emphasises benchmarking potential for applied future research on this emerging model of the prospering insect-livestock sector.

Alteration in the Stylet Probing Behavior and Host Preference of the Vector Matsumuratettix hiroglyphicus (Hemiptera: Cicadellidae) After Infection with Sugarcane White Leaf Phytoplasma.

The leafhopper (Matsumuratettix hiroglyphicus (Matsumura) (Hemiptera: Cicadellidae)) is a crucial insect vector of the phytoplasma associated with sugarcane white leaf (SCWL) disease. The aim of this study was to compare the stylet probing behaviors of M. hiroglyphicus on healthy sugarcane plants, asymptomatic, and symptomatic SCWL-infected sugarcane plants, using DC electropenetrography. We also used host-selection preference (free-choice) assays to identify the preferred types of host plants, and scanning electron microscopy to observe stylet puncture holes and salivary flanges after leafhopper probing. According to a quantitative analysis of M. hiroglyphicus stylet probing, mean durations per insect of both phloem ingestion (waveform D; the phytoplasma-acquisition behavior) and phloem salivation (waveform C; the phytoplasma-inoculation behavior) were significantly longer on both types of infected sugarcane than on healthy plants. These longer overall durations were mainly because the same number of significantly longer-duration C and D events was performed on infected sugarcane compared with healthy plants. On free-choice tested plants, M. hiroglyphicus displayed a significantly greater preference to settle on the infected plants (both types) than the healthy sugarcane. These results provide the first empirical evidence that acquiring the SCWL phytoplasma alters the host selection and stylet probing behaviors of its main vector (M. hiroglyphicus). Our study thus contributes to a better understanding of the interactions between the insect vector and SCWL phytoplasma-infected plants, and will aid in developing novel disease management tactics for sugarcane.

Phylogeny and Strain Typing of Wolbachia from Yamatotettix flavovittatus Matsumura Leafhoppers.

Wolbachia is a maternally inherited bacterium of insects that can alter the reproduction, biology, and fitness of the hosts. It was detected in natural populations of the Yamatotettix flavovittatus Matsumura leafhoppers, the vector of phytoplasma, which is responsible for sugarcane white leaf disease. Wolbachia infection prolongs the longevity of female leafhoppers and promotes a strong reproductive incompatibility; importantly, highly maternal transmission rate was observed. However, limited data on the diversity or strain typing of Wolbachia in Y. flavovittatus are available. We aimed here to detect the presence of Wolbachia in different populations by amplification of the wsp gene, which was then sequenced. Multilocus sequence typing (MLST) was also performed to explore the diversity of the Wolbachia strains. Based on the wsp sequences, Wolbachia in the Y. flavovittatus leafhoppers belonged to supergroup B, and formed a distinct evolutionary lineage; therefore, we designated this new specific strain as wYfla. The MLST profiles revealed ten potential new sequence types (STs) in different leafhopper populations. Multiple STs were detected in individual leafhoppers, among which the ST-wYfla1 strain was predominant. Furthermore, we obtained congruent results for the phylogenetic analyses using the wsp gene and MLST loci. To the best of our knowledge, this is the first study characterizing Wolbachia strains in Y. flavovittatus. Our results reveal a novel strain and multiple STs of Wolbachia, and these data may prove useful in the exploitation of Wolbachia as a biological Y. flavovittatus control agent.

Effect of Wolbachia infection states on the life history and reproductive traits of the leafhopper Yamatotettix flavovittatus Matsumura.

Wolbachia is a maternally inherited bacterium of insects that can affect host reproduction and fitness. We examined the effect of Wolbachia infection on the life history and reproductive traits of the leafhopper Yamatotettix flavovittatus, which is a vector of the phytoplasma that causes white leaf disease in sugarcane. This investigation was performed using Wolbachia-infected and uninfected leafhopper lineages. Results revealed that Wolbachia infection did not significantly affect the survival of nymphal stages, male longevity, and sex ratio. However, Wolbachia-infected lineages had prolonged immature development periods and female longevity. In intrapopulation crosses, Wolbachia infection had no significant effects on occupation success, number of eggs laid, and female offspring, but the effect on egg-hatching varied. In interpopulation crosses, Wolbachia infection had no significant effect on occupation success and female offspring, but it did affect the number of eggs laid and egg-hatching rates. Assortative pairings regarding infection status resulted in normal egg deposition and hatching, whereas disassortative pairings resulted in lower egg deposition and no hatching. Wolbachia was thus shown to be highly vertically transmitted (>98% of the tested individuals). Our findings provide additional data on the interactions between Wolbachia in insect hosts. This evidence of perfect maternal transmission and strong reproductive incompatibility highlights the importance of further studies on the use of Wolbachia as a biological control agent for the leafhopper vector.

