Glycoprotein Hormone Receptor Knockdown Leads to Reduced Reproductive Success in Male Aedes aegypti

Abstract

Glycoprotein hormone receptors mediate a diverse range of physiological functions in vertebrate and invertebrate organisms. The heterodimeric glycoprotein hormone GPA2/GPB5 and its receptor LGR1, constitute a recently discovered invertebrate neuroendocrine signaling system that remains to be functionally characterized. We previously reported that LGR1 is expressed in the testes of adult Aedes aegypti mosquitoes, where its immunoreactivity is particularly regionalized. Here, we show that LGR1 immunoreactivity is associated with the centriole adjunct of spermatids and is observed transiently during spermatogenesis in mosquitoes, where it may act to mediate the regulation of flagellar development. RNA interference to downregulate LGR1 expression was accomplished by feeding mosquito larvae with bacteria that produced LGR1-specific dsRNA, which led to defects in spermatozoa, characterized with shortened flagella. LGR1 knockdown mosquitoes also retained ∼60% less spermatozoa in reproductive organs and demonstrated reduced fertility compared to controls. To date, the endocrine regulation of spermatogenesis in mosquitoes remains an understudied research area. The distribution of LGR1 and detrimental effects of its knockdown on spermatogenesis in A. aegypti indicates that this heterodimeric glycoprotein hormone signaling system contributes significantly to the regulation of male reproductive biology in this important disease-vector.

Expression Profiling, Downstream Signaling and Subunit Interactions of GPA2/GPB5 in the Adult MosquitoAedes aegypti

“…Using gene-specific primers (Table S1), A. aegypti GPA2 and GPB5 sequences were amplified from previously prepared constructs (14) that contained the GPA2 and GPB5 complete open reading frames (ORFs). Sense and antisense probes were then generated following a similar protocol as recently reported (19). Briefly, cDNA fragments were ligated to pGEM T Easy vector (Promega, Madison, WI, USA) and used to transform NEB 5-α competent Escherichia coli cells (New England Biolabs, Whitby, ON, Canada).…”

“…In vertebrates, the function of GPA2/GPB5 has been implicated, or at least suggested, to be involved in reproduction (6), thyroxine production (3, 7), skeletal development (8), immunoregulation (9, 10) and the proliferation of ovarian cancer cell lines (11). For invertebrate species, GPA2/GPB5 has been implicated or demonstrated to function in development (12–15), hydromineral balance (13–18) as well as in reproduction (18, 19).…”

“…In adults, LGR1 was found localized to epithelia throughout the gut where GPA2/GPB5 could regulate feeding-related processes and hydromineral balance (18). Notably, LGR1 transcript expression was also observed in the reproductive organs of males and females (19). In adult male mosquitoes, knockdown of LGR1 expression led to abnormal spermatogenesis with spermatozoa displaying malformations such as shortened flagella and consequently, LGR1-knockdown males had 60% less spermatozoa as well as significantly reduced fecundity relative to control mosquitoes (19).…”

Expression Profiling, Downstream Signaling, and Inter-subunit Interactions of GPA2/GPB5 in the Adult Mosquito Aedes aegypti

“…In vertebrates, the function of GPA2/GPB5 has been implicated, or at least suggested, to be involved in reproduction (6), thyroxine production (4, 7), skeletal development (8), immunoregulation (9,10) and the proliferation of ovarian cancer cell lines (11). For invertebrate species, GPA2/GPB5 has been implicated or demonstrated to function in development (12)(13)(14)(15), hydromineral balance (13)(14)(15)(16)(17)(18) as well as in reproduction (15,17,19).…”

“…In adults, LGR1 was found localized to epithelia throughout the gut where GPA2/GPB5 could regulate feeding-related processes and hydromineral balance (17). Notably, LGR1 transcript expression was also observed in the reproductive organs of males and females (19). In adult male mosquitoes, knockdown of LGR1 expression led to abnormal spermatogenesis with spermatozoa displaying malformations such as shortened flagella and consequently, LGR1-knockdown males had 60% less spermatozoa as well as significantly reduced fecundity relative to control mosquitoes (19).…”