Coconut rhinoceros beetle digestive symbiosis with potential plant cell wall degrading microbes.

Coconut rhinoceros beetle (CRB, Oryctes rhinoceros) is an invasive palm pest whose larvae eat wood, yet lack the necessary digestive enzymes. This study confirmed endogenous CRB cellulase is inactive, suggesting microbial fermentation. The inner lining of the CRB hindgut has tree-like structures covered with a conspicuous biofilm. To identify possible symbionts, 16 S rRNA amplicon sequencing was used on individuals from across Taiwan. Several taxa of Clostridia, an anaerobic class including many cellulolytic bacteria, were highly abundant in most individuals from all locations. Whole metagenome sequencing further confirmed many lignocellulose degrading enzymes are derived from these taxa. Analyses of eggs, larvae, adults, and soil found these cellulolytic microbes are not transmitted vertically or transstadially. The core microbiomes of the larval CRB are likely acquired and enriched from the environment with each molt, and enable efficient digestion of wood.

A practical guide to collections-based research on ecogeographic rules.

Ecogeographic research into how species' forms vary across space, time, and climate has taken on new urgency due to contemporary global climate change. Research using museum specimens and other records to study biological rules like Bergmann's, Allen's, and Gloger's Rules has a long history and continues to generate publications and robust scientific debates. Despite the prevalence and history of the field, however, no simple guide on how to carry out such work has ever been published. To lower the barriers of entry for new researchers, this review was created as a practical guide on how to perform ecogeographic research. The guide consolidates disparately published methodologies into a single, convenient document that reviews the history and present of the field of ecogeographic rule research, and describes how to generate appropriate hypotheses, design experiments, gather, and analyze biotic and geographic data, and interpret the results in an ecologically meaningful manner. The result is a semi-standardized guide that enables scientists at all levels from any institution to carry out an investigation from start to finish on any biological rule, taxon, and location of their choice.

Chryseobacterium oryctis sp. nov., isolated from the gut of the beetle Oryctes rhinoceros, and Chryseobacterium kimseyorum sp. nov., isolated from a stick insect rearing cage.

Two strains of Chryseobacterium identified from different experiments are proposed to represent new species. Strain WLa1L2M3T was isolated from the digestive tract of an Oryctes rhinoceros beetle larva. Strain 09-1422T was isolated from a cage housing the stick insect Eurycantha calcarata. Sequence analysis of the 16S rRNA and rpoB genes found both strains to be similar but not identical to other Chryseobacterium species. Whole-genome sequencing suggested the isolates represent new species, with average nucleotide identity values ranging from 74.6 to 80.5 %. Genome-to-genome distance calculations produced values below 25.3 %, and digital DNA-DNA hybridization values were 13.7-29.9 %, all suggesting they are distinct species. The genomic DNA G+C content of WLa1L2M3T is approximately 32.53 %, and of 09-1422T is approximately 35.89 %. The predominant cellular fatty acids of strain WLa1L2M3T are C15 : 0 iso, summed feature 9 (C16 : 0 10OH or C17 : 1 iso ω6c), C17 : 0 iso 3OH, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C15 : 0 iso 3OH, C15 : 0 anteiso and C13 : 0 iso, and those of strain 09-1422T are C15 : 0 iso, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C17 : 0 iso 3OH, C15 : 0 anteiso, C15 : 0 iso 3OH, C16 : 1 ω7c, C17 : 0 2OH and C18 : 0. In addition, physiological and biochemical tests revealed phenotypic differences from related Chryseobacterium type strains. These cumulative data indicate that the two strains represent novel species of the genus Chryseobacterium for which the names Chryseobacterium oryctis sp. nov. and Chryseobacterium kimseyorum sp. nov. are proposed with WLa1L2M3T (=BCRC 81350T=JCM 35215T=CIP 112035T) and 09-1422T (=UCDFST 09-1422T=BCRC 81359T=CIP 112165T), as type strains, respectively.

Anatomical changes of the beetle digestive tract during metamorphosis correspond to dietary changes.

During pupation, the tissues of holometabolous insects change in preparation for the adult lifestyles, although little literature exists examining this hidden process in detail. Using beetles as a model, we hypothesized that species where the adult and larva have the same diets will show less pronounced changes of the digestive tract during metamorphosis than species where the adults diets differ. We also wanted to observe these changes and document them at a level of detail missing from the current record. We compared the structure of the digestive tracts of scarab beetles Oryctes rhinoceros, Thaumastopeus shangaicus, and Protaetia spp. (Coleoptera: Scarabaeidae)-where the larvae eat wood, soil, or compost while the adults feed on soft plant matter, tree sap, and rotting fruits-with the tortoise beetle, Cassida circumdata (Coleoptera: Chrysomelidae), which feeds on leaves as both larva and adult. In the scarab beetles we observed considerable changes in the digestive tracts during the pupal stage, which we could divide into distinct stages, while in the leaf beetle pupae, the gut did not change. This information can provide new insight into metamorphosis, and the illustrations of what occurs during pupation are novel contributions to this field that will facilitate future work.