Wolbachia (Rickettsiales: Alphaproteobacteria) Infection in the Leafhopper Vector of Sugarcane White Leaf Disease.

Wolbachia is a maternally inherited bacterium ubiquitous in insects that has attracted interest as a prospective insect pest-control agent. Here, we detected and characterized Wolbachia in the leafhoppers Matsumuratettix hiroglyphicus (Matsumura) (Cicadellidae: Hemiptera) and Yamatotettix flavovittatus Matsumura (Cicadellidae: Hemiptera), insect vectors of the phytoplasma that cause white leaf disease in sugarcane. The 16S rRNA and wsp gene markers revealed that Wolbachia was not present in the M. hiroglyphicus but naturally occurs in Y. flavovittatus. Additionally, the infection rates in adult leafhoppers ranged from 0 to 100% depending on geographic location. Moreover, Wolbachia was detected in the eggs and first- to fifth-instar nymphs of Y. flavovittatus. A phylogenic tree of Wolbachia indicated that it resided in the monophyletic supergroup B clade and clustered in the Ori subgroup. Furthermore, fluorescence in situ hybridization revealed that Wolbachia localized to the egg apices, randomly distributed in the egg cytoplasm, and was concentrated in the nymph and adult bacteriomes, as well as occasional detection in the thorax and abdomen. To the best of our knowledge, the present study is the first to demonstrate the prevalence of Wolbachia in the leafhopper Y. flavovittatus. The obtained results would provide useful information for the future development of Wolbachia as a biological control agent for the leafhopper vectors.

Sugarcane White Leaf Disease Incidences and Population Dynamic of Leafhopper Insect Vectors in Sugarcane Plantations in Northeast Thailand.

The work consisted of two experiments, i.e. Experiment 1 was conducted under controlled environments where sugarcane plants were used as hosts. This investigation aimed to monitor the occurrence of the Sugarcane White Leaf disease and the abundance of Leafhopper insect vectors and also the work aimed to provide useful information in understanding some aspects on epidemiology of the Sugarcane White Leaf disease. A Completely Randomized Design with three replications was used to justify growth and development of Leafhopper insects as affected by different temperatures: 20 (T1), 25 (T2), 30 (T3) and 35 degrees C (T4). Experiment 2 was carried out to determine the numbers of Leafhopper insects with the use of light traps in the sugarcane Field 1 (ratoon plants), Field 2 (newly planted), Field 3 (newly planted) and Field 4 (ratoon plants). The results of Experiment 1 showed that growth and development of Leafhopper insects were highly affected by temperatures i.e. the higher the environmental temperature the faster the growth and development of the insects to reach its full adulthood. At 20 degrees C, Leafhopper insects took 12 days to lay eggs whereas at 25 degrees C the insects took only 6 days. Male reached its adulthood approximately 9 days earlier than female when cultured at 25 degrees C and became approximately one week at 30 degrees C or higher. The results of Experiment 2 showed that the majority of Leafhopper insects were found within the months of June and July for both newly planted and ratoon crops. A small amount was found in May and August with an exceptional case of Field 4 where the highest number of Leafhopper insects was found in April followed by June and July. For Sugarcane White Leaf disease, the disease was found in all months of the year except February for Fields 2 and 3. Newly planted sugarcane plants attained much smaller percentages of disease than those of the ratoon plants.

Candidates for symbiotic control of sugarcane white leaf disease.

The leafhopper Matsumuratettix hiroglyphicus (Matsumura) is the most important vector of a phytoplasma pathogen causing sugarcane white leaf (SCWL) disease. The purpose of this study was to evaluate candidate bacterial symbionts for possible use as vehicles in the control of the disease. 16S rRNA bacterial genes were amplified from whole bodies of M. hiroglyphicus leafhoppers and analyzed by cloning and sequencing. Two dominant groups were found: one belonged to the Betaproteobacteria that did not closely match any sequences in the database and was named bacterium associated with M. hiroglyphicus (BAMH). Another one found to be abundant in this leafhopper is "Candidatus Sulcia muelleri" in the order Bacteroidetes, which was previously reported in the insect members of the Auchenorrhyncha. Most M. hiroglyphicus leafhoppers carry both BAMH and "Ca. Sulcia muelleri." Fluorescent in situ hybridization showed that BAMH and "Ca. Sulcia muelleri" colocalized in the same bacteriomes. BAMH was present in the midgut and ovaries of the leafhopper and was found in all developmental stages, including eggs, nymphs, and adults. Because BAMH appears to be specific for the SCWL vector, we evaluated it as a candidate for symbiotic control of sugarcane white leaf disease.