Functional Role of AsAP in the Reproduction of Adelphocoris suturalis (Hemiptera: Miridae)

Abstract

Adelphocoris suturalis Jakovlev (Hemiptera: Miridae) is an omnivorous agricultural pest that has severe economic impacts on a diverse range of agricultural crops. Although the targeted disruption of reproductive development among insects has been proposed as a novel control strategy for pest species, the current understanding of the physiology and molecular mechanisms of A. suturalis reproduction is very limited. In this study, we isolated a putative A. suturalisaspartic protease (AsAP) gene that is highly expressed in the fat body and ovaries of sexually mature females. The double-stranded RNA (dsRNA)-mediated knockdown of AsAP suppressed ovarian development and negatively impacted female fertility, which suggested that it plays an essential role in A. suturalis reproduction. The results of this study could help to expand our understanding of A. suturalis reproductive development and have the potential to facilitate the development of effective strategies for the better control of this pest species.

Keywords: Adelphocoris suturalis Jakovlev; RNAi; aspartic protease; ovarian development; reproduction.

Figure 1

The structural domains and sequence of Adelphocoris suturalis aspartic proteases (AsAP): (A) the nucleotide and deduced amino acid sequence of AsAP with the putative signal peptide indicated by red letters, the potential N-glycosylation sites indicated by stars, the amino acids (DTG) comprising conserved catalytic sites indicated by solid squares and the putative polyadenylation signal (AATAAA) indicated by the underline; (B) a schematic diagram illustrating the functional domains of AsAP. SP: signal peptide; Asp: aspartic domain.

Figure 2

The phylogenetic relationships in the Adelphocoris suturalis aspartic protease (AsAP). A neighbor-joining tree was constructed using the JTT model for amino acids with confidence values (>50%) shown on the branches, which were based on 1000 rapid bootstrap replicates. Species abbreviations are: Asut, Adelphocoris suturalis; Hlon, Haemaphysalis longicornis; Ppla, Pristhesancus plagipennis; Cflo, Camponotus floridanus; Hill, Hermetia illucens; Dper, Dysdercus peruvianus; Dmoj, Drosophila mojavensis; Zcuc, Zeugodacus cucurbitae; Ppyr, Photinus pyralis; Btab, Bemisia tabaci; Fexs, Formica exsecta; Rpom, Rhagoletis pomonella; Rzep, Rhagoletis zephyria; Ccap, Ceratitis capitate; Tcur, Temnothorax curvispinosus; Waur, Wasmannia auropunctata; Dmax, Dipetalogaster maximus; Rmic, Rhipicephalus microplus; IRic, Ixodes Ricinus; Bmor, Bombyx mori; Sman, Schistosoma mansoni; Acan, Ancylostoma caninum; Hsap, Homo sapiens; Cnuc, Cocos nucifera; Hhal, Halyomorpha halys; and Clec, Cimex lectularius.

Figure 3

The tissue- and stage-dependent expression profiles of AsAP from the RT-qPCR-based analysis of AsAP transcript levels across different (A) developmental stages or (B) tissues. The whole bodies of 1st, 2nd, 3rd, 4th and 5th instar nymphs, sexually immature (1-day-old) male and female adults and sexually mature (8-day-old) male and female adults were collected separately to determine the RNA distribution profiles. Samples from the head, thorax, ovary, gut and fat body were collected separately from 8-day-old females. Elongation factor-1γ (EF1γ) was used as a reference gene for the AsAP transcript normalization. The values are expressed as the means ± SEM, based on three independent biological replicates. Different letters show significant differences (p < 0.05, one-way ANOVA followed by Tukey’s HSD test).

Figure 4

AsAP knockdown suppressed ovarian development. Newly emerged females were micro-injected with dsAsAP or dsGFP (control): (A) the AsAP transcript levels at 5, 10, 14 and 18 days post-injection; (B) the number of oocytes per ovary pair; (C) the ovaries from females that were injected with dsGFP; (D) the ovaries from females that were injected with dsAsAP. (C,D) were imaged at 10 days post-injection using a stereo microscope. All values are expressed as means ± SEM, based on three independent biological replicates. Asterisks indicate statistical significance (** p < 0.01 and *** p < 0.001, according to Student’s t-tests).

Figure 5

AsAP knockdown impacted female fertility. Newly emerged females were micro-injected with dsAsAP or dsGFP (control) and four reproductive parameters evaluated: (A) lifetime fecundity; (B) adult longevity; (C) egg hatching rate; (D) pre-oviposition period. All values are expressed as means ± SEM, based on three independent biological replicates. Asterisks indicate statistical significance (* p < 0.05 and ns represents non-significant results, According to Student’s t-tests).