Biocontrol Potential of Chitin and Chitosan Extracted from Black Soldier Fly Pupal Exuviae against Bacterial Wilt of Tomato

Globally, Ralstonia solanacearum (Smith) is ranked one of the most destructive bacterial pathogens inducing rapid and fatal wilting symptoms on tomatoes. Yield losses on tomatoes vary from 0 to 91% and most control measures are unaffordable to resource-poor farmers. This study investigated the antimicrobial activities of chitin and chitosan extracted from black soldier fly (BSF) pupal exuviae against R. solanacearum. Morphological, biochemical, and molecular techniques were used to isolate and characterize R. solanacearum for in vitro pathogenicity test using disc diffusion technique. Our results revealed that BSF chitosan significantly inhibited the growth of R. solanacearum when compared to treatments without chitosan. However, there was no significant difference in the antibacterial activities between BSF and commercial chitosan against R. solanacearum. Soil amended with BSF-chitin and chitosan demonstrated a reduction in bacterial wilt disease incidence by 30.31% and 34.95%, respectively. Whereas, disease severity was reduced by 22.57% and 23.66%, when inoculated tomato plants were subjected to soil amended with BSF chitin and chitosan, respectively. These findings have demonstrated that BSF pupal shells are an attractive renewable raw material for the recovery of valuable products (chitin and chitosan) with promising ability as a new type of eco-friendly control measure against bacterial wilt caused by R. solanacearum. Further studies should explore integrated pest management options that integrate multiple components including insect-based chitin and chitosan to manage bacterial wilt diseases, contributing significantly to increased tomato production worldwide.

Corrections and Additions to Descriptions of Some Species of the Subgenus Orthocladius s. str. (Diptera, Chironomidae, Orthocladiinae)

The larvae of some species of the subgenus Orthocladius s. str. (Diptera, Chironomidae) are here described for the first time with corrections and additions to the descriptions of adult males and pupal exuviae. The identification of larvae is generally not possible without association with pupal exuviae and/or adult males, so the descriptions here are based only on reared material or on pupae with the associated larval exuviae. Usually, Chironomidae larvae can be separated on the basis of morphometric characters, the most discriminant ones are: (1) the ratio between the width of median tooth of mentum (Dm) and the width of the first lateral tooth (Dl) = mental ratio (DmDl), (2) the ratio between the length of the first antennal segment (A1) and the combined length of segments 2–5 (A2–5) = antennal ratio (AR). The shape of mandible, maxilla, and other body parts are almost identical in all the species considered in this study. The larva of Orthocladius (Symposiocladius) lignicola is very characteristic and can be separated by the shape of mentum and the larvae of all the known species of Symposiocladius are characterized by the presence of large Lauterborn organs on antennae and of tufts of setae on abdominal segments. The larvae of Orthocladius (Orthocladius) oblidens and Orthocladius (Orthocladius) rhyacobius can be distinguished from other species basing on their large Dm and to each other by AR. A principal component analysis was carried out using 5 characters: (1) Dm, (2) Dl, (3) length of A1, (4) width of A1 (A1W), (5) combined length of segments 2–5 (A2–5). The most discriminant characters were Dm and A1, confirming that DmDl and AR can be used to separate species at larval stage, but the large superposition of morphometric characters in different species confirms that association with pupal exuviae is in any case needed to identify larvae. In future perspective, the development of reference DNA barcodes from specimens identified by specialists is recommended since possibly the best tool for larvae identification, but association of barcodes with morphotypes is in any case fundamental.

