Table 5.

Methodological parameters extracted from pyrethroid + PPF net biological durability monitoring methods, which scored ovary development following dissection. Methods were compared and a consensus value was proposed for each parameter. Justification for this choice regarding each parameter is listed. Superscript numbers = Study ID.

	CREC, Benin SOP BL/159/01-S v01 4	KCMUCO, Tanzania SOP 008v02 5	Proposed for Consensus SOP	Justification
Author	Syme	Martin, Matowo, and Furnival-Adams	Lees and Lissenden	-
Method of exposure	Not included in SOP	Cone test	Cone test	The cone test has been used in several studies to evaluate PPF nets and seems to be a suitable method of exposure.
Exposure time	Not included in SOP	3 min	3 min	This is the standard exposure time used in WHO cone bioassays [6]. Preliminary validation testing will be conducted to look at effect of exposure time.
Age of mosquitoes	Unknown	2–5 days old	3–5 days	This age range falls within the range of standard cone test (2–5 days, [6]) but allows an extra day for mating to increase likelihood of insemination. Effect of age for PPF is unknown and could be validated, but should be held constant until it is.
Blood feeding timing	‘Blood-fed at the time of collection/testing’.	Females ‘freshly blood fed’ for exposure.	3–9 h before net exposure. Blood fed using method of feeding standard for the test population (e.g., Hemotek membrane feeding system, arm feed, animal feed).	There is little data available and some contradiction on the impact of time of blood feeding, and this could be validated. Consensus was that this was a suitable, and logistically possible, method.
Mosquitoes per replicate	N/A	5	5 per cone	This is the standard number used in WHO cone bioassays [6].
Replicates per piece of net	N/A	20–25 replicates (n = 100–150); 4 per piece; 30 nets per treatment.	2 replicates per piece (8 cones per net)	Consensus was that this was a feasible number for testing. Numbers will be confirmed during multi-center validation.
Replicate nets per treatment	N/A		A minimum of 30 nets of each treatment at each time point.	WHO guidelines [6] recommend a minimum of 30 nets (at time points 0–24 months), and a minimum of 50 nets at 36 months testing.
Species/strain	Anopheles mosquitoes (generic SOP for dissection).	An. gambiae s.s. Muleba kis (kdr east and mixed-function oxidize resistance), or wild blood-fed resistance mosquitoes of unknown age with species id at time of dissection.	Lab-reared pyrethroid-susceptible strain. Lab-reared pyrethroid-resistant strain. Lab strains characterized before and after the bioassays for each time point as per strain characterization guidelines (Lees et al. in prep).	Pyrethroid-susceptible strain to monitor pyrethroid durability. Pyrethroid-resistant strain to monitor durability of PPF.
Time of dissection	72 h post-exposure	72 h post-exposure	72 h post-exposure	This allows 3 days for bloodmeal digestion and egg maturation.
Blinded samples	No	Yes	Yes	Controls for scorer subjectivity.
Number of scorers	2, in case of discrepancy calculate the average (only for egg count).	2, using slide or photograph if slide cannot be counted on the same day. 3 scorers in case of discrepancy.	2, using slide or photograph if slide cannot be counted on the same day. 3rd scorer in cases of discrepancy.	Controls for scorer subjectivity.
Microscope details	Can use dissecting microscope, better a compound microscope at 4× or 10×.	0.7× magnification, stereomicroscope.	Microscope details not critical. However, we recommend using a magnification of ×4 or ×10 for dissections and ×40 for observation of eggs.	-
Entomological endpoints measured	Live/dead and gravid/semi-gravid at time of collection, egg development stage, and fertility status of each mosquito, total number of eggs present in ovary (1/2 per female?).	KD: 60 min. Mortality: 24 h. Mortality: 48 h. Mortality: 72 h. % of dissected females with under-developed ovaries 72 h post-feeding. Proportion of dissected females with deformed eggs. Average number of eggs in the ovaries 72 h post-feeding.	Primary endpoint: Fertility inhibition (fertility rate/fertility rate in the negative control). Additional measures: KD: 60 min. 24-h mortality. Egg development stage. Fertility rate (proportion with developed ovaries/total).	A preliminary validation test will be conducted to establish if other endpoints should be included, e.g., number of eggs in each dissected ovary.
Definition of Fertility	Christophers’ scale to score development stage of eggs (I–V); female is fertile if eggs are V and sterile if eggs are I–IV.	Christophers’ stages to score development stage of eggs (I–V); female is fertile if eggs are V and sterile if eggs are I–IV. Inconclusive if both are present.	Score development stage of eggs (1–5) [15]. Female is classed as fertile if all eggs are 5 and sterile if eggs are 1–4. If both classes 4 and 5 are present, the results are inconclusive.	This is a well-established method for scoring fertility
Controls	-	Untreated net. Standard LN: Interceptor.	Negative control: Untreated control net. Positive control 1: New pyrethroid + PPF net of the same brand.	Untreated net controls for handling procedure and checks for contamination, and provides denominator for measuring oviposition inhibition. New pyrethroid + PPF net provides ‘baseline’ and allows us to monitor the suitability of test mosquito strains.
Notes on the protocol	Dissect all mosquitoes left alive at 72 h post-collection, but if there are not adequate numbers, also dissect dead mosquitoes at this time. Photographs taken of eggs.	Method from Detinova et al. 1962. Photographs taken of eggs.	If the testing site has the capacity to photograph dissected ovaries, then this should be conducted. Photographs can then be used in future training, and machine learning activities.