Use of a Novel Feeding System to Assess the Survival of a Very Stable Mammalian Virus, Porcine Parvovirus, Within Black Soldier Fly (Hermetia illucens) Larvae: A Comparison with Mealworm (Tenebrio molitor) Larvae

Abstract

Insect larvae production offers the potential for large-scale synthesis of high-quality protein that can be used as feed or food. However, currently, there are limitations on the source of substrates for the insect larvae to use. One concern is the potential survival of animal pathogens within insect larvae if their feed is contaminated. In this study, the survival of a very stable virus, porcine parvovirus (PPV), within mealworm (Tenebrio molitor) and black soldier fly (BSF) (Hermetia illucens) larvae has been analyzed after oral ingestion of the virus. PPV genomic DNA could be readily detected by PCR in both species of larvae up until 9 days post ingestion (DPI), the end of the study period. Furthermore, infection of susceptible PK15 cells by PPV from homogenized mealworm larvae could be detected until at least 3 DPI, using an immunoperoxidase staining method and, up until 9 DPI, with a more sensitive real time PCR assay. Thus, PPV can remain infectious within mealworm larvae during their main growth phase through to their harvesting. However, it may be considered that PPV is exceptional in this respect since it displays unusual stability, e.g., to heat.

Keywords: insect larvae; virus infectivity assays; virus ingestion; virus survival.

Figure 2

Inactivation of PPV in EMEM incubated at different temperatures. PPV was incubated at 5 °C, 20 °C (A), 35 °C, 40 °C (B) and 45 °C, 50 °C and 55 °C (C) in EMEM for the indicated times and residual infectious virus was assayed by titration in primary swine kidney cells and is presented as log₁₀ TCID₅₀/50 μL. The minimum level of virus detection in the assays is indicated by the horizontal dashed line. Note that samples were collected at different time points at the various temperatures to cover the relevant periods for virus survival.

Figure 1

Feeding of black soldier fly larvae with growth medium or virus. (A) Aliquots (5 µL) of virus suspension or growth medium, were placed into 0.2 mL PCR Tube Strips. (B) BSF larvae were placed with mouthparts facing downwards until they had visibly consumed the liquid within a maximum period of 30 min.

Figure 3

Maintenance of PPV DNA in mealworm (T. molitor) larvae. Two separate experiments were performed using different batches of larvae. Typically, 10 larvae were assayed from each time point and the results are plotted individually and as an average. In Study 1 (A) and Study 2 (B) at 0 days post ingestion (DPI) and subsequently at 1, 2, 3, 4, 6, 7, 8 and 9 DPI the PPV DNA was quantified by qPCR and values were converted to log₁₀ genome copy numbers/larva using a standard curve. Levels below 10^3.1 PPV genomes/larva were below the detection limit (indicated by dashed line). PPV incubated in EMEM at the same temperature (27 °C) was assayed at the same time points.

Figure 4

The presence of PPV DNA in individual BSF (H. illucens) larvae (typically 10 larvae were assayed at each time point) at 0 days post ingestion (DPI) and subsequently, at the indicated DPI, was quantified by qPCR and values were converted to log₁₀ genome copy numbers/larva using a standard curve. Levels below 10^3.1 PPV genomes/larva were below the detection limit (indicated by the dashed line). PPV incubated in EMEM at the same temperature (27 °C) was assayed at the same time points. Negative controls were larvae fed on EMEM, without virus.

Figure 5

Detection of infectious PPV in PK-15 cells. The cells were treated with the indicated samples, incubated for 72 h and stained for the presence of PPV antigens. (A), positive control, PPV virus stock, diluted 1:100; (B), positive control virus stock (diluted 1:100) following incubation at 27 °C for 9 days. (C), filtered and 10x diluted mealworm homogenate, prepared from a PPV-fed larva frozen on day 0, was added to cells and, after absorption, incubation was continued for 72 h prior to staining. (D), filtered and 10x diluted mealworm homogenate, prepared from a larva frozen on day 3 after PPV ingestion, was added to cells and, after absorption, incubation was continued for 72 h prior to staining. (E), the cells were treated as for panels C and D, but the mealworm homogenate was derived from a larva that had been fed on EMEM without PPV. A summary of the staining results for each of the larvae tested is shown in Table 1. No staining was detectable from the larvae incubated for 6 or 9 days following feeding on the virus. The scale bar indicates 100 µm.