Purification of Chitin from Pupal Exuviae of the Black Soldier Fly

Purpose Chitin purification from remains (pupal exuviae after metamorphosis to adult flies) of Hermetia illucens farming was optimized performing demineralization, deproteinization and bleaching under different conditions. The optimal parameters to obtain high-purity chitin were determined. Methods Dried and ground pupal exuviae, whose composition was initially determined, were demineralized using six different acids. Proteins were removed with a NaOH treatment in which temperature, molarity and duration were varied in a randomized experiment. Bleaching was carried out testing ten different chemicals, including NaOCl, H2O2, solvent mixtures and enzymes. The efficiency of each step was determined to assess the optimal conditions for each of them. The resulting chitin was subjected to spectroscopic characterization. Results The highest demineralization efficiency (90%) was achieved using 0.5 M formic acid for 2 h at 40 °C, confirming the validity of organic acids as a more sustainable alternative to inorganic acids. The treatment with 1.25 M NaOH at 90 °C for 4 h showed the highest deproteinization efficiency, removing 96% of the proteins. Temperature and NaOH concentration were the significant parameters for deproteinization efficiency. The most efficient bleaching treatment was with 6% NaOCl at 60 °C for 1 h (67% efficiency). H2O2 could also be a valid alternative to avoid environmental risk related to chlorine-containing compounds. At the end of the purification process 17% of the original biomass was retained with a chitin content of 85%, corresponding to a chitin yield of 14% related to the initial biomass. Solid-state nuclear magnetic resonance showed that the purified chitin had a degree of acetylation of 96% and X-ray powder diffraction gave a crystallinity index of 74%. Conclusion This investigation shows an optimized method for extraction of high-purity chitin from H. illucens pupal exuviae, supporting the validity of insect-farming remains as source of this versatile biopolymer. Graphical Abstract

Corrections and Additions to Descriptions of Some Species of the Subgenus Orthocladius s. str. (Diptera, Chironomidae, Orthocladiinae)

The larvae of some species of the subgenus Orthocladius s. str. (Diptera, Chironomidae) are here described for the first time with corrections and additions to the descriptions of adult males and pupal exuviae. The identification of larvae is generally not possible without association with their pupal exuviae and/or adult males, so the descriptions here are based only on reared material or on pupae with the associated larval exuviae. Usually, Chironomidae larvae can be separated on the basis of morphometric characters, and the most discriminant characters ones are: 1- the ratio between the width of median tooth of mentum (Dm) and the width of the first lateral tooth (Dl) = mental ratio (DmDl), 2- the ratio between the length of the first antennal segment (A1) and the combined length of segments 2-5 (A(2-5)2-5) = antennal ratio (AR). The shape of mandible, maxilla, and other body parts are almost identical in all the species considered in this study. The larva of Orthocladius (Symposiocladius) lignicola is very characteristic and can be separated by the shape of mentum and the larvae of all the known species of Symposiocladius are characterized by the presence of large Lauterborn organs on antennae and of tufts of setae on abdominal segments. The larvae of Orthocladius (Orthocladius) oblidens and Orthocladius (Orthocladius) rhyacobius can be distinguished from other species basing on their large Dm and to each other by AR. A principal component analysis was carried out using 5 characters: 1- Dm, 2- Dl, 3- length of A1, 4- width of A1 (A1W), 5- combined length of segments 2-5 (A2-5). The most discriminant characters were Dm and A1, confirming that DmDl and AR can be used to separate species at larval stage, but the large superposition of morphometric characters in different species confirms that association with pupal exuviae is in any case needed to identify larvae. In future perspective, the development of reference DNA barcodes from specimens identified by specialists is recommended since possibly the best tool for larvae identification, but association of barcodes with morphotypes is in any case fundamental.

Antimicrobial Activity of Chemically and Biologically Treated Chitosan Prepared from Black Soldier Fly (Hermetia illucens) Pupal Shell Waste

Globally, the broad-spectrum antimicrobial activity of chitin and chitosan has been widely documented. However, very little research attention has focused on chitin and chitosan extracted from black soldier fly pupal exuviae, which are abundantly present as byproducts from insect-farming enterprises. This study presents the first comparative analysis of chemical and biological extraction of chitin and chitosan from BSF pupal exuviae. The antibacterial activity of chitosan was also evaluated. For chemical extraction, demineralization and deproteinization were carried out using 1 M hydrochloric acid at 100 °C for 2 h and 1 M NaOH for 4 h at 100 °C, respectively. Biological chitin extraction was carried out by protease-producing bacteria and lactic-acid-producing bacteria for protein and mineral removal, respectively. The extracted chitin was converted to chitosan via deacetylation using 40% NaOH for 8 h at 100 °C. Chitin characterization was done using FTIR spectroscopy, while the antimicrobial properties were determined using the disc diffusion method. Chemical and biological extraction gave a chitin yield of 10.18% and 11.85%, respectively. A maximum chitosan yield of 6.58% was achieved via chemical treatment. From the FTIR results, biological and chemical chitin showed characteristic chitin peaks at 1650 and 1550 cm−1—wavenumbers corresponding to amide I stretching and amide II bending, respectively. There was significant growth inhibition for Escherichia coli, Bacillus subtilis,Pseudomonas aeruginosa,Staphylococcus aureus, and Candida albicans when subjected to 2.5 and 5% concentrations of chitosan. Our findings demonstrate that chitosan from BSF pupal exuviae could be a promising and novel therapeutic agent for drug development against resistant strains of bacteria.

