Leidynema appendiculata confirmed in the hindgut of B. discoidalis
This work confirmed that the adult nematodes observed during exploratory dissections in the hindgut of B. discoidalis were L. appendiculata. Colorimetric LAMP found L. appendiculata in the hindgut of 96% of the sampled population regardless of diet treatment. This is consistent with studies done in pest cockroach species, which found L. appendiculata colonization in the majority of the cockroaches sampled [1; 9; 18]. Leidynema appendiculata colonization spreads through coprophagy, which is a B. discoidalis feeding strategy [1; 18]. When all members of a colony are practicing coprophagy in a contained environment, L. appendiculata colonization appears widespread.
Sex and diet did not impact colonization
Logistic regression showed that none of the measured variables (sex, diet treatment, cage, and if the cockroach was gravid) significantly influenced L. appendiculata colonization. The lack of sex-based differences to colonization is consistent with studies done on L. appendiculata abundance in other cockroach species [3; 5]. However, the lack of diet-based differences seen in L. appendiculata colonization patterns was contrary to expectations. Previous research has shown that diet has an impact on host gut microbiome [9] and that L. appendiculata consume digestion end-products and gut bacteria [19]. Despite this, L. appendiculata appears able to tolerate all the diet treatments performed in this study.
LAMP is a quick and effective method for nematode detection
The samples tested using the LAMP assay had a 96% positive rate (n = 126) for nematode presence whereas only 64% of the samples tested with endpoint PCR using universal primers confirmed presence of L. appendiculata (n = 126) (Fig. 2). A two-tailed z-test showed this variance to be statistically significant (p-value = 7.259e-10). This difference is likely due to amplification bias of host rDNA, also amplified with the D2a/D3b universal primers. Host 28S rRNA gene amplification was confirmed by Sanger sequencing and BLAST completed on endpoint PCR products. In addition, the LAMP assay is much less time consuming than endpoint PCR. Colorimetric LAMP produces results within 30–40 minutes. In contrast, PCR amplification (30 cycles) requires just over 1 hour to amplify samples. This must be followed by visualization, which involves downstream analysis such as gel electrophoresis. This speed is one of the benefits of LAMP. However, this shorter amplification time can lead to false negatives in samples with low copy numbers.
Limitations
LAMP has been shown to be highly effective for use in detecting certain pathogens such as SARS-CoV-2 [20]. However, some studies in parasite colonization have shown false negative results [21]. LAMP efficacy is highly dependent on primer design [21], which may be a contributing factor to studies where LAMP was found to be ineffective. Additionally, colorimetric LAMP methods may be less effective than real-time methods used in SARS-CoV-2 detection. In this study, LAMP was more effective than endpoint PCR using the universal D2a/D3b primers. However, we did observe cases of false negatives in the LAMP assay. A subset of the samples (n = 24), including all samples that tested negative (n = 13) using the LAMP assay, were done in triplicate to confirm the efficacy of LAMP as a detection method. All samples that tested positive for nematode presence in the initial LAMP assay also tested positive in subsequent runs. Of the 13 samples that tested negative for Leidynema presence in the initial LAMP run, eight samples tested positive in subsequent replications of the LAMP assay. All 13 samples initially identified as negative for nematode presence were also amplified with endpoint PCR using the L. appendiculata-specific LAMP outer primers in triplicate followed by Sanger sequencing to confirm nematode presence. Five samples consistently tested negative for nematode presence using the LAMP assay. Of these, three samples tested positive for L. appendiculata when using the nematode-specific outer primer endpoint PCR. These results demonstrate that false negatives can result when using the LAMP assay. We recommend performing replications on any samples that test negative using the LAMP assay and verifying using endpoint PCR with the parasite-specific LAMP outer primers to ensure the detection of false negatives. False positives are also a concern in this study. Nematode eggs are commonly found in the environment [22], and the presence of nematode eggs is not indicative of a parasitic infection. Because LAMP is a highly sensitive assay, some of the positives observed in this study may indicate DNA from eggs, not from an active infection, but from ingestion; cockroaches often participate in coprophagy as a feeding strategy [1; 18]. For this reason, any studies using LAMP for rapid identification of pathogens require the subsequent use of abundance determining techniques such as qPCR to quantify the abundance of the nematodes in the host gut.