High COI barcode diversity
Our data reveal many unexpected results for this widely used workhorse of insect protein production. They highlight that there is currently a surprising lack of background information for a species that is globally extensively used and shared for scientific research, animal feed production and organic waste recycling.
Our study revealed very high COI barcode variability, but this variability is not mirrored by the two invariant nuclear markers that were included in our study. This high COI diversity also does not translate into reproductive incompatibility although the divergence levels (close to 5%) well exceeds what is typical for flies [28]. This implies that the lack of genotyping of cultures may not have done as much scientific and commercial harm in BSF as it did for leeches belonging to Helobdella and Hirudo. The high COI diversity is structured in both expected and unexpected ways. The species is considered native to the New World; i.e., the high genetic diversity we recorded for the New World samples corresponds well with expectations, while the remaining regions should mostly have subsets of the New World genetic diversity. This is indeed the case for the Afrotropical and the Western Palearctic populations, which contain a limited number of haplotypes. It appears likely that these populations were indeed introduced. However, it was surprising to find several distinct, highly diverged haplotypes in Australasian, Oriental and Eastern Palaearctic populations, and this diversity remains unexplained if the species indeed originated in the New World and only recently dispersed to these regions. Possible explanations include still insufficient sampling of the Neotropical diversity or a broader geographic origin of the species.
BSF has not only unusually high levels of genetic diversity for COI, but it is also morphologically very variable with regard to body size, wing and abdominal coloration, and the shape of translucent abdominal windows. Wing coloration varies and ranges from almost black with a blueish-metallic shine over a clear brownish-opaque insignia in the inner part of the wing to almost pale-translucent. The amount of white versus black coloration on the head shows great variation, while the black and white pattern on the legs seem to be more consistent. There is a tendency for males to have a reddish to red abdomen, while females rarely display red coloration on the abdomen. Much of this variation can be found within the same laboratory cultures and is thus likely due to polyphenism, but more systematic study across cultures representing different haplotype groups is needed.
Global trade and the distribution of BSF
We documented that the distinct haplotypes of the commercial US breeding strain (Fig. 1 & 2A: the haplotypes of the commercial ‘Phoenix Worms’ indicated with arrows) can be found in flies from natural and urban environments in the Afrotropical (South Africa, Kenya), Oriental (Bhutan, China, Indonesia) and the Palaearctic (Portugal, Russia, Spain) regions, but it cannot be ascertained whether the flies originate from (repeated) introductions or if flies are escapees from rearing cultures. Similarly, haplotypes of flies recorded in commercial and/or research cultures in Asia and Australasia are also found in field-collected flies from these and other biogeographical regions (Fig. 1: II & III; Table 1). Barcodes of single flies sampled from Benin, Ghana, Malawi and Portugal were most similar to BSF barcodes from the Neotropical region (Figs 1 & 2A). These patterns are compatible with human-mediated introduction across biogeographic regions, but it will require population-level nuclear markers to confirm whether the introductions were sufficiently recent to be due to global trade.
Arguably, the most convincing evidence for human-mediated introductions come from specimens collected at latitudes that are unlikely to support breeding populations [13,29,30,31]. The lower threshold for BSF larval survival was observed to be 15-19 °C depending on study [32,33], and some Canadian BSF localities are unlikely to meet this requirement. Marshall et al. [13] thus hypothesized that the annual occurrence of BSF in Ontario (Canada) is due to the disposal of unused fishing baits (larvae) by local fishermen or the accidental release of flies into nature by owners of pet reptiles. However, the evidence is not entirely conclusive because BSF flies, prepupae, and pupae are resistant to cold and can survive low temperatures (5°C) for several weeks [34]. This may explain why there is an established BSF population in Northwestern Switzerland that has been regularly encountered for one decade. In addition, wild BSF are occasionally caught in Western and Central European countries such as Germany [29] and the Czech Republic [31]. Overall, more research is needed in order to fully understand the thermal tolerance of BSF and its diapausing abilities. This and all other research needs to be based on genotyped cultures because it is likely that populations from different parts of the (native) range will differ with regard to thermal tolerance. One way or another, it appears likely that BSF will be able to increase its current range as global warming takes hold. We would suggest that urban populations will most likely spearhead the range expansion because they benefit readily available organic waste and benefit from higher temperatures in urban environments [32]. Range expansion may also be facilitated through population homogenization as more cultures are shared over long distances and escaped flies cross-breed with native or previously established non-native populations.
The BSF is now very actively domesticated worldwide [35], but the field lacks ‘herdbook’ keeping that track the fly strains (see international Drosophila stock centers). The scientific utility for the genetic characterization and source documentation in commercial breeding of domestic livestock (e.g. pigs and cattle) is obvious, and genomics has not only proven useful for reconstructing the domestication history of the different breeds, but also for understanding the molecular mechanisms underlying traits of interest [36]. A tremendous amount of phenotypic plasticity among three different BSF strains in the ability of their larvae to reduce dry matter and associated nutrients in three different animal manures was demonstrated in a study by Zhou et al. [37], and additional evidence is emerging more recently [38]. Knowing the link between genes and trait has improved target-based breeding of domestic livestock [39], and it seems obvious that it can also be achieved for ‘mini-livestock’ like the BSF. Linked characterization of both the nuclear and mitochondrial genomes could prove useful, as the mtDNA COI barcode could provide sufficient initial and inexpensive information for avoiding, for example potential reduced fitness resulting from the hybridization of highly diverged strains.
Puzzling phylogeography
This study screened a comprehensive number of both cultured and wild caught BSF samples from six continents and 39 countries, including numerous from the putatively native New World and continents where BSF is an alien species. Our findings clearly highlight the need for additional sampling of New World populations as well as the Australasian and Oriental regions. In particular, the discovery of the haplotypes which are currently only known from Australasian and Oriental populations would strengthen the hypothesis of a New World origin of the species.
If a divergence rate of 2.3% per million years for insect mitochondrial DNA [40] were used as a guide, one would have to conclude that the most distant samples of the BSF haplotype network (Fig. 1) have been separated for 0.74-1.35 million years. This would imply that the Oriental and Australasian wild samples (Fig. 1: II & III) had already diverged 1.04 MY ago. These findings could either challenge the assumption of a New World origin or the spread of BSF was not human-mediated. Arguably, the strongest argument against the latter is that black soldier flies are conspicuous and should have been collected by early naturalists in Asia and Australasia if they had been present. Instead, the earliest records are from the 1940s.