Authors

Discussion

Poultry red mites (PRM) are blood feeding ectoparasites that live off-host, only seeking a bird to rapidly engorge every few days. Three of the five lifestages are hematophagous and are highly mobile making them difficult to contain in a controlled experimental environment. In vitro feeding techniques have been previously been devised to overcome containment issues (e.g. McDevitt et al., 2006; Bartley et al., 2015) for the preliminary screening of PRM vaccines. Mite feeding rates can be highly variable after storage and here we describe steps towards optimising egg laying and evaluating feeding rates of mites stored in different conditions.

 Recently we described utilising Baudruche membrane in an in vitro device to feed adult females (Nunn et al 2020) using goose blood, which led to improved and reproducible feeding rates and fewer animal procedures due to the increased blood volume per procedure. We evaluated the device to feed the hematophagous nymph stages of PRM and demonstrated significant correlation between the feeding rates of deutonymph stages and adult females (Spearman rs. =0.54, p = 0.0008, n=48) and protonymphs and adult females (Spearman rs. = 0.60, p = 0.00001,=48). A highly significant correlation was demonstrated between the proportion of the two nymph stages (Spearman rs. = 0.919,p = >0.00001,n=48) fed adults with no significant feeding of nymph stages in the absence of adult females.  To obtain reasonable in vitro feeding rates, mites are generally starved (conditioned) to allow digestion of their last blood meal, moulting and egg laying. Traditionally, mites are conditioned at room temperature(RT) before being stored at 4-8 C,briefly brought to RT before a feeding assay. Mite feeding can be variable under these conditions and are generally only useful for 2-4 weeks post conditioning at RT. Based on a study by Wang et al (2020) we performed a study to establish feeding rates of mites kept over longer periods of time after two different conditioning regimes. Three collections of mites (1-3) were each divided into two, with one cohort stored at 5C until conditioning at RT for one week prior to feeding (1a-3a) and with the other cohort (1b-3b) conditioned at RT for one week followed by storing at 5C until feeding. The percentage feeding rate of adult females in cohorts 1a-3a, fell by less than 10% over the course of the study (10 weeks), demonstrating feeding rates of 2.6, 1.26 and 1.4 times that of cohorts 1b-3b at 6weeks and 6.75, 2.1 and 4.75 respectively at 8weeks.To see if egg laying of mites was improved by being kept in groups of fed females, we compared numbers of offspring per fed mite on two occasions. Fed mites were incubated individually (n=60,n=41 respectively)and 5 replicate groups of 5, 15 and 30 mites and counting offspring after incubation for 7 days. No difference in offspring/fed mite was demonstrated across the different experimental groups. Repeated feeding of adult female mites was then performed, with 6 flasks of 100 adult females fed on four consecutive days and 6 flasks of 100 adult females fed only once. Flasks were incubated for 5 further days to allow eggs to hatch and for larvae to moult into protonymphs. They were then counted and progeny per adult female was calculated. Using an unpaired t-test with Welch’s correction, a significant increase (P = 0.02; t=2.844, df=6) in progeny/mite was demonstrated in those mites fed on four consecutive days (range 0.49-1.9, mean 1.115, SEM 0.21) compared to those fed once (range 0.23-0.49, mean 0.485, SEM 0.08).In summary, PRM feeding rates are stable (+/- 10%) up to ten weeks post collection if kept at 5C prior to conditioning at RT before in vitro feeding.  Nymph feeding is greatly enhanced by feeding all hematophagous life stages together and fecundity was improved by repeated feeding of females but not by housing groups of fed females together.