# In vitro molting of Dirofilaria immitis third-stage larvae derived from microfilariae collected from doxycycline-treated dogs

## TL;DR

This study examines how doxycycline affects the development of heartworm larvae, finding that it reduces Wolbachia levels but does not prevent larval molting.

## Contribution

The study identifies that doxycycline reduces Wolbachia in heartworm larvae without affecting their ability to molt.

## Key findings

- Doxycycline treatment significantly reduces Wolbachia levels in microfilariae and L3 larvae.
- Larval molting from L3 to L4 is not impacted by doxycycline-induced Wolbachia reduction.
- Wolbachia levels remain below five percent of controls throughout doxycycline treatment.

## Abstract

Dirofilaria immitis, also known as canine heartworm, contains an endosymbiont, Wolbachia, in all life stages. The antibiotic, doxycycline, has been incorporated into heartworm treatment protocols to eliminate Wolbachia. Previous studies indicate that subsequent infection cannot be established using viable third-stage larvae (L3) developed from doxycycline-treated microfilariae (mf). The stages in which the development of larvae is impacted by doxycycline remain unknown. We examined the impact of doxycycline on the third-stage to fourth-stage larval molt, as it is the first molt of D. immitis after it invades the vertebrate host. Microfilaremic blood was collected weekly from D. immitis–infected dogs with or without doxycycline treatment at 10 mg/kg as recommended by the American Heartworm Society. Blood was collected weekly until the end of doxycycline treatment. The blood was used for L3 production and mf isolation. Wolbachia levels in mf and L3 were measured using real-time quantitative PCR. L3 were cultured in vitro for 9 days to assess whether molting occurred. The Fisher’s exact test and Bonferroni correction were used for statistical analysis. The molting of L3 from the doxycycline-treated groups did not show a significant difference compared to the L3 from the control group at weeks 0, 1, 2, 3, and 4. The Wolbachia levels in mf and L3 decreased starting from 7 days post-treatment and remained less than five percent of controls throughout the treatment. Doxycycline treatment can eliminate Wolbachia in both mf and subsequently developed L3. The molts of the mf to L3 in the mosquito and the L3 to L4 molt in vitro do not appear to be impacted by the reduction or elimination of Wolbachia.

The online version contains supplementary material available at 10.1007/s00436-025-08506-z.