New genus and species of Schizomyiina (Diptera, Cecidomyiidae) from Brazil

A new gall midge genus, Distinctamyia gen. nov., and a new species Distinctamyia matogrossensis sp. nov. (Diptera, Cecidomyiidae) are herein described and illustrated (larvae, pupal exuviae, male and female). The new species induces conical, green, hairy and one chambered galls on leaf and bud of Simarouba amara Aubl. (Simaroubaceae). Gall-inducer specimens, samples of gall and host plant were collected at Parque Nacional da Chapada dos Guimarães, in the state of Mato Grosso (Midwestern Brazil).

Evidence of parthenogenetic populations from the Paratanytarsus laccophilus species group (Diptera: Chironomidae) in the Alaskan Arctic

Parthenogenesis, reproduction without fertilization, is not common in the Chironomidae (Diptera), a family of insects with more than 6,000 described species. Nonetheless, parthenogenetic species and strains have been documented in at least three subfamilies (the Chironominae, Orthocladiinae, and Telmatogoninae), spanning 17 genera and ~30 species. One such species, Paratanytarsus laccophilus Edwards 1929, is known to be parthenogenetic in a small portion of its range in Finland, with most other European populations of this species showing evidence of sexual reproduction. We present evidence of parthenogenetic populations from the Paratanytarsus laccophilus species group in the Nearctic, specifically a High Arctic site near Utqiaġvik (formerly Barrow), Alaska. During May-July of 2015 and 2016, we sampled emerging adult chironomids and pupal exuviae daily to document insect emergence phenologies. Across 15 local populations, all 623 pupal exuviae collected from the P. laccophilus species group were female. Larvae reared from two populations under controlled temperature treatments emerged as female adults (N=37). When isolated, these reared female adults oviposited, and eggs hatched successfully. These progeny were reared for another 12-13 days, reaching second instar larvae when they were preserved at the end of our field season. Taken together, this evidence strongly indicates parthenogenesis from the P. laccophilus species group at this location. This species was not previously documented at Utqiaġvik. Although parthenogenetic, their emergence at this location was highly synchronized. In the harsh environment of arctic Alaska, the fitness rewards of parthenogenesis are likely great. Indeed, chironomid parthenogenesis in the northern hemisphere is most commonly documented from far-northern extremes and in extreme habitats.

Twenty-one new species of the Simulium (Gomphostilbia) asakoae species group (Diptera, Simuliidae) in Thailand, with their genetic relationships

Females and males reared from pupae, their pupal exuviae and cocoons, and mature larvae of the Simulium (Gomphostilbia) asakoae species group from various localities in Thailand were morphologically examined. A total of 25 species was identified, including two of four known species (Simulium asakoae Takaoka & Davies and S. chiangdaoense Takaoka & Srisuka), one newly confirmed species (S. myanmarense Takaoka, Srisuka & Saeung, originally described from Myanmar), one newly transferred species (S. inthanonense Takaoka & Suzuki formerly of the S. ceylonicum species group), and 21 new species. Descriptions of all 21 new species are given, and the first full description of the male of S. inthanonense, together with the revised descriptions of its female, pupa, and larva, is also provided. Keys to identify all 27 members of this species group from Thailand are given for females, males, pupae, and larvae. The genetic relationships of all but one species were resolved using COI gene sequence-based analysis. All 26 species were divided into nine subgroups, I–IX, each consisting of two, one, four, nine, one, three, two, one and three species, respectively.