microRNA profile of Hermetia illucens (black soldier fly) and its implications on mass rearing.

The growing demands on protein producers and the dwindling available resources have made Hermetia illucens (the black soldier fly, BSF) an economically important species. Insights into the genome of this insect will better allow for robust breeding protocols, and more efficient production to be used as a replacement of animal feed protein. The use of microRNA as a method to understand how gene regulation allows insect species to adapt to changes in their environment, has been established in multiple species. The baseline and life stage expression levels established in this study, allow for insight into the development and sex-linked microRNA regulation in BSF. To accomplish this, microRNA was extracted and sequenced from 15 different libraries with each life stage in triplicate. Of the total 192 microRNAs found, 168 were orthologous to known arthropod microRNAs and 24 microRNAs were unique to BSF. Twenty-six of the 168 microRNAs conserved across arthropods had a statistically significant (p < 0.05) differential expression between Egg to Larval stages. The development from larva to pupa was characterized by 16 statistically significant differentially expressed microRNA. Seven and 9 microRNA were detected as statistically significant between pupa to adult female and pupa to adult male, respectively. All life stages had a nearly equal split between up and down regulated microRNAs. Ten of the unique 24 miRNA were detected exclusively in one life stage. The egg life stage expressed five microRNA (hil-miR-m, hil-miR-p, hil-miR-r, hil-miR-s, and hil-miR-u) not seen in any other life stages. The female adult and pupa life stages expressed one miRNA each hil-miR-h and hil-miR-ac respectively. Both male and female adult life stages expressed hil-miR-a, hil-miR-b, and hil-miR-y. There were no unique microRNAs found only in the larva stage. Twenty-two microRNAs with 56 experimentally validated target genes in the closely related Drosophila melanogaster were identified. Thus, the microRNA found display the unique evolution of BSF, along with the life stages and potential genes to target for robust mass rearing. Understanding of the microRNA expression in BSF will further their use in the crucial search for alternative and sustainable protein sources.

Building mean field ODE models using the generalized linear chain trick & Markov chain theory.

The well known linear chain trick (LCT) allows modellers to derive mean field ODEs that assume gamma (Erlang) distributed passage times, by transitioning individuals sequentially through a chain of sub-states. The time spent in these sub-states is the sum of k exponentially distributed random variables, and is thus gamma distributed. The generalized linear chain trick (GLCT) extends this technique to the broader phase-type family of distributions, which includes exponential, Erlang, hypoexponential, and Coxian distributions. Phase-type distributions are the family of matrix exponential distributions on [0,∞) that represent the absorption time distributions for finite-state, continuous time Markov chains (CTMCs). Here we review CTMCs and phase-type distributions, then illustrate how to use the GLCT to efficiently build ODE models from underlying stochastic model assumptions. We introduce two novel model families by using the GLCT to generalize the Rosenzweig-MacArthur predator-prey model, and the SEIR model. We illustrate the kinds of complexity that can be captured by such models through multiple examples. We also show the benefits of using a GLCT-based model formulation to speed up the computation of numerical solutions to such models. These results highlight the intuitive nature, and utility, of using the GLCT to derive ODE models from first principles.

The puzzling mitochondrial phylogeography of the black soldier fly (Hermetia illucens), the commercially most important insect protein species.

BACKGROUND: The black soldier fly (Diptera: Stratiomyidae, Hermetia illucens) is renowned for its bioconversion ability of organic matter, and is the worldwide most widely used source of insect protein. Despite varying extensively in morphology, it is widely assumed that all black soldier flies belong to the same species, Hermetia illucens. We here screened about 600 field-collected and cultured flies from 39 countries and six biogeographic regions to test this assumption based on data for three genes (mitochondrial COI, nuclear ITS2 & 28S rDNA) and in order to gain insights into the phylogeography of the species. RESULTS: Our study reveals a surprisingly high level of intraspecific genetic diversity for the mitochondrial barcoding gene COI (divergences up to 4.9%). This level of variability is often associated with the presence of multiple species, but tested nuclear markers (ITS2 and 28S rDNA) were invariant and fly strain hybridization experiments under laboratory conditions revealed reproductive compatibility. COI haplotype diversity is not only very high in all biogeographic regions (56 distinct haplotypes in total), but also in breeding facilities and research centers from six continents (10 haplotypes: divergences up to 4.3%). The high genetic diversity in fly-breeding facilities is mostly likely due to many independent acquisitions of cultures via sharing and/or establishing new colonies from field-collected flies. However, explaining some of the observed diversity in several biogeographic regions is difficult given that the origin of the species is considered to be New World (32 distinct haplotypes) and one would expect severely reduced genetic diversity in the putatively non-native populations in the remaining biogeographic regions. However, distinct, private haplotypes are known from the Australasian (N = 1), Oriental (N = 4), and the Eastern Palearctic (N = 4) populations. We reviewed museum specimen records and conclude that the evidence for introductions is strong for the Western Palearctic and Afrotropical regions which lack distinct, private haplotypes. CONCLUSIONS: Based on the results of this paper, we urge the black soldier fly community to apply molecular characterization (genotyping) of the fly strains used in artificial fly-breeding and share these data in research publications as well as when sharing cultures. In addition, fast-evolving nuclear markers should be used to reconstruct the recent invasion history of the species.

