‘We like it wet’: a comparison between dissection techniques for the assessment of parity in Anopheles arabiensis and determination of sac stage in mosquitoes alive or dead on collection

BACKGROUND: The determination of parous rates in mosquitoes, despite numerous shortcomings, remains a tool to evaluate the effectiveness of control programs and to determine vectorial capacity in malaria vectors. Two dissection techniques are used for this. For one, the tracheoles of dried ovaries are examined with a compound microscope and in the other the follicular stalk of ovaries is examined, wet, with a stereomicroscope. The second method also enables the sac stage of parous insects (which provides information on the duration of the oviposition cycle) and the mated status of insects to be determined. Despite widespread use the two techniques have not previously been compared. METHODS: We compared the two dissection techniques using Anopheles arabiensis, collected with a tent-trap in Eritrea. The paired ovaries were removed in water and one was examined by each method. From a separate set of dissections from Tanzania, we also determined if the sac stages of Anopheles gambiae s.l. (83% of 183 identified by PCR being Anopheles arabiensis the remainder being A. gambiae) that were alive on collection were different to those that died on collection and what the implications for vectorial capacity estimation might be. RESULTS: Seven per cent of the dry ovaries could not be classified due to granulation (yolk) in the ovariole that obscured the tracheoles. The sensitivity of the dry dissection was 88.51% (CI [79.88–94.35%]) and the specificity was 93.55% (CI [87.68–97.17%]) among the 211 ovaries that could be classified by the dry technique and compared to the ovaries dissected wet. 1,823 live and 1,416 dead from Furvela tent-traps, CDC light-trap and window-trap collections were dissected ‘wet’ from Tanzania. In these collections parous insects were more likely to die compared to nulliparous ones. The proportion of parous mosquitoes with ‘a’ sacs (indicative of recent oviposition) was significantly greater in insects that were dead (0.36) on collection in the morning compared to those that were alive (0.12) (Chi square 138.93, p < 0.001). There was a preponderance of newly emerged virgin insects in the outdoor collection (Chi sq = 8.84, p = 0.003). CONCLUSIONS: In anophelines the examination of mosquito ovaries using transmitted light in a ‘wet’ dissection is a more useful and informative technique than examination of dry ovaries. In order to correctly estimate the duration of the oviposition cycle mosquitoes should be dissected as soon as possible after collection. Younger insects were more likely to attempt to feed outdoors rather than indoors.