Cytochrome P450 Genes Expressed in Phasmatodea Midguts.

Cytochrome P450s (CYPs) are xenobiotic detoxification genes found in most eukaryotes, and linked in insects to the tolerance of plant secondary chemicals and insecticide resistance. The number and diversity of CYP clans, families, and subfamilies that an organism produces could correlate with its dietary breadth or specialization. This study examined the CYP diversity expressed in the midguts of six species of folivorous stick insects (Phasmatodea), to identify their CYP complement and see if any CYPs correlate with diet toxicity or specialization, and see what factors influenced their evolution in this insect order. CYP genes were mined from six published Phasmatodea transcriptomes and analyzed phylogenetically. The Phasmatodea CYP complement resembles that of other insects, though with relatively low numbers, and with significant expansions in the CYP clades 6J1, 6A13/14, 4C1, and 15A1. The CYP6 group is known to be the dominant CYP family in insects, but most insects have no more than one CYP15 gene, so the function of the multiple CYP15A1 genes in Phasmatodea is unknown, with neofunctionalization following gene duplication hypothesized. No correlation was found between CYPs and diet specialization or toxicity, with some CYP clades expanding within the Phasmatodea and others likely inherited from a common ancestor.

Critical commentary on "The potential of a site-specific delivery of thiamine hydrochloride as a novel insect repellent exerting long-term protection on human skin: In-vitro, ex-vivo study and clinical assessment".

This letter comments on a recent article by Halawani et al. (10.1016/j.xphs.2021.07.017), which claimed a complex hydrogel formulation of thiamine nanospheres is a topical insect repellent. The authors did not thoroughly review the extensive prior literature on the subject that found no evidence of repellency for thiamine, and the experiment described lacked negative controls. Its results are not conclusive.

Increasing Body Sizes in Anomala expansa expansa (Coleoptera: Scarabaeidae) Populations in Response to Rising Temperatures Over Time.

Many insects have been studied over wide geographical areas to determine whether they follow Bergmann's Rule, which predicts that animal clades or populations should have smaller body sizes in warmer climates. While this ecogeographic rule is well supported in mammals and birds, insect latitudinal sizes can show Bergmann, converse Bergmann, or no size clines at all. Museum collections are typical sources of data for insect clines, and long-term collections should reflect rising global temperatures and shifting climates, possibly producing temporal size clines along with any geographical clines. We hypothesize that insects with Bergmann clines geographically will show Bergmann-like clines temporally as well, and that the converse and inverse of this rule are also true. By looking at museum samples going back a century, we tested whether Anomala expansa expansa (Bates, 1866), a species of Scarabaeidae beetle common in lowland Taiwan, was experiencing long-term changes in body size in response to rising temperatures. We found that overall, the size of these beetles increased over time. Within Taipei populations, this increase was correlated with rising average yearly temperatures. The impact of this pest species' rising size with time will need to be monitored, and temporal size clines in other pests need to be investigated.

Thiamine (vitamin B1) as an insect repellent: a scoping review.

While the desire for systemic repellents is high, ineffective repellents put one at risk of insect-vectored pathogens. Vitamin B1, or thiamine, has been touted as a systemic insect repellent since 1943, and denounced as an ineffective placebo for just as long. This paper presents a scoping review of 104 relevant case reports, research studies, and review articles to trace the evolution of this idea and identify an evidence-based, scientific consensus. Reports of thiamine's systemic repellency are primarily anecdotal and based on uncontrolled trials and/or used bite symptoms as a proxy for reduced biting. Controlled experiments on insect landing and feeding found no evidence of repellency. Of the 49 relevant review papers, 16 insect bite prevention guidelines, and 4 government documents, none after the 1990s claimed thiamine is a repellent. The findings of this review are that thiamine cannot repel arthropods in any dosage or route of administration. Due to limited available evidence, the possibility that thiamine reduces the subjective symptoms of insect bites cannot currently be ruled out. Unfortunately, many medical professionals and travelers today still believe thiamine may be effective despite the evidence stating otherwise. Continued promotion of debunked repellents on the commercial market poses a serious risk in countries with the endemic, mosquito-vectored disease.

Evolution of CP2 transcription factors in Hexapoda.

The CP2 transcription factors are highly conserved in metazoans, where they are divided into two groups: grainyhead and late SV40 factor (LSF). We traced their evolutionary history in the Hexapoda using over 500 insect transcriptomes, to test the hypothesis that the evolution of holometaboly involved novel isoforms of these genes. All insects appear to express at least one grainyhead and one LSFlike gene, regardless of life cycle, as in most known metazoa. No major evolutionary events in these gene families occurred during the evolution of insects.

