Histological confinement of transglutaminase-mediated nit sheath crosslinking is essential for proper oviposition and egg coating in the human head louse, Pediculus humanus capitis

Abstract

Background: Head louse females secrete liquid gel, which is mainly composed of the louse nit sheath protein 1 (LNSP1) and LNSP2, when they lay eggs. The gel is crosslinked by transglutaminase (TG) to form the nit sheath, which covers most of the egg except the top operculum area where breathing holes are located. Knowledge on the selective mechanism of nit sheath solidification to avoid uncontrolled crosslinking could lead to designing a novel method of louse control, but no information is available yet.

Methods: To elucidate the crosslinking mechanisms of nit sheath gel inside the reproductive system of head louse females, in situ hybridization in conjunction with microscopic observation of the oviposition process was conducted.

Results: Histochemical analysis revealed that LNSP1 and LNSP2 are expressed over the entire area of the accessory gland and uterus, whereas TG expression site is confined to a highly localized area around the opening of posterior oviduct. Detailed microscopic observations of oviposition process uncovered that a mature egg is positioned in the uterus after ovulation. Once aligned inside the uterus, the mature egg is redirected so that its operculum side is tightly held by the ventral end of the uterus being positioned toward the head again and its pointed bottom end being positioned toward the dorsal end of the uterus, which functions as a reservoir for the nit sheath gel.

Conclusions: Physical separation of the TG-mediated crosslinking site from the ventral end of the uterus is necessary to avoid uncontrolled crosslinking inside the uterus and to ensure selective crosslinking over only the lower part of egg without any unwanted crosslinking over the operculum during oviposition.

Keywords: Egg sheath; Head louse; LNSP; Nit; Oviposition; Pediculus humanus capitis; Transglutaminase.

Fig. 1

Histological sites of LNSP1, LNSP2 or TG transcription in the accessory glands and uterus. Representative microscopic images of the accessory glands and uterus of head louse females following in situ hybridization with LNSP1 (a), LNSP2 (b) or TG (c) probe. a, b FITC-labeled probes were used for LNSP1 and LNSP2. Both LNSP1 and LNSP2 were mainly detected in the entire areas of accessory gland with reduced expression being detected in the uterus (green signal). Images of nuclear staining (blue signal) and LNSP1 or LNSP2 were merged. c For TG, DIG-labeled probes were used for signal amplification. TG was exclusively expressed at the focal area around the opening of posterior oviduct (dark purple). A dotted circle indicates the opening of posterior oviduct where an egg passes through for oviposition. ag accessory gland, vu ventral end of uterus, du dorsal end of uterus, po posterior oviduct

Fig. 2

Schematic diagram of the oviposition processes of head louse females. For better view, accessory gland was graphically removed, and connecting area of accessory gland was marked with a yellow circle. Stage I, all mature eggs are still inside the ovary (~ 170 min); stage II, mature eggs are ovulated into the anterior oviduct (~ 40 min). During ovulation, a mature egg first moves to the other side of lateral oviduct, thereby changing its position to upside down, and then to the anterior oviduct; stage III, mature eggs stay inside the uterus and are coated with nit sheath gel materials in the dorsal end of the uterus (~ 80 min); stage IV, coated eggs move to the posterior oviduct where crosslinking begins. Actual images corresponding to each stage were provided in the bottom row. ov, ovary; ao, anterior oviduct; vu, ventral end of uterus; du, dorsal end of uterus; ag, accessory gland; po, posterior oviduct; v, vulva

Fig. 3

Detailed ovulation processes during the oviposition stages I and II. A mature egg first moves to the other side of lateral oviduct, thereby changing its position to upside down, and then to the anterior oviduct. The red arrows indicate the movement direction of mature eggs inside the oviduct during ovulation. The yellow arrowheads indicate operculum of an egg

Fig. 4

Image of a mature egg with its operculum attached to the ventral end of the uterus. The operculum stays attached to the ventral end of the uterus even after surgically removing the dorsal part of the uterus. The ventral uterus turns inside out in this picture

Fig. 5

Reproductive system of a female head louse during oviposition. a, b A mature egg is aligned inside the uterus with the operculum being tightly covered by the ventral end of the uterus (see red circle) and bottom side covered by the dorsal end of the uterus (see blue circle) during the stage III. Black arrows indicate the nit sheath gel reservoir in the dorsal end of the uterus. ov, ovary; ao, anterior oviduct; vu, ventral end of uterus; du, dorsal end of uterus; ag, accessory gland; po, posterior oviduct; v, vulva. c An egg dissected out from uterus. A white arrow indicates nit sheath gel coated on the egg