Isolation and identification of Aspergillus spp. from brown kiwi (Apteryx mantelli) nocturnal houses in New Zealand

Aspergillosis, a disease caused by infection with Aspergillus spp., is a common cause of death in birds globally and is an irregular cause of mortality of captive kiwi (Apteryx spp.). Aspergillus spp. are often present in rotting plant material, including the litter and nesting material used for kiwi in captivity. The aim of this study was to survey nocturnal kiwi houses in New Zealand to assess the levels of Aspergillus currently present in leaf litter. Samples were received from 11 nocturnal kiwi houses from throughout New Zealand, with one site supplying multiple samples over time. Aspergillus was isolated and quantified by colony counts from litter samples using selective media and incubation temperatures. Isolates were identified to the species level by amplification and sequencing of ITS regions of the ribosomal. Aspergillus spp. were recovered from almost every sample; however, the levels in most kiwi houses were below 1000 colony-forming units (CFU)/g of wet material. The predominant species was Aspergillus fumigatus, with rare occurrences of Aspergillus niger, Aspergillus nidulans, and Aspergillus parasiticus. Only one site had no detectable Aspergillus. The limit of detection was around 50 CFU/g wet material. One site was repeatedly sampled as it had a high loading of A. fumigatus at the start of the survey and had two recent clinical cases of aspergillosis diagnosed in resident kiwi. Environmental loading at this site with Aspergillus spp. reduced but was not eliminated despite changes of the litter. The key finding of our study is that the background levels of Aspergillus spores in kiwi nocturnal houses in New Zealand are low, but occasional exceptions occur and are associated with the onset of aspergillosis in otherwise healthy birds. The predominant Aspergillus species present in the leaf litter was A. fumigatus, but other species were also present. Further research is needed to confirm the optimal management of leaf litter to minimize Aspergillus spore counts. However, in the interim, our recommendations are that leaf litter should be freshly collected from areas of undisturbed forest areas and spread immediately after collection, without interim storage

An improved method to produce adults of Costelytra zealandica White (Coleoptera: Melolothinae) from field-collected larvae

Rearing techniques provide a unique opportunity to study aspects of insect ecology, behaviour and physiology. Both the larval and adult stages in Melolonthinae scarabs have important impacts on crop and pasture yields worldwide. Rearing techniques for this group of phytophagous beetles usually results in a low survival rate from larva to adult, varying from 10% to 50%. Here, the current rearing method used for the New Zealand grass grub (Costelytra zealandica) was improved by increasing the pupation weight threshold, as well as by changing the container type used to rear the larvae. This improved method produced an 83% increase in the survival rate from larva to adult, and the technique developed here may help increase the laboratory survival rate of other Melolonthinae species worldwide

Purification of high molecular-weight antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium belonging to the Brevibacillus brevis phylogenetic cluster. Globally, insect pathogenic strains of the bacterium have been isolated, characterised, and some activities patented. Two isolates, Bl 1821L and Bl 1951, exhibiting pathogenicity against the diamondback moth and mosquitoes, are under development as a biopesticide in New Zealand. However, due to the suspected activity of putative antibacterial proteins (ABPs), the endemic isolates often grow erratically. Various purification methods including size exclusion chromatography, sucrose density gradient centrifugation, polyethylene glycol precipitation, and ammonium sulphate precipitation employed in this study enabled the isolation of two putative antibacterial proteins of ~30 kD and ~48 kD from Bl 1821L and one putative antibacterial protein of ~30 kD from Bl 1951. Purification of the uninduced cultures of Bl 1821L and Bl 1951 also yielded the protein bands of ~30 kD and ~48 kD on SDS-PAGE which indicated their spontaneous induction. Disc diffusion assay was used to determine the antagonistic activities of the putative ABPs. Subsequent transmission electron microscope (TEM) examination of purified putative antibacterial protein-containing solution showed the presence of encapsulin (~30 kD) and polysheath (~48 kD) like structures. Although only the ~30 kD protein was purified from Bl 1951, both structures were seen in this strain under TEM. Furthermore, while assessing the antibacterial activity of some fractions of Bl 1951 against Bl 1821L in size exclusion chromatography method, population of Bl 1821L persister cells was noted. Overall, this work added a wealth of knowledge for the purification of the HMW proteins (bacteriocins) of the Gram-positive bacteria including Bl

Genome sequence of the entomopathogenic Serratia entomophila isolate 626 and characterisation of the species specific itaconate degradation pathway