Pseudomonas schmalbachii sp. nov., isolated from the gut of a millipede (Trigoniulus corallinus) from a coconut tree.

During an investigation of microbes associated with arthropods living in decaying coconut trees, a Pseudomonas isolate, Milli4T, was cultured from the digestive tract of the common Asian millipede, Trigoniulus corallinus. Sequence analysis of 16S rRNA and rpoB genes found that Milli4T was closely related but not identical to Pseudomonas panipatensis Esp-1T, Pseudomonas knackmussi B13T and Pseudomonas humi CCA1T. Whole genome sequencing suggested that this isolate represents a new species, with average nucleotide identity (OrthoANIu) values of around 83.9-87.7% with its closest relatives. Genome-to-genome distance calculations between Milli4T and its closest relatives also suggested they are distinct species. The genomic DNA G+C content of Milli4T was approximately 65.0 mol%. Phenotypic and chemotaxonomic characterization and fatty acid methyl ester analysis was performed on Milli4T and its related type strains. Based on these data, the new species Pseudomonas schmalbachii sp. nov. is proposed, and the type strain is Milli4T (=BCRC 81294T=JCM 34414T=CIP 111980T).

Detection of deformed wing virus (DWV) in the Vietnamese walking stick Medauroidea extradentata (Phasmatodea).

Deformed wing virus (DWV) is a single-stranded positive sense RNA virus that mainly infects honey bees (Apis mellifera) and can have devastating impacts on the colony. Recent studies have shown the presence of this virus in several species of Apis spp. and some other Hymenoptera, but our knowledge of their host range is very limited. We screened previously sequenced RNAseq libraries from different tissues of Vietnamese Walking Stick, Medauroidea extradentata (Phasmatodea) for DWV. We only found this virus in six libraries from anterior and posterior midgut tissue. From the midgut libraries we were able to construct a complete DWV genome sequence, which consisted of 10,140 nucleotides and included one open reading frame. Pairwise genome comparison confirmed strong similarity (98.89 %) of these assembled sequences with only 113 SNPs to the original DWV genome. We hypothesize the M. extradentata acquired this virus via a foodborne transmission by consuming DWV-infected material such as pollen or leaves contaminated with virus infected bee faeces.

Syzygium samarangense Leaf Infusion as Aedes albopictus (Diptera: Culicidae) Ovitrap Bait.

Adding lures can improve the efficiency of mosquito ovitraps used for monitoring or in attract-and-kill pest management. Easily produced, low-to-no cost bait would be ideal for remote field sites and community-run vector management. Plant infusions are popular ovitrap baits for their low cost and potent attractiveness, attributed either to the plants or their microbiomes. We tested fermented leaf infusions of the wax apple tree, Syzygium samarangense (Blume) Merr. & Perry, as bait in lethal ovitraps in urban Taipei with Bti larvicide. All trapped insects were inferred to be Asian tiger mosquito, Aedes albopictus (Skuse). The bait significantly increased the yield of the traps, with no negative interactions with the larvicide in either direction. Syzgium samarangense leaves are readily available across Taiwan, making their infusion an easy bait to greatly improve ovitrap efficiency.

Culturing-Enriched Metabarcoding Analysis of the Oryctes rhinoceros Gut Microbiome.

Wood-feeding insects should have a source of enzymes like cellulases to digest their food. These enzymes can be produced by the insect, or by microbes living in the wood and/or inside the insect gut. The coconut rhinoceros beetle, Oryctes rhinoceros, is a pest whose digestive microbes are of considerable interest. This study describes the compartments of the O. rhinoceros gut and compares their microbiomes using culturing-enriched metabarcoding. Beetle larvae were collected from a coconut grove in southern Taiwan. Gut contents from the midgut and hindgut were plated on nutrient agar and selective carboxymethylcellulose agar plates. DNA was extracted from gut and fat body samples and 16S rDNA metabarcoding performed to identify unculturable bacteria. Cellulase activity tests were performed on gut fluids and microbe isolates. The midgut and hindgut both showed cellulolytic activity. Bacillus cereus, Citrobacter koseri, and the cellulolytic fungus Candida xylanilytica were cultured from both gut sections in most larvae. Metabarcoding did not find Bacillus cereus, and found that either Citrobacter koseri or Paracoccus sp. were the dominant gut microbes in any given larva. No significant differences were found between midgut and hindgut microbiomes. Bacillus cereus and Citrobacter koseri are common animal gut microbes frequently found in Oryctes rhinoceros studies while Candida xylanilytica and the uncultured Paracoccus sp. had not been identified in this insect before. Some or all of these may well have digestive functions for the beetle, and are most likely acquired from the diet, meaning they may be transient commensalists rather than obligate mutualists. Broader collection efforts and tests with antibiotics will resolve ambiguities in the beetle-microbe interactions.