Effects of storage temperature on the change in size of Calliphora vicina larvae during preservation in 80% ethanol.

The size of immature blowflies is a common measure to estimate the minimum time between death and the discovery of a corpse, also known as the minimum post-mortem interval. This paper investigates the effects of preservation, in 80% ethanol, on the length and weight of first instar, second instar, feeding third instar, and post-feeding third instar Calliphora vicina larvae, at three different storage temperatures. For each larval stage, the length of larvae was recorded after 0 h, 3 h, 6 h, 9 h, 12 h, 24 h, 72 h, 7 days, 14 days, 30 days, 91 days, 182 days, 273 days, and 365 days of storage in 80% ethanol, at -25°C, 6°C and 24°C. Storage temperature had no statistically significant effect on the change in larval length and weight for all larval stages, but larval length and weight were significantly affected by the duration of preservation for first, second, and feeding third instar larvae, but not for post-feeding larvae. Generally, first and second instar larvae reduced in size over time, while feeding third instar larvae increased slightly in size, and post-feeding larvae did not change in size over time. The length of blowfly larvae preserved in 80% ethanol is not affected by constant storage temperatures between -25°C and +24°C, but we recommend that forensic entomologists should use the models provided to correct for changes in larval length that do become apparent over time.

Decomposed liver has a significantly adverse affect on the development rate of the blowfly Calliphora vicina.

The development rate of immature Calliphora vicina reared on decomposed liver was significantly slower, by as much as 30 h (55.4 % of total development time) for mid-sized larvae, and 71 h (35.0 %) and 58 h (14.6 %) if using times to the onset of pupariation and eclosion, respectively, than those of immatures that developed on fresh whole pig's liver. Development rates of larvae reared on decomposed liver were also slower than those of larvae reared on minced pig's liver and frozen/thawed pig's liver. These results suggest that any estimate of minimum post-mortem interval may result in an over estimate if the blowflies used were developing on an already decomposed body.

Virtual forensic entomology: improving estimates of minimum post-mortem interval with 3D micro-computed tomography.

We demonstrate how micro-computed tomography (micro-CT) can be a powerful tool for describing internal and external morphological changes in Calliphora vicina (Diptera: Calliphoridae) during metamorphosis. Pupae were sampled during the 1st, 2nd, 3rd and 4th quarter of development after the onset of pupariation at 23 °C, and placed directly into 80% ethanol for preservation. In order to find the optimal contrast, four batches of pupae were treated differently: batch one was stained in 0.5M aqueous iodine for 1 day; two for 7 days; three was tagged with a radiopaque dye; four was left unstained (control). Pupae stained for 7d in iodine resulted in the best contrast micro-CT scans. The scans were of sufficiently high spatial resolution (17.2 µm) to visualise the internal morphology of developing pharate adults at all four ages. A combination of external and internal morphological characters was shown to have the potential to estimate the age of blowfly pupae with a higher degree of accuracy and precision than using external morphological characters alone. Age specific developmental characters are described. The technique could be used as a measure to estimate a minimum post-mortem interval in cases of suspicious death where pupae are the oldest stages of insect evidence collected.

Factors affecting accuracy and precision of thermal summation models of insect development used to estimate post-mortem intervals.

This paper investigates the effects that different summary statistics (minimum, median, mean, or maximum), temporal sampling resolutions (duration between sampling events), and sample sizes (number of individuals sampled per sampling event) had on the accuracy and precision of the regression coefficients of a typical thermal summation model used to calculate minimum post-mortem interval (PMI). No significant differences were found in the values of the developmental constants calculated from different summary statistics of the duration of development. Sample size was found to affect the precision of measurement of the duration of development but had little overall influence on thermal summation constant (K) and developmental threshold (D0) calculations (and therefore, subsequent PMI estimates), but temporal sampling resolution had a direct influence on the accuracy of K and D0 calculations. These data suggest that when numbers of experimental maggots are limited, it is more important to sample more frequently using smaller sample sizes than to sample less frequently with large sample sizes. Furthermore, we suggest that the median is the most representative summary measure of the duration of development and should be used preferentially.

Estimating the age of immature Chrysomya albiceps (Diptera: Calliphoridae), correcting for temperature and geographical latitude.

Developmental curves for Chrysomya albiceps (Wiedemann) (Diptera: Calliphoridae) were established at 13 different constant temperatures using developmental landmarks and length as measures of age. The thermal summation constants (K) and developmental zeros (D (0)) were calculated for five developmental landmarks using the method described by Ikemoto and Takai (Environ Entomol 29:671-682, 2000). Comparison with the K and D (0) values of our findings to those of three previously published studies of C. albiceps suggests that K is directly proportional to geographic latitude, and D (0) is inversely proportional to both K and geographic latitude. Body size and developmental landmarks have a complex relationship because of trade-offs between mortality risk and female fecundity (as measured by body size) at non-optimal temperatures. This relationship can be summarized using superimposed isomorphen and isomegalen diagrams, which can then be used to make forensic estimates of postmortem intervals from larval body lengths. Finally, we recommend that future studies providing data for precise forensic estimates of postmortem intervals should use a relative temporal precision of about 10% of the total duration being measured. For many blowflies, this translates into a sampling interval of approximately every 2 h before hatching, 3 h before first ecdysis and 6 h before second ecdysis.