Background: Isolates of Serratia entomophila and S. proteamaculans (Yersiniaceae) cause disease specifc to the endemic New Zealand pasture pest, Costelytra giveni (Coleoptera: Scarabaeidae). Previous genomic profling has shown that S. entomophila isolates appear to have conserved genomes and, where present, conserved plasmids. In the absence of C. giveni larvae, S. entomophila prevalence reduces in the soil over time, suggesting that S. entomophila has formed a host-specifc relationship with C. giveni. To help define potential genetic mechanisms driving retention of the chronic disease of S. entomophila, the genome of the isolate 626 was sequenced, enabling the identifcation of unique chromosomal properties, and defining the gain/loss of accessory virulence factors relevant to pathogenicity to C. giveni larvae. Results: We report the complete sequence of S. entomophila isolate 626, a causal agent of amber disease in C. giveni larvae. The genome of S. entomophila 626 is 5,046,461 bp, with 59.1% G+C content and encoding 4,695 predicted CDS. Comparative analysis with five previously sequenced Serratia species, S. proteamaculans 336X, S. marcescens Db11, S. nematodiphila DH-S01, S. grimesii BXF1, and S. ficaria NBRC 102596, revealed a core of 1,165 genes shared. Further comparisons between S. entomophila 626 and S. proteamaculans 336X revealed fewer predicted phage-like regions and genomic islands in 626, suggesting less horizontally acquired genetic material. Genomic analyses revealed the presence of a four-gene itaconate operon, sharing a similar gene order as the Yersinia pestis ripABC complex. Assessment of a constructed 626::RipC mutant revealed that the operon confer a possible metabolic advantage to S. entomophila in the initial stages of C. giveni infection. Conclusions: Evidence is presented where, relative to S. proteamaculans 336X, S. entomophila 626 encodes fewer genomic islands and phages, alluding to limited horizontal gene transfer in S. entomophila. Bioassay assessments of a S. entomophila-mutant with a targeted mutation of the itaconate degradation region unique to this species, found the mutant to have a reduced capacity to replicate post challenge of the C. giveni larval host, implicating the itaconate operon in establishment within the host

The number of larval instars in the flax weevil (Anagotus fairburni) (Coleoptera: Curculionidae)

The flax weevil Anagotus fairburni is a large flightless beetle, that is one of the members of the endemic insect ‘megafauna’ of New Zealand. It is a protected species that currently persists only on predator-free islands or in remote and difficult to access alpine areas. Little is documented about the ecology of the flax weevil. In this study we estimated the number of instars in the A. fairburni life cycle by measuring the head capsule widths of larvae collected in the field on Mana Island Scientific Reserve. We used kernel density function estimates to predict average head-capsule widths and the number of larval instars. We then used Brooks-Dyar’s law on the head capsule width data and analysed Brooks and Crosby indexes to refine the estimated number of instars based on imperfect data. Results from sampling of 86 larvae suggested four instar groupings, but further analysis based on Brooks-Dyar’s law found that A. fairburni likely passes through 6 or 7 larval stages prior to pupation, with some uncertainty for smaller instars. Our method provides new data on ecology of an endemic species and provides a framework for further work on similar endangered species where data is imperfect or difficult to gather

Serratia spp. bacteria evolved in Aotearoa-New Zealand for infection of endemic scarab beetles

The Melolonthinae branch of the beetle family Scarabaeidae has evolved in isolation in Aotearoa, radiating into >100 endemic species, since Aotearoa separated from Gondwanaland 82 million years ago. The group includes important pasture pests, such as the New Zealand grass grub Costelytra giveni and the manuka beetle Pyronota festiva. These beetles, like other organisms, host their own distinctive microflora including beneficial microbial symbionts and pathogens. A wide range of microbial pathogens infect the Scarabaeidae, but in Aotearoa the bacteria Serratia entomophila, S. proteamaculans and S. quinivorans (Enterobacteriaceae) are frequently found causing natural disease epizootics in C. giveni. S. entomophila is widespread in Aotearoa pasture soils, with only rare isolations of S. entomophila documented in other countries. In contrast S. proteamaculans and S. quinivorans are globally ubiquitous, and are widely distributed within Aotearoa, with some isolates active against either C. giveni or Pyronota spp. larvae, or both. Virulence determinants that impart differential host specificity and potency are located on variants of the amber disease associated plasmid (pADAP). The host specificity of the Serratia-scarab system and the absence of similar systems in other geographies, suggests that the relationship between Serratia spp. and endemic scarabs has evolved in Aotearoa

A response to Pennisi - “How do gut microbiomes help herbivores”, a hint into next-generation biocontrol solutions

In a world where invasive invertebrate species can significantly compromise food security and where a dwindling range of synthetic pesticides remains our principal line of defence, testing a new invasion ecology hypothesis and understanding what makes a phytophagous insect invasive should be regarded as high priority research. Recent advances in microbiology strongly support the crucial and effective role of the gut microbiome in insect growth, development and, most importantly, environmental adaptation to their host plants. On the basis of recent literature, and following Elizabeth Pennisi’s article published in the journal Science, we hypothesis that gut microbiome could be a critical determinant of invasion success in phytophagous insects, and that the uncovering of common traits in the gut microbiome of invasive insects, a “gut microbiome invasiveness signature”, would open new avenues of research towards next-generation biocontrol solutions

Isolation, purification, and characterisation of a phage tail-like bacteriocin from the insect pathogenic bacterium Brevibacillus laterosporus