Identification of a Novel Picorna-like Virus in Coconut Rhinoceros Beetles (Oryctes rhinoceros).

A novel Picorna-like virus, tentatively named Oryctes rhinoceros Picorna-like virus 1 (OrPV1), was identified in coconut rhinoceros beetle (Oryctes rhinoceros) larvae in Taiwan. The complete genome sequence consisted of 9,665 nucleotides with a polyA tail and included one open reading frame. Conserved structural domains such as Picornavirus capsid protein, RNA helicase, Peptidase and RNA-dependent RNA polymerase (RdRp) were identified through Pfam domain searches. The genome shares approximately 27-28% identity with other unclassified Picornavirales that infect honey bees (Darwin bee virus 2, Bundaberg bee virus 5, and Sacbrood virus) and a recently reported virus from Asian lady beetle (Harmonia axyridis virus 1). We did not detect this virus in any other geographical populations of O. rhinoceros collected from the South Pacific Islands and the Philippines. Analysis of the deduced RdRp amino acid sequences showed that the virus clustered with other Picorna-like viruses and separated from other members of family Dicistroviridae and Iflaviridae.

Potential of Black Soldier Fly Production for Pacific Small Island Developing States.

Organic waste such as food waste and livestock manure is a serious concern in the Pacific Islands, where landfills are overflowing and illegal dumping of waste threatens the fragile ecosystems. Organic waste also attracts filth flies, some of which are vectors for pathogens that cause human disease. The black soldier fly, Hermetia illucens, has tremendous potential for the Pacific Islands. Capable of digesting almost any organic matter and converting it into insect biomass, black soldier flies are already being used around the world to process organic waste into larvae. The system can be adapted to large-scale municipal composting as well as small sizes for individual livestock farms or even urban households. The larvae can be fed live to fish or poultry, processed into feed comparable to fishmeal or soy meal, or even used to generate biofuel. Thus, the fly not only eliminates waste, but also can improve the sustainability of livestock production. The Pacific Small Island Developing States stand to benefit immensely from black soldier fly bioconversion facilities, used primarily as a means to compost organic waste; however, several knowledge gaps must first be addressed. We reviewed the state of black soldier flies in the Pacific and identified where their use shows the most promise. Research priorities for the field include fly surveys and bioconversion assays using Pacific crop waste.

Who's afraid of DEET? Fearmongering in papers on botanical repellents.

DEET (N,N-Diethyl-meta-toluamide) is considered the gold standard in mosquito repellents, not only for its effectiveness, but also for its safety. DEET has been more extensively studied for safety than any other repellent, and is accepted as completely safe when used correctly (i.e. not consumed or bathed in). Researchers studying botanical repellents, however, often paint DEET as far more toxic than it really is, falsely claiming it is a menace to the public health or even the environment. These claims are unfounded, and often the only evidence given by such publications are references to other publications also studying botanical repellents. Such publications are biased, and may be attacking DEET's excellent safety record to justify their existence and the need for their research. The inconvenient yet undisputable fact is that no botanical repellent has been proven to be as safe as DEET, and the majority never had any safety testing whatsoever. The automatic assumption that botanical repellents are safer than DEET is the 'appeal to nature fallacy,' which also drives most of the market for "natural" repellents, yet natural repellents have side effects and even a body count. Finding a botanical repellent that works as well as DEET and is equally safe is a legitimate research goal on its own, and need not be justified by fear-mongering and irrational chemophobia. Researchers studying these alternatives should strive for integrity, raising the real issue of the lack of safety testing for botanical repellents rather than denying the proven safety of DEET.

Microbes Associated With Black Soldier Fly (Diptera: Stratiomiidae) Degradation of Food Waste.

Black soldier fly (Hermetia illucens L.) larvae are capable of valorizing waste by converting it into insect biomass that can be used as animal feed, leaving undigested residue that can be used as soil enrichment. Evidence is conflicting over whether larvae fed substrate containing pathogenic microbes emerge uncontaminated. Studies also differ on which clades comprise the species' gut microbiome, and on whether and how diet affects these microbes. Using culturing and metabarcoding, the bacterial microbiota of black soldier fly larvae reared on two different kinds of food waste (postproduction soy pulp and postconsumer cafeteria waste) were analyzed, along with the microbes of their substrates. Little to no overlap was found between the wastes, the larvae, and the residues, but the larvae fed different foods had a significant percentage of their microbes in common. The data, in line with other works on this species, suggest the larvae have a conserved microbiota whose components vary geographically.