Honey Bee Larval Hemolymph as a Source of Key Nutrients and Proteins Offers a Promising Medium for Varroa destructor Artificial Rearing

Abstract

Varroa destructor, a major ectoparasite of the Western honey bee Apis mellifera, is a widespread pest that damages colonies in the Northern Hemisphere. Throughout their lifecycle, V. destructor females feed on almost every developmental stage of their host, from the last larval instar to the adult. The parasite is thought to feed on hemolymph and fat body, although its exact diet and nutritional requirements are poorly known. Using artificial Parafilm™ dummies, we explored the nutrition of V. destructor females and assessed their survival when fed on hemolymph from bee larvae, pupae, or adults. We compared the results with mites fed on synthetic solutions or filtered larval hemolymph. The results showed that the parasites could survive for several days or weeks on different diets. Bee larval hemolymph yielded the highest survival rates, and filtered larval plasma was sufficient to maintain the mites for 14 days or more. This cell-free solution therefore theoretically contains all the necessary nutrients for mite survival. Because some bee proteins are known to be hijacked without being digested by the parasite, we decided to run a proteomic analysis of larval honey bee plasma to highlight the most common proteins in our samples. A list of 54 proteins was compiled, including several energy metabolism proteins such as Vitellogenin, Hexamerin, or Transferrins. These molecules represent key nutrient candidates that could be crucial for V. destructor survival.

Keywords: artificial dummy; hemolymph; insect physiology; mite; nutrition; parasite; protein.

Figure 1

Artificial feeding chambers used to maintain mature female mites. Chambers consist of Petri dishes (Ø 5 cm) covered with Parafilm™. Two dummies containing an artificial feeding solution were placed in the chamber before 10 to 12 females V. destructor were inserted.

Figure 2

Survival curves and confidence intervals of mites fed on hemolymph of larvae (Hemlar, red curve), pupae (Hempu, light blue curve), or adult bees (Hemadu, green curve). The dashed black line shows the median survival for the three groups. Colored dashed lines surrounding the survival curves represent the 95% confidence interval. p-value resulting from the log rank test is shown in the top right corner of the graph along with pairwise p-values comparing the different feeding groups.

Figure 3

Survival of mites fed on larval hemolymph under different treatments (in % ± CI95). Prior to its insertion in Parafilm™ dummies, hemolymph was heated (Stored-Hemlar), filtered (Hemlar-F), or both heated and filtered (Hemlar-FH for the filtered then heated condition and Hemlar-HF for the heated then filtered condition). No significant difference was highlighted either on day 7 (dark blue) or on day 14 (light blue).

Figure 4

Pictures showing the coloration of V. destructor guts through the cuticle (upper panels) or after dissection (lower panels). (A,D): Mites that feed on a natural uncolored diet; (B,E): Mites that feed on a synthetic Blue-FCF colored diet; (C,F): Mite that feed on Blue-FCF colored larval hemolymph.

Figure 5

Feeding success of mites fed on synthetic solutions in comparison with mites fed on larval hemolymph (Stored-Hemlar). Synthetic solutions consisted of PBS (control group), PBS supplemented with glucose and fructose (Sugar), or with glucose, fructose, and yeast extract (SugarYE). The coloration of the gut was checked 24 h after the beginning of rearing. No significant difference was detected between the four groups (Larval hemolymph N = 30; PBS control N = 31; Sugar N = 92; SugarYE N = 31).

Figure 6

Survival curves and confidence intervals of mites fed synthetic solutions or larval hemolymph. Negative control (yellow curve) = starved mites without feeding dummies; PBS Control (blue curve) = mites with PBS-filled Parafilm™ dummies; Sugar (red curve) = mites fed on a sugar-supplemented PBS solution included in dummies; SugarYE (green curve) = mites fed on sugar and yeast extract supplemented PBS solution included in dummies. Colored dashed lines surrounding the survival curves represent the 95% confidence interval. The p-value resulting from the log rank test is shown in the bottom right corner of the graph along with pairwise p-values comparing the different feeding groups.

Figure 7

(A) Number of proteins detected in filtered larval hemolymph and systematic origin of the matching protein sequence. (B) Number of identified proteins that were found only once or shared between two, three, or four hemolymph samples from our three different colonies.