The Gram-positive and spore-forming bacterium Brevibacillus laterosporus (Bl) belongs to the Brevibacillus brevis phylogenetic cluster. Isolates of the species have demonstrated pesticidal potency against a wide range of invertebrate pests and plant diseases. Two New Zealand isolates, Bl 1821L and Bl 1951, are under development as biopesticides for control of diamondback moth and other pests. However, due to the often-restricted growth of these endemic isolates, production can be an issue. Based on the previous work, it was hypothesised that the putative phages might be involved. During investigations of the cause of the disrupted growth, electron micrographs of crude lysate of Bl 1821L showed the presence of phages’ tail-like structures. A soft agar overlay method with PEG 8000 precipitation was used to differentiate between the antagonistic activity of the putative phage and phage tail-like structures (bacteriocins). Assay tests authenticated the absence of putative phage activity. Using the same method, broad-spectrum antibacterial activity of Bl 1821L lysate against several Gram-positive bacteria was found. SDS-PAGE of sucrose density gradient purified and 10 kD MWCO concentrated lysate showed a prominent protein band of ∼48 kD, and transmission electron microscopy revealed the presence of polysheath-like structures. N-terminal sequencing of the ∼48 kD protein mapped to a gene with weak predicted amino acid homology to a Bacillus PBSX phage-like element xkdK, the translated product of which shared >90% amino acid similarity to the phage tail-sheath protein of another Bl published genome, LMG15441. Bioinformatic analysis also identified an xkdK homolog in the Bl 1951 genome. However, genome comparison of the region around the xkdK gene between Bl 1821L and Bl 1951 found differences including two glycine rich protein encoding genes which contain imperfect repeats (1700 bp) in Bl 1951, while a putative phage region resides in the analogous Bl 1821L region. Although comparative analysis of the genomic organisation of Bl 1821L and Bl 1951 PBSX-like region with the defective phages PBSX, PBSZ, and PBP 180 of Bacillus subtilis isolates 168 and W23, and Bacillus phage PBP180 revealed low amino acids similarity, the genes encode similar functional proteins in similar arrangements, including phage tail-sheath (XkdK), tail (XkdO), holin (XhlB), and N-acetylmuramoyl-L-alanine (XlyA). AMPA analysis identified a bactericidal stretch of 13 amino acids in the ∼48 kD sequenced protein of Bl 1821L. Antagonistic activity of the purified ∼48 kD phage tail-like protein in the assays differed remarkably from the crude lysate by causing a decrease of 34.2% in the number of viable cells of Bl 1951, 18 h after treatment as compared to the control. Overall, the identified inducible phage tail-like particle is likely to have implications for the in vitro growth of the insect pathogenic isolate Bl 1821L

Identification of genes involved in exoprotein release using a high-throughput exoproteome screening assay in Yersinia entomophaga

Bacterial protein secretion is crucial to the maintenance of viability and pathogenicity. Although many bacterial secretion systems have been identified, the underlying mechanisms regulating their expression are less well explored. Yersinia entomophaga MH96, an entomopathogenic bacterium, releases an abundance of proteins including the Yen-Tc into the growth medium when cultured in Luria Bertani broth at ≤ 25˚C. Through the development of a high-throughput exoproteome screening assay (HESA), genes involved in MH96 exoprotein production were identified. Of 4,080 screened transposon mutants, 34 mutants exhibited a decreased exoprotein release, and one mutation located in the intergenic region of the Yen-Tc operon displayed an elevated exoprotein release relative to the wild-type strain MH96. DNA sequencing revealed several transposon insertions clustered in gene regions associated with lipopolysaccharide (LPSI and LPSII), and N-acyl-homoserine lactone synthesis (quorum sensing). Twelve transposon insertions were located within transcriptional regulators or intergenic regions. The HESA will have broad applicability for identifying genes associated with exoproteome production in a range of microorganisms

Biological control of Diamondback moth—Increased efficacy with mixtures of Beauveria fungi

Diamondback moth (DBM) is an important horticultural pest worldwide as the larvae of these moths feed on the leaves of cruciferous vegetables. As DBM has developed resistance to more than 100 classes of synthetic insecticides, new biological control options are urgently required. Beauveria species are entomopathogenic fungi recognized as the most important fungal genus for controlling a wide range of agricultural, forestry, and veterinary arthropod pests. Previous research, aimed at developing new Beauveria-based biopesticides for DBM, has focused on screening single isolates of Beauveria bassiana. However, these fungal isolates have individual requirements, which may limit their effectiveness in some environments. This current study separately assessed 14 Beauveria isolates, from a range of habitats and aligned to four different species (Beauveria bassiana, B. caledonica, B. malawiensis, and B. pseudobassiana), to determine the most effective isolate for the control of DBM. Further assays then assessed whether selected combinations of these fungal isolates could increase the overall efficacy against DBM. Six Beauveria isolates (three B. bassiana and three B. pseudobassiana) achieved high DBM mortality at a low application rate with the first documented report of B. pseudobassiana able to kill 100% of DBM larvae. Further research determined that applications of low-virulent Beauveria isolates improved the control of DBM compared to mixtures containing high-virulent isolates. This novel approach increased the DBM pest mortality and shortened the time to kill